Heparanase expression correlates with malignant potential in human colon cancer.

PURPOSE: Heparanase cleaves carbohydrate chains of heparan sulphate proteoglycans and is an important component of the extracellular matrix. This study was designed to determine the relation between heparanase expression and prognosis of patients with colon cancer. METHODS: The study included 54 patients (35 males and 19 females) who underwent colorectal resection for colorectal cancer between January 1992 and December 1994. Expression of heparanase protein and mRNA were determined and correlated with various clinicopathological parameters. In vitro studies were also performed to examine tumor invasion and to test the effects of heparanase inhibition, and in vivo studies were performed to examine tumor metastasis and prognosis. RESULTS: Heparanase expression was detected in the invasion front of the tumor in 37 of 54 (69%) colon cancer samples, whereas 17 of 54 (31%) tumors were negative. Expression of heparanase was significantly more frequent in tumors of higher TNM stage (P=0.0481), higher Dukes stage (P=0.0411), higher vascular infiltration (P=0.0146), and higher lymph vessel infiltration (P=0.0010). Heparanase expression in colon cancers correlated significantly with poor survival (P=0.0361). Heparanase-transfected colon cancer cells exhibited significant invasion compared with control-transfected colon cancer cells (P=0.001), and the peritoneal dissemination model also showed the malignant potential of heparanase-transfected cells, as assayed by number of nodules (P=0.017) and survival (P=0.0062). Inhibition of heparanase significantly reduced the invasive capacity of cancer cells (P=0.003). CONCLUSIONS: Heparanase is a marker for poor prognosis of patients with colon cancer and could be a suitable target for antitumor therapy in colon cancer.

Esophageal cancer associated with right aortic arch: report of two cases.

Esophageal carcinoma associated with a right aortic arch is very rare. In such cases, the dissection of right paratracheal lymph nodes is difficult. Herein, we report two cases of thoracic esophageal carcinoma with right aortic arch, for which the left door open method was used to provide a good surgical view. Postoperative chemotherapy and radiotherapy were used for both cases and no evidence of recurrence or metastasis has been noted in the 24-month postoperative period.

Two cases of superficial basaloid squamous carcinoma of the esophagus.

. Basaloid squamous carcinoma of the esophagus is very rare. We report two cases of basaloid squamous carcinoma of the esophagus. Both tumors histologically consisted of solid cell nests with intervening fibromyxoid stroma. In some tumor nests were comprised of pseudoglandular structures containing myxoid matrix, and displayed focal immunoreactivity for laminin. Thoracic esophagectomy with lymph node dissection was followed by intrathoracic esophagogastrostomy in both patients. The patients had uneventful postoperative courses. Regular periodic follow-up showed no evidence of recurrence or metastasis in the 22-month postoperative period.

TNF combined with IFN-alpha accelerates NF-kappaB-mediated apoptosis through enhancement of Fas expression in colon cancer cells.

Immunostaining and EMSA revealed that NF-kappaB was activated strongly by TNF/IFN-alpha compared to TNF alone in a human colon adenocarcinoma cell line, RPMI4788. Although inhibition of activated NF-kappaB, by using an NF-kappaB decoy, reduced cell viability after treatment with TNF only, NF-kappaB decoy resulted in recovery of cell viability after TNF/IFN-alpha treatment. Caspase-3 activity was increased in cells induced by TNF/IFN-alpha, while suppression of caspase-3 activity was observed in cells transfected with NF-kappaB decoy and then treated by TNF/IFN-alpha. On the other hand, Fas expression was strongly enhanced by TNF/IFN-alpha, and inhibition of TNF/IFN-alpha-induced NF-kappaB activation, by using NF-kappaB decoy, decreased Fas expression. Cell viability and caspase-3 activity decreased in cells treated with TNF/IFN-alpha and anti-FasL antibody. Taken together, our findings suggest that activated NF-kappaB induced by the crosstalk between TNF and IFN-alpha is a novel pro-apoptotic signal acting via enhancement of Fas expression.

A new generic column switching system for quantitation in cassette dosing using LC/MS/MS.

Cassette dosing is a method in which multiple drugs are administered to a single animal at the same time, and the plasma concentrations of the individual compounds are simultaneously determined. This method enables high-throughput rapid screening for pharmacokinetic assessment of new drug candidates. An available gradient method was modified for cassette dosing analysis to attain the advantages of high sensitivity and applicability to a wide range of compounds. However, two problems arose; (1). the time-consuming optimization of mobile phases for each compound group, which limited applicability and (2). the remarkable suppression of ionization by polyethyleneglycol, which is commonly used in intravenous administration. To resolve these problems, a new column switching method was established to attain wider applicability and avoid the ionization suppression. This column switching system is very simple because the trap column and the analytical column are specified and the mobile phase is selected from only two species. Method optimization requires only the selection of the mobile phase and takes only a few hours. About 200 compounds, which were administered as about 50 cassettes, were analyzed using this column switching system. Assay validation of one cassette was carried out, and good accuracy and precision were obtained. About 90% of the compounds could be determined within 20% bias. These results showed that this new column switching system for cassette dosing is accurate enough for the screening of drug candidates and offers wide applicability for various compounds. This system was shown to be very useful for the determination of cassette dosing samples, containing multiple compounds.

Rapid LC-TOFMS method for identification of binding sites of covalent acylglucuronide-albumin complexes.

A method for rapid identification of binding sites of covalent adducts was developed using delta bilirubin as a model compound. Delta bilirubin, containing intact human serum albumin (HSA), was digested with trypsin and the peptide fragments were monitored at 436 nm, but no predominant peaks were detected indicating the instability of the digested peptides containing bilirubin-related compounds. Therefore, the high-performance liquid chromatography time-of-flight mass spectrometer (LC-TOFMS) data of digested fragments of delta bilirubin were compared with those of control digests of HSA, revealing a characteristic peptide in the digest mixture of delta bilirubin. This peptide was sequenced by high-performance liquid chromatography time-of-flight tandem mass spectrometry (LC-TOFMS/MS) and identified as LDELRDEGKASSAK (Leu182 to Lys195) with a modification of a 178 Da increase at Lys190. This indicated the Lys190 to be a predominant covalent binding site of BGs on HSA via the imine mechanism and the binding between the bilirubin moiety and the glucuronic acid moiety to be unstable to digestion with trypsin. The method of comparing LC-TOFMS data requires no specific detection such as fluorescence or radioactivity for every compound. This should accelerate the structure elucidation of covalent adducts and be helpful for studying the relationship between the structure of ligands and specific binding sites.

CD57+ T cells augment IFN-gamma production in a one-way mixed lymphocyte reaction and their expansion after stem cell transplantation in paediatric patients.

To clarify the immune response of CD57+ T cells (most of them are CD8+) in peripheral blood (PB) against alloantigens in order to elucidate the T helper 1 (Th 1) immune response, we assessed the role of CD57+ T cells in IFN-gamma (one of the representative Th 1 cytokines) production in a one-way mixed lymphocyte reaction (MLR). In this study, we showed that CD57+ T cells in responder cells were essential for effective IFN-gamma production in allogeneic MLR due partly to the augmentation of the alloresponse of regular T cells. Furthermore, IFN-gamma production in MLR correlated with the proportions of CD57+ T cells in PB regardless of the responders' age. We also showed that the extent of the expansion of CD57+ T cells in paediatric patients after haematopoietic stem cell transplantation (HSCT) was markedly lower than that in adult patients. In addition, CD57+ T cells purified and activated with a combination of cytokines showed a greater cytotoxicity than regular T cells against human umbilical vein endothelial cells. Because IFN-gamma production in one-way MLR is a useful predictor of graft-versus-host disease (GVHD), especially in the acute phase that occurs after allogeneic HSCT, our findings suggested that CD57+ T cells play a role in the development of GVHD and thus may explain the reason as to why a higher donor age is associated with an increased risk of developing GVHD while, in addition, the incidence of severe GVHD in paediatric patients is lower than that in adult patients.

Activation of human T cells with NK cell markers by staphylococcal enterotoxin A via IL-12 but not via IL-18.

We have reported recently that mouse liver NK cells and NK1 x 1+ T cells were activated by bacterial superantigens via the IL-12 production from Kupffer cells. In the present study, we examined the effect of staphyloccoccal enterotoxin A (SEA) on human T cells with NK cell markers, CD56 or CD57 (NK-type T cells). After stimulating peripheral blood mononuclear cells (PBMC) with SEA, PBMC produced a large amount of IFN- and acquired a potent antitumour cytotoxicity. The in vitro depletion of either CD56+ TCR NK cells, CD56+ T cells or 57+ T cells from PBMC significantly inhibited the IFN- production from PBMC. When purified NK-type T cells, NK cells and regular T cells were cultured with monocytes and SEA they all produced IFN-, while the IFN- amounts produced by both NK-type T cells were greater than those produced by NK cells. NK cells as well as CD56+ T cells showed cytotoxicity against NK-sensitive K562 cells, whereas both NK-type T cells showed a more potent cytotoxicity against NK-resistant Raji cells than did NK cells. The IFN- production from each population as well as from whole PBMC was greatly inhibited by anti-IL-12 antibody but not by anti-IL-18 antibody. The antitumour cytotoxicity of whole PBMC was also significantly inhibited by anti-IL-12 antibody while the SEA-induced proliferation of PBMC was not affected by anti-IL-12 antibody. Furthermore, SEA-activated NK-type T cells as well as NK cells showed cytotoxicities against vascular endothelial cells. Our findings suggest that human NK-type T cells are thus involved in bacterial superantigen-induced immune response.

Autographa californica M nucleopolyhedrovirus ProV-CATH is activated during infected cell death.

V-CATH, a cathepsin L-like cysteine protease encoded by the baculovirus Autographa californica M nucleopolyhedrovirus, has been shown to play an essential role in host liquefaction. Similar to cellular cathepsin L, V-CATH is synthesized as an inactive proenzyme and is activated by cleavage of the propeptide. Previous studies indicated that removal of the propeptide was rapid, occurring as soon as the protein could be detected by Western blot, 22 h postinfection. We found, however, that these results reflected artifactual processing of the proenzyme. When the protease inhibitor E-64 was used to prevent this aberration, we found that proV-CATH accumulated in infected cells and activation did not begin until the onset of cell death, at approximately 80 h postinfection. Western blot analysis of fractions of live and dead cells isolated by fluorescence-activated cell sorting revealed that mature V-CATH was found only in dead cells. The regulation of activation of proV-CATH, therefore, was quite different from that of cellular cathepsins. Acridine orange staining revealed that lysosome integrity was lost in dead cells, an occurrence that could lead to the activation of proV-CATH by lysosomal proteases.

Standardization of nitric oxide aqueous solutions by modified Saltzman method.

Nitric oxide (NO) aqueous solutions were prepared by saturating pure NO gas and hydrolyzing 1 mM 1-hydroxy-2-oxo-3-(N-methyl-3-aminoethyl)-3-methyl-1-triazene (NOC-7), a NO donor, under anerobic conditions. The modified Saltzman method was employed for standardization of the NO aqueous solutions. NO and NO(2) in the solutions were driven with nitrogen gas stream into the first Saltzman solution to measure NO(2) and the leaked NO was driven with air stream through an oxidizing solution into the second Saltzman solution to measure NO, and NO(-)(2) and NO(-)(3) in the residual solutions were determined directly and after reduction with nitrate reductase, respectively. The concentrations of nitrogen oxide species in the NO solutions were about 1.8 mM NO/0.01 mM NO(2)/0.1 mM NO(-)(2)/0.1 mM NO(-)(3), and unchanged during keeping at 20 degrees C for 1 h under anerobic conditions but became 0.05 mM NO/0.01 mM NO(2)/1.7 mM NO(-)(2)/0.1 mM NO(-)(3) by keeping at 20 degrees C for 10 min under aerobic conditions. Instability of NO under aerobic conditions was supported by consumption of 1/4 equivalent amount of dissolved oxygen, and by loss of ability to convert 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) to carboxy-PTI. Simultaneous quantification of nitrogen oxide species by the modified Saltzman method was found to be useful for practical standardization of NO aqueous solutions.

Antisense-mediated suppression of human heparanase gene expression inhibits pleural dissemination of human cancer cells.

Heparan sulfate proteoglycans is a major component of the cell surface and extracellular matrix and functions as a barrier against cationic molecules and macromolecules. Heparanase is an endoglucuronidase capable of specifically degrading heparan sulfate, and its activity is associated with the metastatic potential of tumor cells. To inhibit human heparanase expression in human cancer cells, we constructed an adenoviral vector carrying a full-length human heparanase cDNA in an antisense orientation (Ad-AS/hep). Increased heparanase expression in T.Tn human esophageal cancer cells and A549 human lung cancer cells after infection with an adenovirus vector expressing the human heparanase gene (Ad-S/hep) was specifically inhibited by simultaneous infection with Ad-AS/hep in a dose-dependent manner. A modified Boyden chamber assay demonstrated that infection with Ad-AS/hep significantly inhibited in vitro invasion of A549 cells after Ad-S/hep infection. Moreover, intrathoracic administration of Ad-AS/hep reduced the number and size of heparanase-expressing A549 tumors implanted intrathoracically into BALB/c-nu/nu mice. Our results suggest that heparanase contributes to the invasive phenotype of tumor cells, and that antisense-mediated inhibition of heparanase activity may be efficacious in the prevention of pleural dissemination.

Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells.

We investigated the function of CD56+ CD8+ T cells (CD56+ T cells) and CD56- CD57+ CD8+ T cells (CD57+ T cells; natural killer (NK)-type T cells) and compared them with those of normal CD56- CD57- CD8+ T cells (CD8+ T cells) and CD56+ NK cells from healthy volunteers. After the stimulation with immobilized anti-CD3 antibodies, both NK-type T cells produced much larger amounts of interferon-gamma (IFN-gamma) than CD8+ T cells. Both NK-type T cells also acquired a more potent cytotoxicity against NK-sensitive K562 cells than CD8+ T cells while only CD56+ T cells showed a potent cytotoxicity against NK-resistant Raji cells. After the stimulation with a combination of interleukin (IL)-2, IL-12 and IL-15, the IFN-gamma amounts produced were NK cells > or = CD56+ T cells > or = CD57+ T cells > CD8+ T cells. The cytotoxicities against K562 cells were NK cells > CD56+ T cells > or = CD57+ T cells > CD8+ T cells while cytotoxicities against Raji cells were CD56+ T cells > CD57+ T cells > or = CD8+ T cells > or = NK cells. However, the CD3-stimulated proliferation of both NK-type T cells was smaller than that of CD8+ T cells partly because NK-type T cells were susceptible to apoptosis. In addition to NK cells, NK-type T cells but not CD8+ T cells stimulated with cytokines, expressed cytoplasmic perforin and granzyme B. Furthermore, CD3-stimulated IFN-gamma production from peripheral blood mononuclear cells (PBMC) correlated with the proportions of CD57+ T cells in PBMC from donors. Our findings suggest that NK-type T cells play an important role in the T helper 1 responses and the immunological changes associated with ageing.

Regulation of the H+ pump activity in the plasma membrane of internally perfused Chara corallina.

The role of cytoplasm for the maintenance of the H+ pump activity in Chara corallina internodal cells was examined by the intracellular perfusion technique. Cytoplasm-rich and -poor states were obtained by changing the perfusion time, short-term (less than 2 min) and long-term (more than 5 min), respectively. A large portion of cytoplasm was left by short-term perfusion but most of the cytoplasm was removed by long-term perfusion. The activities of the H+ pump of these two different conditions were examined by measuring current-voltage relation (I-V curve) and conductance-voltage relation (G-V curve) under voltage clamp conditions. The H+ pump conductance decreased to 37%, 9% and zero by short-term, long-term and hexokinase perfusion, respectively, whereas the passive channel conductance decreased to 71%, 39% and 73% by short-term, long-term and hexokinase perfusion, respectively. On the other hand, the electromotive-force of the H+ pump (approximately -260 mV) and the passive channel (approximately -130 mV) were not affected by either short- or long-term perfusion. It is indicated that the cytoplasm plays an essential role to regulate the activity of both the H+ pump and the passive channel together with ATP.

The mechanism of a defective IFN-gamma response to bacterial toxins in an atopic dermatitis model, NC/Nga mice, and the therapeutic effect of IFN-gamma, IL-12, or IL-18 on dermatitis.

NC/Nga (NC) mice raised under conventional conditions (Conv. NC mice) spontaneously develop dermatitis similar to human atopic dermatitis, whereas NC mice raised under the specific pathogen-free conditions do not develop dermatitis. In the present study, we show that the representative Th1 cytokine, IFN-gamma levels in the sera of NC mice, injected with either staphylococcal enterotoxin B or endotoxin (LPS), to be severalfold lower than those of normal mice. The low IFN-gamma response to staphylococcal enterotoxin B was correlated to the lack of regular Vbeta8(+) T cells and Vbeta8(+) NK T cells, and the low IFN-gamma response to LPS was correlated to an impaired IL-18 production of macrophages. The CD3-stimulated IL-4 production from liver and spleen T cells from Conv. NC mice in vitro was greatly augmented. The serum IL-4 levels of untreated Conv. NC mice also were higher than those of normal mice and specific pathogen-free NC mice. Treatment of Conv. NC mice either with IFN-gamma, IL-12, or IL-18 twice a week from 4 wk of age substantially inhibited the elevation of the serum IgE levels, serum IL-4 levels, and dermatitis, and IL-12 or IL-18 treatment also reduced the in vitro IL-4 production from CD3-stimulated liver T cells. The systemic deficiency in the Th1 response to bacterial stimulation thus leads to a Th2-dominant state and may induce an abnormal cellular immune response in the skin accompanied with an overproduction of IgE and a susceptibility to dermatitis in NC mice.

Clinical significance of enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid.

The NC16A domain of the 180-kDa bullous pemphigoid antigen (BP180) is the most immunogenic and, probably, pathogenic region in bullous pemphigoid (BP). In the present study, in order to determine whether serum level of circulating anti-BP180 autoantibodies is a valuable serum marker in BP, the immunoreactivity of sera against the NC16A domain of BP180 was measured using enzyme-linked immunosorbent assay (ELISA) in ten patients with BP. Serum levels of anti-BP180 autoantibodies correlated with the clinical course in BP patients, who received various therapeutic agents. The result suggests that this NC16A-ELISA is a useful method for evaluating the clinical course and efficacy of the therapy in patients with BP.

Release of nitric oxide together with carbon-centered radicals from N-nitrosamines by ultraviolet light irradiation.

Solutions of N-nitrosamines, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosomorpholine and N-nitrosopyrrolidine in phosphate buffer (pH 7.4) were irradiated by ultraviolet (UV) light at room temperature. The N-nitrosamines were extensively degraded due to irradiation for 120 min in a time-dependent fashion as monitored by UV-absorption or high performance liquid chromatographic analysis. Carbon-centered radicals were generated from four N-nitrosamines during the short time irradiation of 10-60 s as monitored by electron spin resonance (ESR) technique using 5,5-dimethyl-1-pyrroline N-oxide and N-tert-butyl-alpha-phenylnitrone as spin traps. Nitric oxide (NO) was generated during the short time irradiation as monitored by ESR technique using cysteine-Fe(II) complex, N-methyl-D-glucamine dithiocarbamate and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Significant amounts of nitrite (4-16%) from four N-nitrosamines and also a significant amount of nitrate (4%) was produced from N-nitrosodimethylamine during the irradiation time of 120 min. Released NO from the N-nitrosamines must be converted into nitrite through intermediary reactive nitrogen oxide species including nitrogen dioxide and dinitrogen trioxide in contact with dissolved oxygen.

Decrease of CD56(+)T cells and natural killer cells in cirrhotic livers with hepatitis C may be involved in their susceptibility to hepatocellular carcinoma.

CD56(+)T cells and CD56(+)natural killer (NK) cells are abundant in the human liver. The aim of this study was the further characterization of these cells in the liver with or without hepatitis C virus (HCV) infection. Liver mononuclear cells (MNC) were isolated from liver specimens obtained from the patients during abdominal surgery. In addition to a flow cytometric analysis, liver MNC and PBMC were cultured with the immobilized anti-CD3 Ab, IL-2, or a combination of IL-2 and IL-12 and their IFN-gamma production and the antitumor cytotoxicity were assessed. The liver MNC of HCV (-) patients contained 20% CD56(+)T cells whereas the same proportions decreased to 11% in chronic hepatitis livers and to 5% in cirrhotic livers. The proportion of NK cells also decreased in the cirrhotic livers. On the other hand, the populations of these cells in PBMC did not significantly differ among patient groups. The IFN-gamma production and the cytotoxicity against K562 cells, Raji cells, and a hepatocellular carcinoma, HuH-7 cells, greatly decreased in the cirrhotic liver MNC. In contrast, the cytotoxicity in PBMC did not significantly differ among the patient groups and was lower than that in the liver MNC of HCV (-) patients. CD56(+)T cells and NK cells but not regular T cells purified from liver MNC cultured with cytokines showed potent cytotoxicities against HuH-7 cells. These results suggest that a decreased number of CD56(+)T cells and NK cells in cirrhotic livers may be related to their susceptibility to hepatocellular carcinoma.