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The histologic activity of associated hepatitis was examined in 285 patients who underwent hepatectomy for hepatocellular carcinoma (HCC), to determine if the histologic activity is an independent risk factor for postoperative mortality due to liver failure. The proportion of patients with liver cirrhosis who died due to liver failure (6/180, 3.3%) was not different from that of patients with chronic hepatitis (2/68, 2.9%). However, mortality was higher in patients with liver cirrhosis and active hepatitis (4/46, 8.7%) than in those with cirrhosis and inactive hepatitis (2/134, 1.5%, P0.05). Such difference was not observed in the chronic hepatitis group. Multivariate analysis showed that clearance of indocyanine green at 15 min (ICGR15) and activity of hepatitis were two independent risk factors for postoperative mortality due to liver failure. In conclusion, histologic activity of associated hepatitis should be taken into account in hepatic resection of HCC in cirrhotic liver, in addition to the functional reserve of the liver.

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action of alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX slowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

Induction of unresponsiveness to graft is one of major interest in xenotransplantation. Two different modalities [direct graft treatment by mitomycin C (MMC) and blockage of the lymphocyte function-associated antigen-1/intracellular adhesion molecule-1 (LFA-1/ICAM-1) pathway in recipients by species-specific mAbs] were tested for their ability to produce unresponsiveness to secondary islet xenografts.Collagenase-digested WS (RT1k) rat islets, purified by Ficoll density gradient, were incubated for 30 min with MMC 10 microg/ml, cultured for 20 hr, and transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (H-2b) mice. Recipient mice were divided into experimental groups according to anti-rat ICAM-1 and/or anti-mouse LFA-1 mAb treatment and transplantation of MMC-treated or nontreated islets.MMC pretreatment alone prolonged graft survival, with a mean survival time (MST) of 23.0+/-7.4 days, compared with that of cultured islets (12.4+/-2.7 days; P0.01). MMC treatment of islets significantly augmented graft survival, compared with that of crude islet grafts under treatment with anti-donor ICAM-1 mAb (MST:41.3+/-30 vs. 16.6+/-5.4 days, P0.01), anti-recipient LFA-mAb (MST:70.3+/-28.9 vs. 30.4+/-10.4 days, P0.001), or both mAbs (MST:88.1+/-24.1 vs. 23+/-7.4 days, P0.0001). One of six, four of nine, and six of eight animals accepted MMC-treated islet xenografts over 100 days after treatment with anti-rat ICAM-1, anti-mouse LFA-1, or both mAbs treatments, respectively, whereas none of the animals accepted nontreated islets under the same treatment. When the mice bearing long-term functioning xenografts were challenged with the secondary graft from the original donor strain, the animals previously treated with anti-recipient LFA-1 and anti-donor ICAM-1 mAbs were more prone to accept it than animals given anti-recipient LFA-1 mAb alone (MST: 55.8+/-25.7 vs. 15+/-2.4 respectively; P0.001), although they rejected the third-party xenograft and allograft acutely.In the xenogeneic islet transplantation model, MMC graft pretreatment and blockage of the ICAM-1/LFA-1 pathway constitute a potent protocol for inducing unresponsiveness to islet xenografts.

Between January 1997 and December 1998, six patients with germ cell tumor were treated with high-dose CEC: carboplatin (1,500 mg/m2), etoposide (1,200 mg/m2) and cyclophosphamide (100 mg/kg), followed by peripheral blood stem cell transplantation (PBSCT) at Nagoya University Hospital. Four patients received one cycle of high-dose CEC and two received two cycles. The reasons why the high-dose CEC was administered included: 1) refractory to the induction chemotherapy (AFP/beta-HCG elevated during the induction chemotherapy or prolonged half-life of each marker) in three patients, 2) relapse in two patients, and 3) consolidation in one with unresectable mediastinal residual tumor. There were no treatment-related deaths and grade 1 hepatotoxicity occurred in one (17%) patient. The median duration (range) from PBSCT until a granulocyte count of 500/microL and a platelet count of 50,000/microL was 8.5 (8-11) and 11 (9-16) days, respectively. Of the six patients studied, 5 responded to the treatment; two achieved a complete response (CR) and three achieved a partial response (PR). One patient achieving a CR and two achieving a PR remained in complete remission after 23 to 24 months of follow-up, while the remaining patients with a CR, a PR and an incomplete response died of the disease. High-dose CEC could be administered without serious toxicity but the effectiveness of high-dose CEC for the poor-risk patients with germ cell tumor needs to be further investigated.

Arteriovenous malformation (AVM) of the scalp is uncommon, and a subtype which has connection with the intracranial dural sinus is extremely rare. Only 3 cases have been reported. We present a case of congenital AVM of the scalp which was connected with the intracranial venous sinus. A 27-year-old woman had been noted as having a pulsatile soft mass in the midline of the occipital region since her birth. She visited our hospital because of pain and enlargement of the mass. The patient had had no history of trauma. Physical examination revealed a pulsatile scalp mass in the midline of the occipital region, measuring 3.5 x 3.5 x 1 cm. A loud bruit was ausculated. Tenderness was noted. The skin over the mass was slightly reddish. No focal neurological deficits were noted. Plain skull films demonstrated a round defect in the midline of the occipital bone. Magnetic resonance imaging (MRI) demonstrated a subcutaneous mass with low signal intensity and an infratentorial mass with flow void. 3D CT angiograms demonstrated a subcutaneous vascular mass with a single large vein draining into the right transverse sinus. External carotid angiograms revealed a vascular lesion within the scalp with supply from the branches of the bilateral occipital arteries and the meningeal arteries. The nidus penetrated the skull and connected to a dilated varix, which had a draining vein shunting into the right transverse sinus. After embolization of the right meningeal feeding arteries, surgery was performed. The vascular lesion penetrated the skull and the dura. The infratentorial mass was in the epiarachnoid space and was fed by a small pial artery. The mass was excised completely after interruption of the pial artery and the draining vein. Postoperative course was uneventful. Histologically, the subcutaneous mass and infratentorial vascular mass were shown to be AVM and varix, respectively.

There is a general agreement that a preferential accumulation of alloantigens within the liver could induce hyporesponsiveness to the inoculated antigens. Entrapment of antigens in the liver may evoke an unique immune response in the organ and play a key role in determination of the fate of the transplanted grafts. To understand the immune response in the liver after inoculation of allogeneic donor antigens, we examined the immune response to systemically inoculated alloantigen in rats whose sensitized liver was replaced with that of naive rats or in naive rats whose liver was replaced with that of sensitized rats.Using implantation of syngeneic liver (alloantigen-accumulated/naive) in rats (naive/alloantigen-sensitized), we compared the immune responses to alloantigen between rats with hepatic/extrahepatic alloantigen at 24 hr after alloantigen inoculation. This was called sensitized-liver-grafted (SLG)/sensitized-liver-removed (SLR) treatment. The immune response to donor alloantigen in this model was evaluated by survival of skin or heart grafts, complement-dependent cytotoxicity (CDC) titer and delayed-type hypersensitivity (DTH) response.Compared with the mean survival time (MST) in donor spleen cell inoculated (DSI) rats (skin and heart, MST: 8.2+/-1.1 and 10.7+/-2.3 days), SLG rats rejected allografts in an accelerated fashion (skin and heart, MST: 5.5+/-0.5 and 4.2+/-0.8 days), associated with higher CDC titer and DTH response. In contrast, allograft survival was moderately prolonged in SLR (skin and heart, MST: 16.5+/-2.6 and 29.5+/-3.7 days) associated with suppressed CDC titer and DTH response. The survival of third-party allograft after SLG or SLR treatment (skin, MST: 9.3+/-1.5 or 9.7+/-0.6 days) indicated that immunological hyper/hyporesponsiveness was donor-specific.A strong anti-donor immune response was induced by the transfer of donor antigen-baring liver to naive rats 24 hr after alloantigen inoculation, whereas removal of the liver suppressed alloimmune response. Our results indicate that vigorous anti-alloimmune response occurred in the liver after systemic inoculation of donor spleen cells.

We evaluated the role of magnetic resonance (MR) imaging with gadolinium-ethoxybenzyl-diethylenetriamine-pentaacetic acid (Gd-EOB-DTPA), in estimating regional liver function in a rat ischemia-reperfusion model. Ischemic liver damage was induced in the right lobe by vascular clamping for 0 (sham), 30 (I-30), 60 (I-60), and 90 min (I-90 group). The ischemic lobes in the I-60 and I-90 groups was clearly visualized as a high intensity area in the T1 images at late phase of Gd-EOB-DTPA enhancement, Moreover, the T1/2 of signal intensity in ischemic lobes significantly correlated with the duration of vascular clamping. We also observed significant correlation between T1/2 and ATP concentration in the liver tissue (r = -0.719, P = 0.04). Our results indicate that MR imaging with Gd-EOB-DTPA is useful for evaluation of regional liver function in rats.

TAK-778 [(2R,4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4, 5-tetrahydro-4-methyl-7, 8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxyamide; mw 505.53], a novel osteoblast differentiation promoting compound, was characterized in vitro and in vivo models. TAK-778 at doses of 10(-6) M and higher promoted potently bone-like nodule formation in the presence of dexamethasone in rat bone marrow stromal cell culture. This was accompanied by increases in cellular alkaline phosphatase activity, soluble collagen release, and osteocalcin secretion. Under the culture conditions, TAK-778 also stimulated the secretion of transforming growth factor-beta and insulin-like growth factor-I, indicating that TAK-778 may exert regulatory effects on osteoblast differentiation via autocrine/paracrine mechanisms. Furthermore, the in vivo osteogenic potential of TAK-778 was studied in bony defect and osteotomy animal models, using sustained release microcapsules consisted of a biodegradable polymer, poly (dl-lactic/glycolic) acid (PLGA). Single local injection of TAK-778/PLGA-microcapsules (PLGA-MC) (0.2-5 mg/site) to rat skull defects resulted in a dose-dependent increase in new bone area within the defects after 4 weeks. When the pellet containing TAK-778/PLGA-MC (4 mg/pellet) was packed into place to fill the tibial segmental defect in rabbit, this pellet induced osseous union within 2 months, whereas the placebo pellet did not. In addition, single local application of TAK-778/PLGA-MC (10 mg/site) to rabbit tibial osteotomy site enhanced callus formation accompanied by an increase in breaking force after 30 days. These results reveal for the first time that a nonendogenous chemical compound promotes potently osteogenesis in vitro and enhances new bone formation during skeletal regeneration and bone repair in vivo and should be useful for the stimulation of fracture healing.

A 79-year-old male was admitted to our hospital for further examination on gastric carcinoma (1' type) in the cardia. The histology of biopsied tissue was moderately differentiated adenocarcinoma (tub2). The patient refused a gastrectomy and received three cycles of local injection therapy with OK-432 + Beriplast into the tumor. However, the tumor showed no decrease in size. Considering the quality of life, the patient was given out patient treatment with 5'-DFUR (Furtulon, 800 mg/day). Three months later, the patient showed a partial response (PR) on the basis of gastric X-ray and endoscopic findings. No adverse reactions to the drug were seen. The patient has been receiving the same drug since then, and has continued to show PR for 15 months. Biopsied tissues were checked immunohistochemically for expression of thymidine phosphorylase (TdRPase), and changes in tissue TdRPase level were examined by ELISA. The TdRPase level decreased with shrinking of the tumor. These results suggest that the shrinking of tumor following 5'-DFUR therapy is closely related to TdRPase.

Rejection of pancreatic islet grafts is still a serious problem. We evaluated the effect of mitomycin C (MMC) on the survival of crude islets grafts after xenogeneic islet transplantation.WS (RT1k) rat islets pretreated with various concentrations of MMC (0, 1, 3.2, 10, 32, 50, 100, 320, and 1,000 microg/ml) were transplanted into C57BL/6 mice with streptozotocin-induced diabetes. In vivo graft function was assessed by a daily measurement of nonfasting blood glucose concentration in each animal. We also examined the separate effect of MMC on purified islets and contaminants present in the crude islet preparation.MMC at doses of 10, 32, 50, and 100 microg/ml resulted in a significant prolongation of the mean graft survival time from a control of 12.4+/-2.5 days to 23+/-7.4, 17.5+/-5.4, 25.5+/-14.7, and 26.7+/-8.9 days, respectively. Deterioration of glucose metabolism was noted when the dose exceeded 32 microg/ml, whereas at 320 microg/ ml, MMC failed to restore normoglycemia. Prolongation of survival time of crude islets was the result of its effect on islets and contaminant components of the crude islet preparation. In vitro study showed that MMC treatment at a higher concentration than 10 microg/ml reduces the stimulatory as well as proliferative capacity of lymph node cells.Pretreatment of pancreatic islets with MMC at 10 microg/ml prolongs xenograft survival without deterioration of in vivo graft function. This novel treatment modality represents a new strategy for the modulation of immunity of islets and contaminants in crude islet preparations.

Macroscopic synovitis of the glenohumeral joint is frequently seen during arthroscopy in patients with anterior instability. Interleukin-1beta is known to be expressed in inflamed tissue, to correlate with the magnitude of inflammation, and to affect articular cartilage in the joint. We hypothesized that chronic synovitis may occur in the glenohumeral joint in patients with anterior instability. The purpose of this study was to examine the expression of interleukin-1beta in the synovium of the glenohumeral joint with anterior instability and to discuss its clinicopathologic significance. Specimens of synovial tissue around the greater tuberosity in the subacromial synovium (as controls) and around the rotator interval in the glenohumeral synovium were obtained from 10 patients who had anterior instability without signs of subacromial impingement. Semiquantitative reverse transcriptase-polymerase chain reaction was used to compare the levels of interleukin-1beta mRNA expression in the glenohumeral joint with those in the subacromial bursa. We also employed immunohistochemistry and in situ reverse transcriptase-polymerase chain reaction to detect the cells producing interleukin-1beta protein and mRNA. The levels of interleukin-1beta mRNA expression were significantly higher in the glenohumeral joint than in the subacromial bursa (p0.01). Histology showed nonspecific inflammation in all 10 samples of glenohumeral synovium, whereas no inflammation was seen in seven of 10 samples of subacromial synovium. Immunohistochemistry identified interleukin-1beta protein in the vessels and inflammatory and synovial cells (from lining to sublining layers) in synovium of the glenohumeral joint, whereas immunoreactivity was negative in seven subacromial bursa. The remaining three synovial specimens of subacromial bursa, however, showed positive immunoreactivity that was unremarkable and confined around the vessels. In situ reverse transcriptase-polymerase chain reaction was exclusively performed in the synovial specimens of the glenohumeral joint, which exhibited a positive reaction (in the same kinds of cells as seen with immunohistochemistry) in the lining and sublining layers and to a lesser extent in the stroma. Thus, our data confirmed the increased production of interleukin-1beta in the synovium of the glenohumeral joint in patients with anterior instability, suggesting the presence of chronic inflammation at the site. We conclude that this chronic synovitis may be partly associated with the development of dislocation arthropathy in the long term.

Three females with urinary retention caused by uterine leiomyoma are reported. The patients were 35, 48 and 50 years old. In each patient, transabdominal ultrasonography showed a large and homogeneous mass located in the retrovesical space, compressing the bladder. Pre-operative computed tomographic (CT) scan and magnetic resonance (MR) imaging revealed uterine leiomyoma which severely compressed the bladder from the posterior wall to the urethra. The urinary symptoms completely resolved in all patients following total hysterectomy, and postoperative uroflowmetry demonstrated normal voiding.

We performed retroperitoneal lymph node dissection (RPLND) on 14 patients (IIA: 1, IIB: 4, IIIA: 3, IIIB2: 2, IIIC: 4) with testicular and one with retroperitoneal germ cell tumor at the Nagoya University Hospital between 1986 and 1997. According to the international germ cell consensus classification, 4 patients were classified as "good-prognosis", three as "intermediate-prognosis" and eight as "poor-prognosis". RPLND was performed on 12 patients with the tumor marker levels normalized preoperatively and on three without the marker normalization. The mean surgical time was 510 (195-1, 125) minutes and the mean bleeding volume was 3,806 (100-12,598) g. The surgical time and bleeding volume were correlated with the size of the tumor in the body axis. Intraoperative complications occurred in 5 (33%) out of 15 patients: injury of renal artery (2), renal vein (1), ureter (1) and common iliac artery (2). Postoperative complications occurred in 2 patients: ileus (2) and lower extremity edema resulting from resection of the inferior vena cava (1) and would dehiscence (1). Of the 8 patients whose completely resected retroperitoneal tumors were necrosis/fibrosis or teratoma (psCR), 6 achieved survival with no evidence of disease (NED). Among 4 patients, whose tumor was not completely resected but pathologically diagnosed as necrosis/fibrosis or teratoma (pCR), NED without recurrence was achieved in 2 and also in one with resection of relapsed teratoma 2.5 years after RPLND. All three patients with cancer tissues pathologically retained in the resected tumors (sCR or psIR), consequently died of the disease. In six patients with relapse, the initial sign was elevation of the tumor marker levels, which was noted more than 30 days postoperatively in 2 patients with psCR and 7 to 15 days in 4 patients without psCR. We believe that RPLND is needed to examine the pathology and to predict the prognosis of the poor-risk patients with NSGCT. Careful dissection of vessels is needed to reduce vascular complications.

In this study, preoperative mitomycin-C- (MMC) treated donor-specific transfusion (DST) was examined for its ability to induce unresponsiveness to cardiac allografts in rats.DA (RT1a) rats were used as donors, BUF (RT1b) or WS (RT1k) rats as recipients, and Lew (RT1l) rats as third party donors. BUF or WS rats were given i.v. injection of DA spleen cells (SPCs) suspension (5x10(7)/l ml) with or without MMC treatment 10 days before cardiac transplantation. Delayed-type hypersensitivity and complement-dependent cytotoxicity assays were carried out in these animals separately to examine in vivo immunosuppressive effect. Suppressor assay was also examined to determine in vitro immunosuppressive effects in allogeneic mixed leukocyte culture.In the full allogeneic DA-to-BUF rat strain combination, preoperative i.v. administration of MMC-treated donor SPCs led to a significant prolongation of graft survival over the control (110+/-66 versus 7.2+/-0.8 days: P0.01), although administration of nontreated donor SPCs did not (9.3+/-1.0 days). This beneficial effect of MMC treatment was also seen in the DA-to-WS rat combination (31+/-16 days versus donor-specific transfusion alone; 11+/-1.5 days or untreated control; 12+/-1.5 days; P0.05). However, injection of third party DA SPCs in the Lew-to-BUF combination induced no significant prolongation of cardiac allograft survival compared with the untreated control (11+/-0.6 versus 11+/-2.0 days; NS), indicating that this prolongation effect was induced in an antigen-specific manner. The immunosuppressive effect was also secured for both delayed-type hypersensitivity response and anti-donor cytotoxic antibody production. Moreover, addition of MMC-treated SPCs to mixed lymphocyte culture led to antigen-specific suppression.Preoperative i.v. injection of MMC-treated donor SPCs is promising for inducing unresponsiveness in rat cardiac allograft model.

Cytokines are potentially active biological peptides that are known to play an important role in several immune responses. Several studies have reported the existence of a variety of cytokines in the nasal mucosa of patients with allergic rhinitis. However, there are few reports on cytokines released into the nasal secretion. In the present study, we investigated the sources, and levels of three key proinflammatory cytokines namely, IL-6, IL-8, and GM-CSF in the nasal secretion, as well the mechanisms of their release, by ELISA and immunohistochemistry. Firstly, we examined the levels of IL-6, IL-8, and GM-CSF in the nasal secretion after in vivo nasal challenge with methacholine (MC), histamine (HI) and allergen (Ag) in patients with nasal allergy to house dust mite (HDMAR). Next, we examined the levels of IL-6, IL-8, and GM-CSF released, in vitro, after Ag challenge of nasal scrapings from patients with HDMAR. Finally, we examined the sources of these cytokines in the nasal mucosa, by immunohistochemistry. After MC challenge in patients with HDMAR, the concentration of IL-6, but not IL-8, and GM-CSF, was significantly greater on the challenged side than on the contralateral side. Ag and HI provocation induced significantly greater levels of IL-6 and IL-8 secretion in patients with HDMAR, on the challenged side than on the contralateral side. GM-CSF was only detected in the nasal secretion after Ag challenge. Immunoreactivity for IL-6 and IL-8 was very similar in that it was predominantly localised to the apical portion of epithelial cells, the superficial lamina propria, gland cells, and migrating cells. The immunoreactivity for GM-CSF varied slightly from that of IL-6 and IL-8: strong immunoreactivity was detected in the basal part of epithelial cells, basement membrane, glandular ducts, and migrating cells. These results suggest that the levels, sources, and mechanisms of release of IL-6, IL-8, and GM-CSF in the nasal secretion of patients with HDMAR do vary, but are important in the manifestation of the allergic reaction.

Intrathymic microchimerism (MC) is thought to be responsible for inducing allograft tolerance. However, the role of MC in the thymus gland after transplantation, particularly in the rejection response, is unknown. We investigated serial changes in intrathymic cytokine production associated with MC and allograft rejection.Donor-specific cell injection (DSI) and heterotopic heart transplantation (HTx) were performed in the fully allogeneic combination using DA rats (RT1a) as donors and WS rats (RT1k)as recipients. MC was checked by polymerase chain reaction (PCR) using a donor RT1.Bbeta domain 1 region sequence-specific primers. Reverse transcription (RT)-PCR analysis of cytokine (interleukin [IL]-2, interferon-gamma, IL-4, and IL-10) profiles of the thymus was performed in animals given DSI, HTx, or DSI/HTx.DSI alone resulted in an immediate development of MC, detected by PCR, in various organs including the thymus, spleen, liver, and blood, of most rats, lasting for over 2 months. However, DSI-induced MC selectively disappeared in the thymus on day 7 after grafting, several days before the rejection of cardiac allograft. RT-PCR analysis of cytokine profiles showed that the levels of Th1 (IL-2 and interferon-gamma) cytokines transcribed in the thymus were higher than in the spleen. MC reappeared in the thymus on day 21 after grafting, but was not associated with elevation of Th1 cytokine transcription when allograft was replaced by fibrosis.Intrathymic MC does not always confer unresponsiveness to alloantigen, but can be eliminated after anti-donor response.

We previously showed that extracellular levels of N-acetylaspartate (NAA) increase when a medium with reduced NaCl concentration is perfused through a microdialysis probe, and proposed that NAA may be released during hypoosmotic swelling. Here, we demonstrate that this effect is due to hypoosmolarity of the perfusion medium, and not to low NaCl. NAA changes in the dialysate were compared with those of taurine as the osmoregulatory role of this amino acid is established. Reduction of the NaCl concentration in the perfusion medium increased the dialysate levels of NAA and taurine, but this effect was abolished when NaCl was replaced by sucrose to maintain isosmolarity. The NAA response to hypoosmolarity was smaller than that of taurine, but it may still be important to neurons as NAA is predominantly neuronal in the mammalian CNS.

We investigated the presence of K-ras gene mutation in plasma DNA and assessed its clinical value in patients with pancreatic adenocarcinoma. Mutations in codon 12 of the K-ras gene were examined by mutant allele-specific amplification method using DNA extracted from surgical specimens and plasma samples of 21 patients with pancreatic adenocarcinoma. K-ras gene mutation was detected in 15 of 21 (71%) primary tumors. In 9 of 15 (60%) patients with K-ras gene mutation-positive tumors, an identical mutation was detected in the plasma DNA. None of four patients with chronic pancreatitis or five healthy subjects had such mutations in plasma DNA. Tumors positive for K-ras gene mutation in plasma DNA were significantly larger (P = 0.04) and less likely to result in a curative cure after surgical resection (P = 0.09) than those negative for the mutation. Other clinicopathological features, including age, sex, histological type, mode of invasion, and metastasis, did not correlate with K-ras gene mutations in plasma DNA. Treatment resulted in disappearance of K-ras gene mutations in plasma DNA in six of nine (67%) patients. Three patients with a persistently positive K-ras gene mutation in pre- and post-treatment plasma samples were likely to show early recurrence or have a progressive disease. Our findings suggest that K-ras gene mutation can be detected in plasma DNA of patients with pancreatic adenocarcinoma. Detection of K-ras mutations in plasma may be clinically useful for evaluating tumor burden and efficacy of treatment.

Cellular interactions that lead to graft rejection were examined in a rat-to-mouse xenogeneic combination using species-specific monoclonal antibodies (mAbs) against donor and recipient intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) molecules, respectively. Although both mAbs displayed moderate blocking activity in an in vitro mixed lymphocyte response assay, strong suppression was observed when anti-donor (rat) ICAM-1 mAb was combined with anti-recipient (mouse) LFA-1 mAb. Likewise, significant prolongation of islet xenograft survival was observed with these mAbs. Thus, 0.05 mg of anti-mouse LFA-1 mAb and anti-rat ICAM-1 mAb given on days 0 and 1 produced significant prolongation of graft survival over the control (51+/-20 days vs. 10+/-3 days, P0.0001), but not when anti-mouse ICAM-1 mAb was combined with anti-mouse LFA-1 mAb (13+/-3 days). In this species combination, mouse T cells were able to proliferate in the presence of rat antigen-presenting cells (APCs) in a cell number-dependent manner, but not in the presence of mouse APCs. The binding assay showed that LFA-1 molecules on mouse T cells can bind immobilized rat ICAM-1 molecules. These results suggest that rat ICAM-1 molecules on APCs can interact with mouse LFA-1 molecules on T cells across a species barrier and that this binding generates the consequent immune responses leading to rejection. mAb treatment against these adhesion molecules of recipient as well as donor is crucial for preventing rejection in a xenogeneic transplantation model.

We report a rare case of myelopathy caused by compression of the upper cervical cord by the bilateral anomalous vertebral arteries. A 49-year-old man had dragged his right foot for 4 years. He also complained of a tingling sensation in his right arm and occipitalgia. Neurological examination disclosed right hemiparesis, hypalgesia in the right half of the body and hypertonicity of the lower extremities. MRIs showed a flow void area which compressed and distorted the spinal cord bilaterally at the level of the atlas. A vertebral angiogram showed that the bilateral vertebral arteries had pierced the dura matter under the posterior arch of the atlas, turned upward and laterally in the vertebral canal, making vascular loops at the level of the atlas. 3D CT angiography showed the loops convex medially in the anterior part of the vertebral canal. With these findings, we diagnosed the patient as suffering compression of the cervical cord by the bilateral anomalous vertebral arteries. Suboccipital craniectomy and C1 laminectomy were performed. When the dura mater was opened, the dorsolateral aspect of the spinal cord was found to be compressed and indented markedly by the vertebral arteries. To decompress the spinal cord, the vertebral arteries were retracted dorsolaterally by means of Gore-tex tape and anchored to the spinous process of the axis. Postoperatively, his neurological symptoms improved. Postoperative MRIs showed that the spinal cord was decompressed and had recovered its contour. A review of the literature revealed that only 3 such cases as this one have been reported. The clinical features of these rare cases are nonspecific myelopathy and concomitant occipitalgia or neck pain. The main cause of this rare entity seemed to be the compression of the vertebral artery through its course when it enters the vertebral canal between the atlas and the axis.

Antiadhesion therapy using monoclonal antibodies (mAbs) to adhesion molecules in vivo has been shown to produce significant prolongation of graft survival in various transplantation models. However, it remains unclear whether antiadhesion therapy operates by merely blocking adhesion between antigen-presenting cells and T cells physically and/or by blocking costimulatory signals while preserving signals mediated through T-cell receptors in vivo. We examined antigen-specific T-cell responses during and after antiadhesion therapy.BALB/c islets were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 mice given anti-lymphocyte function-associated antigen (LFA)-1 and/or anti-intercellular adhesion molecule-1 mAb treatment. The animals bearing surviving islet allografts were challenged with BALB/c or third-party islets on day 7 or more than 100 days after transplantation.Islet allografts were acutely rejected in untreated animals, with a mean survival time (MST) of 19+/-8 days. Administration of anti-LFA-1 mAb induced significant prolongation of graft survival with a mean survival time of 72+/-33 days, and half of the allografts showed indefinite survival. The animals given anti-LFA-1 mAb alone 7 days before transplantation showed acute rejection of BALB/c islets, whereas a significant number of animals given anti-LFA-1 mAb and the BALB/c islet allograft simultaneously accepted secondary BALB/c islets, but rejected third-party islets. Likewise, most of the animals bearing long-term functioning BALB/c allografts for more than 100 days accepted secondary BALB/c islets, but rejected C3H islets acutely. Interestingly, the spleen cells from these animals transferred unresponsiveness to BALB/c islets into the 2.5-Gy x-irradiated recipients, whereas those from naive animals induced acute rejection.These results indicate that anti-LFA-1 mAb treatment prevents T-cell activation leading to rejection, but results in a T-cell receptor engagement leading to antigen-specific unresponsiveness maintained by transferrable suppressor cells.

Surgical resection, transcatheter arterial embolization (TAE) and percutaneous ethanol injection therapy (PEIT) are effective for hepatocellular carcinoma (HCC), but the recurrence rate is high. We have devised a new therapy of transarterial immuno-embolization (TIE) with OK-432, fibrinogen and thrombin, and 2 cases are reported. Case 1: A 78-year-old Japanese male with HCC (diameter, 4 cm in subsegment 5) received TIE. The tumor size was markedly decreased, and the patient survived for more than 3 years without recurrence. Case 2: A 61-year-old Japanese male with HCC (diameter, 4.5 cm in segment 5) received hepatic subsegmentectomy following TIE. Histological examination of resected specimens following TIE showed massive infiltration of mononuclear cells in the main tumor. Tumor recurrence had developed three times thereafter, but was effectively treated by TIE. TIE may be an effective therapy for HCC.

N-Acetylaspartate (NAA) is characterized by a high tissue-to-extracellular concentration ratio under normal conditions and is released from neurons during hypoosmotic cell swelling. As cell volume regulation and acid-base homeostasis share common processes, we have examined by microdialysis whether the extracellular concentration of NAA is altered by various acidotic challenges. Twenty-minute perfusion of 50 mM NH4+ through the microdialysis probe progressively lowered dialysate pH by 0.18, followed by a sudden, additional reduction after NH4+ removal. The latter effect indicated extrusion of cellular H+ because it was suppressed by blockade of Na+/H+ exchange with 5-(N,N-dimethyl)amiloride (1 or 5 mM in perfusion medium). NH4+ increased dialysate levels of NAA and lactate by approximately two- and threefold their initial values, respectively. These data demonstrate that pronounced intracellular acidosis is associated with NAA efflux, presumably from neurons. Whether this effect is linked directly to acid-base homeostasis or is secondary to acidosis-induced cell swelling remains to be clarified. Hypercapnia and perfusion of acid medium failed to increase dialysate NAA, probably because acidosis was not severe enough or the associated cellular swelling was not followed by regulatory volume decrease. As cellular swelling and acidosis are key features of cerebral ischaemia, further investigations into the role of NAA, and the development of sophisticated magnetic resonance spectroscopic methods capable of resolving intra-/extracellular NAA redistribution, would be especially relevant to clinical practice.

We are developing new techniques for detecting point mutations by DNA-DNA hybridization and DNA-protein interaction analysis with an affinity sensor, BIACORE. To detect point mutations by the hybridization method using synthetic oligonucleotides, we already found that the length of the probe and the location of mismatches were important. The PCR products of the N-ras gene derived from Hep G2 cells, which have heterozygous point mutations at codon 61 in the gene, were analyzed and the point mutations were detected with 13-mer probes. We suggest that the detection method using DNA-DNA hybridization is useful for detecting known point mutations. However, detect unknown mutations, E. coli mismatch recognition protein, MutS, was employed. All mismatches in immobilized 20 base pairs of double-strand DNA could be detected by MutS binding. We have started to apply of the MutS to the detection of point mutations in PCR products.

We investigated the influence of different buffers (Tris-buffer and phosphate buffered saline (PBS)/Tween-20 buffer) on anti-prothrombin antibody (aPT) measurement by enzyme-linked immunosorbent assay (ELISA), employing a gamma-ray-irradiated plate. We found considerable discrepancies in aPT positivity between each buffer, and we suggest that the use of Tris-buffer is not suitable for aPT measurement with a gamma-ray-irradiated plate to measure aPT.

Aberrant tyrosine phosphorylation of beta-catenin inactivates the E-cadherin-mediated cell adhesion and invasion suppressor system in cancer cells. Elucidation of the association between beta-catenin and c-erbB-2 protein prompted us to investigate whether interference with this interaction can change the invasive phenotype. In a human gastric cancer cell line, TMK-1, N-terminally deleted beta-catenin, which binds to c-erbB-2 but not to cadherin, inhibited the association between endogenous beta-catenin and c-erbB-2 protein, and suppressed the tyrosine phosphorylation of beta-catenin. Cells expressing truncated beta-catenin exhibited markedly reduced invasiveness in vitro and peritoneal metastasis in vivo, and developed an epithelial morphology. These results suggest that tyrosine phosphorylation of beta-catenin regulated by c-erbB-2 protein may play an important role in the invasion, metastasis and morphogenesis of cancer cells and that inhibition of the aberrant tyrosine phosphorylation of beta-catenin effectively prevents invasion and metastasis of cancer cells.

Hepatocellular carcinoma (HCC) originating from the caudate lobe is rare (about 2%), and prognosis is poor due to difficulties in early diagnosis, transarterial chemoembolization (TAE), and surgical resection. Distant metastasis is also likely to develop. To prevent metastasis during surgical manipulations, we performed microwave coagulation therapy before caudate lobectomy in one patient. Although the efficacy of this procedure remains to be elucidated, the multidisciplinary treatment described in this study is essential for the improvement of prognosis in patients with HCC developing from caudate lobe.

A patient with hepatocellular carcinoma (HCC) who underwent successfully percutaneous ethanol injection therapy (PEIT) with etoposide was reported. A 72-year-old Japanese man with positive HCV-Ab developed multiple tumors of HCC. He underwent transcatheter arterial embolization (TAE) four times and PEIT three times, but the growth of tumors (particularly, S8) could not be controlled by these modalities. Then, the mixture of etoposide (20 mg), ethanol (8.5 ml) and lipiodol (1.5 ml) was injected into S6 tumors. Consistent with the normalization of serum AFP level, CT scan revealed the marked remaining lipiodol in these tumors. PEIT with etoposide is the treatment of choice in patients with HCC refractory to treatment by conventional TAE or PEIT.

We performed the locoregional injection of OK-432/fibrinogen/thrombin to unresectable hepatic tumors metastasized from colorectal cancers, which were hardly controlled by arterial infusion chemotherapy. CEA was markedly decreased following this treatment, although abdominal CT did not show a significant reduction of tumor mass. This immuno-injection therapy may be a choice of treatment for metastatic liver tumors, refractory to treatment by conventional chemotherapy.

The effective treatment of unresectable pancreatic cancer remains to be elucidated. We report herein a patient who underwent a local injection of a mixture of OK-432, fibrinogen and thrombin. Tumor size and tumor markers significantly decreased following this procedure. Local injection therapy with OK-432, fibrinogen and thrombin may be a promising treatment for unresectable pancreatic cancer.

Chemotherapy for pancreatic acinar cell carcinoma has not been well studied. We report herein one patient, in whom arterial chemotherapy with 5-FU, CDDP and MMC was extremely effective. He underwent distal pancreatectomy associated with partial resection of transverse colon. Hepatic recurrence was detected by MRI 8 months after the surgery. 5-FU (250 mg/day), CDDP (20 mg/day), and MMC (10 mg) was injected through a catheter introduced into the proper hepatic artery. His tumor became hardly detectable by MRI, and he was discharged about 1 month after admission. 5-FU (250 mg) and CDDP (20 mg) were injected every two weeks, and recurrence was not observed for 5 months.

We developed a maneuver for holding and lifting the intraperitoneal organs from the extracorporeal side using a new instrument, the leading bar, by which we could guide the tape or the thread to the outside of the abdomen. In the case of cholecystolithiasis with a thickened wall or a stone stuck in the neck, we lifted the round ligament and gallbladder for better exposure of Calot's triangle, using the tape or thread through the abdominal wall to the extracorporeal side, and then clamped them with Kocher's forceps. With this technique, a good view of Calot's triangle was maintained so that the surgeon could expose the cystic duct easily and safely using both hands.

We conducted this study to investigate whether antioxidized low-density lipoprotein (a-oxLDL) is an antibody to cryptic and/or neo-antigen on beta2-glycoprotein I (GPI), which is introduced by binding to anionic phospholipid, similar to that of GPI-dependent anticardiolipin antibody (aCL) employing a-oxLDL ELISA. We found that no significant optical density differences existed among systemic lupus erythematosus patients, including cases with aCL and/or lupus anticoagulant positivity, before and after the addition of GPI. Our results suggest that a-oxLDL is not an antibody to denatured GPI, but rather to oxLDL.

Marcarino and Bassani have proposed the sodium liquid-vapour equilibrium as a means to approximate the ITS-90 between 660 °C and 962 °C with an accuracy of about 10 mK. We have also measured the sodium vapour pressure over this temperature range using a pressure-controlled heat pipe. Our results differ from those of Marcarino and Bassani by approximately 34 mK at 660 °C and 21 mK at 962 °C with a linear temperature dependence of the difference between these values. This relationship may be explained very satisfactorily by a difference in the impurity content of the two samples. We show that an additional 200 × 10-6 mass fraction of potassium in our sample as compared with theirs is sufficient to explain the discrepancy. As sodium of sufficient purity to ensure repeatability of its use as a temperature standard at the millikelvin level is not commercially available, it appears necessary to characterize the pressure/temperature relationship for each sample prior to relying on a measurement of the pressure to determine the temperature of the heat pipe. The alternative is to rely on the heat pipe as an intercomparison device with the temperature given by a calibrated thermometer.

We devised a new therapeutic modality for multiple hepatocellular carcinoma (HCC) consisting of transarterial immuno-embolization (TIE) using OK-432 and fibrinogen, and applied the treatment to 22 patients with advanced HCC who had been insensitive to TAE, and 21 patients who had no treatment. Nineteen patients had a high AFP level of more than 200 ng/ml. The serum AFP level decreased in 15 patients after TIE, and in 7 patients the AFP level decreased to less than 30%. Furthermore, a marked reduction in tumor size was observed in 69% after TIE. We report 61% 1-year, and 3-year survival rate for cases who had been insensitive to TAE, and 86% 2-year survival rate for those who selected TIE as the first choice. A high fever of more than 38 degrees C occurred in all cases, and hypotension less than 80 mmHg was observed in 42% patients. No deterioration of liver function and no disturbance of the coagulation-fibrinolysis system due to TIE except in one patient with liver failure after TIE.

We investigated the prevalence rate of beta 2-glycoprotein I (beta 2-GPI)-dependent antiphospholipid antibodies (aPL)[anti-cardiolipin antibody (aCL), anti-phosphadidylserine antibody (aPS), and anti-phosphatidic acid antibody (aPA)], antinuclear antibody (ANA), anti-deoxyribonucleic acid antibody (aDNA), anti-thyroglobulin antibody (aTG), and anti-thyroid peroxidase antibody (aTPO) in 56 patients with hepatitis C virus (HCV) infection and correlated the results with inteferon-alpha (IFN) treatment.aCL, aPS, and aPA were positive in, respectively, 7/56 (13%), 12/56 (21%), and 13/56 (23%) patients before treatment. aPS and aPA appeared in 6/44 and 9/43 and disappeared in 6/12 and 2/13 patients, respectively, after IFN treatment; the differences were statistically significant. The changes in OD readings were higher in the group of patients who became positive for aPS and aPA than in those who became negative after treatment. The positive rates of aTG and aTPO in the patient group were statistically higher than in the healthy controls, and aTG developed in four patients and disappeared in two. No obvious changes in aTPO were observed; however, the aTPO titer was increased in four previously positive patients. None of the patients positive for any antibodies developed an abnormality associated with these antibodies during an observation period of up to 1 yr.The pathogenesis of production and clinical significance of aPL, ATG/aTPO in this clinical setting are unclear, but immunological disturbances, such as the effects of HCV infection and/or IFN treatment, were considered to be a possibility. Further investigation is needed to clarify whether HCV/aPL- and/or aTG/aTPO-positive patients treated with IFN might develop aPL-associated complications and/or autoimmune disease(es), in the future and to confirm whether IFN treatment justifies the outcome in this clinical setting.

The expression pattern of E- and P-cadherin in human carcinomas has been reported by many laboratories. However, little is known about the involvement of other cadherin types in human carcinomas. cDNA clones for a cadherin molecule were isolated from a cDNA library of human hepatocellular carcinoma cells which lacked E- and P-cadherin expression but exhibited cell aggregation activity mediated by an unknown cadherin, and they were subjected to sequence analysis. The overlapped clones covered 4315 nucleotides and were found to encode a typical cadherin molecule consisting of 790 amino acids. Since the deduced amino acid sequence was identical to a partially available human cadherin-6 sequence except for two amino acid residues, the clones were considered to be human cadherin-6 cDNAs encoding the entire open reading frame. The deduced amino acid sequence also showed extremely high homology with recently reported rat K-cadherin, 97% for the putative mature protein, suggesting that cadherin-6 is the human counterpart of rat K-cadherin. Expression of cadherin-6 in various human normal tissues and carcinoma cells was examined by Northern blot analysis using a specific probe corresponding to the signal and precursor sequence. Among normal tissues examined, brain, cerebellum, and kidney showed strong expression of cadherin-6, whereas lung, pancreas, and gastric mucosa showed weak expression. Transcripts of cadherin-6 were not detected in normal liver, whereas four of six hepatocellular carcinoma cell lines examined expressed cadherin-6 abundantly. As reported for rat K-cadherin, three renal carcinoma cell lines also expressed cadherin-6 strongly. The most interesting finding was obtained for small cell lung carcinoma lines. Among 15 of such cell lines examined, all of 11 cadherin-6-positive lines were classified into the classic type, whereas the negative cell lines were all of the variant type. The present results suggest that besides E- and P-cadherin, other cadherin molecules are expressed in human cancers and are responsible for additional biological properties of the carcinoma cells.

Some researchers claim that lupus anticoagulant-positive plasma may cause a false-positive reaction in the test for activated protein C (APC) resistance, a hereditary thrombophilic state characterized by abnormal factor V, which frequently causes venous thrombosis. We investigated whether anti-beta 2-glycoprotein I antibody (aGPI), which has recently come to be regarded as an anti-cardiolipin antibody (aCL) itself, might have an effect on the APC resistance test.