Radiation therapy in combination with transcatheter arterial chemoembolization for hepatocellular carcinoma with extensive portal vein involvement.

AIM: The aim of this study was to examine the effectiveness and toxicity of radiation therapy in combination with transcatheter arterial chemoembolization (TACE) for hepatocellular carcinoma (HCC) with extensive portal vein tumor thrombus (PVTT). METHODS: The combined therapy was performed in 24 HCC patients with extensive PVTT. External radiation targeted for PVTT (50 Gy in 2 Gy fractions) was performed in combination with repetitive TACE for intralobar lesions using 30-60 mg epirubicin every 3 months, and associations of the following variables with the survival rate were evaluated: gender, age, viral etiology, Child's class, performance status, extrahepatic metastasis, size and number of HCC, and location of PVTT. RESULTS: The local response confined to PVTT was complete response (CR) in four patients, partial response (PR) in eight patients, no change (NC) in eight patients, and progressive disease (PD) in four patients. By using the stepwise Cox's regression analysis, only Child's class was associated with the survival rate. The survival rates after 1 and 2 years were 73 and 21% in Child's A, 10 and 0% in Child B or C, and 61 and 21% in patients in whom the local response was CR or PR, and 19 and 9% in those in whom the local response was NC or PD, respectively. By using the multiple logistic regression analysis, Child's class was the only factor associated with the local response (P = 0.006). CONCLUSIONS: The combined therapy is feasible and may be useful to reverse PVTT in patients with good hepatic function reserve.

[The efficacy of a comprehensive smoking cessation class, combined with nicotine patch and gum replacement therapy].

A six-month comprehensive smoking cessation class was conducted in our hospital, and the results were evaluated after one year. To increase the rate of smoking cessation, a smoking cessation support team composed of a medical doctor, a pharmacist, nurses, a registered dietitian, and a physical therapist was formed. The team provided specialized lectures and comprehensive group counseling programs every two weeks for the first two months and every month for the next four months. Each participant's expired carbon monoxide concentration and body weight were measured at every attendance. The participant continued with beneficial behavioral treatment for smoking cessation for six months, with nicotine replacement therapy (NRT) for the first eight weeks. The protocol of our NRT consisted of both the routine use of nicotine patches (Nicotinell TTS) and the rescue use of nicotine gum (Nicorette). We first ascertained each participant's degree of nicotine dependence, using the Fagerström Tolerance Questionnaire score, and daily nicotine intake was estimated by a detailed questionnaire. We then divided the participants into two NRT groups according to their nicotine dependence. The higher nicotine dependence group consisted of those whose Fagerström Tolerance Questionnaire score was more than 5 points or whose estimated nicotine intake was more than 10 mg/day. This group they used Nicotinell TTS 30 (TTS 30) for the first four weeks, TTS 20 for the next two weeks, and TTS 10 for the last two. In the lower dependence group. TTS 20 and TTS 10 were each given for four weeks. Nicotine gum use was restricted to 4 pieces a day for the first week and reduced by one per day each subsequent week. As a result, there was an 81.3% smoking cessation rate after eight weeks, 70.3% after six months, and 58.2% after one year. In conclusion, two courses of routine nicotine patch use, with the addition of restricted rescue use of nicotine gum, can produce better longterm abstinence results than previously reported NRTs, suggesting that this may be one of the best ways to cease smoking. We also emphasize that intensive group counseling programs and lectures supported by doctors and medical teams, as well as a continuing behavioral treatment component, may be indispensable for enhancing longterm sustained abstinence rates.

Both inhalant and intravenous uroguanylin inhibit leukotriene C4-induced airway changes.

Uroguanylin, a well-known ligand of guanylyl cyclase C receptor in the gastrointestinal tissue, has recently been reported to have pulmonary effects. We investigated the inhibitory effects of uroguanylin against leukotriene C4-induced bronchoconstriction and airway microvascular leakage. Anesthetized guinea pigs, ventilated via a tracheal cannula in a plethysmograph box, were measured by pulmonary mechanics for 10 min after i.v. administering 2 microg/kg leukotriene C4. Airway microvascular leakage was assessed by extravasation of Evans blue dye into airway tissues. Both inhalant and i.v. pretreatment of uroguanylin significantly inhibited leukotriene C4-induced pulmonary changes in a dose-dependent manner, suggesting its effectiveness against an asthmatic condition.

Adrenomedullin inhibits ovalbumin-induced bronchoconstriction and airway microvascular leakage in guinea-pigs.

Human adrenomedullin is a potent vasodilator with bronchodilation properties. The effects of adrenomedullin on antigen-induced bronchoconstriction and airway microvascular leakage in guinea-pigs was investigated. The portion of the adrenomedullin molecule possessing these pulmonary active profiles was also examined, using two truncated adrenomedullin molecules: adrenomedullin (1-25) and adrenomedullin (22-52). Four weeks after sensitization with ovalbumin (0.1 mg x k(-1)), the guinea-pigs were anaesthetized and mechanically ventilated. Respiratory resistance, dynamic compliance and arterial blood pressure were monitored. Airway microvascular leakage was evaluated by extravasation of 20 mg x kg(-1) Evans blue into airway interstitial tissue. In order to enhance the pulmonary effects of adrenomedullin, the active production of endogenous nitric oxide was inhibited by coadministration of a nitric oxide synthase inhibitor, L-N(G)-nitroarginine methethyl ester (10 mg x kg(-1)). Intravenous pretreatment with adrenomedullin (10, 30 and 100 microg x mL(-1)) dose-dependently inhibited ovalbumin-induced bronchoconstriction and airway microvascular leakage in all airway segments. Inhaled adrenomedullin (100 microg.mL(-1), 1 min) also significantly inhibited pulmonary changes induced by ovalbumin inhalation (3 mg x mL (-1) , 3 min). These pulmonary profiles of adrenomedullin were enhanced by inhibiting the active production of endogenous nitric oxide. In conclusion, adrenomedullin has inhibitory effects on antigen-induced microvascular leakage and bronchoconstriction in guinea-pigs. These beneficial effects strongly related to its unique ring structure and N-terminal segment, making it a potential anti-asthma.

Granulocyte colony stimulating factor-producing diffuse malignant mesothelioma of pleura.

We report the rare case of a 61-year-old man with a diffuse malignant mesothelioma of mixed subtype which produced granulocyte colony-stimulating factor (G-CSF). The white blood cell (WBC) was elevated to 85,100/mm3 without any evidence of infection, and the G-CSF level in the pleural effusion was also increased at 13,200 pg/ml. The lobes of the lung were encased in a tumor. Histopathologically, the tumor cells were of a polymorphous morphology with an epithelial and sarcomatoid mixed pattern. Immunohistochemistry showed that the tumor cells were positive for vimentin, cytokeratin, epithelial membrane antigen, thrombomodulin, and G-CSF, and negative for carcinoembryonic antigen (CEA), CD34, and surfactant apoprotein-A.

Primary structure of a 120 kDa protein associated with the fucose sulfate glycoconjugate constituting the acrosome reaction-inducing substance of the sea urchin, Hemicentrotus pulcherrimus.

A fucose sulfate glycoconjugate (FSG), a natural acrosome reaction-inducer, was purified from the egg jelly of the sea urchin Hemicentrotus pulcherrimus. The FSG is composed primarily of four constituents: a 120 kDa protein, a 237 kDa protein, a 258 kDa protein, and a polysaccharide-containing protein. Among them, the 120 kDa protein was thought to play a critical role in the association of other FSG constituent proteins, and therefore was characterized from a structural point of view. The protein was isolated from the carboxymethylated FSG by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and then digested with trypsin to obtain information regarding the primary structure. Based on the partial amino acid sequences of three internal peptides (FSG120KA: LHNNEYGYGDTAAGEPELAQEEID, FSG 120KG: AIDIPAETGHYGR, and FSG120KC: RPTFDLADAVDT) and the N-terminal peptide (LHNNEYGYGDTAAGEPELAQQEID) of the 120 kDa protein obtained from intact FSG, degenerate oligonucleotide primers were synthesized and used to amplify a 297 bp cDNA fragment. This fragment enabled us to obtain the full-length cDNA (3176 bp) by 5'- and 3'-rapid amplification of cDNA ends. The deduced amino acid sequence revealed that the 120 kDa protein is composed of 663 amino acid residues including 72 cysteine residues, and hence, about 40% is presumed to be carbohydrate by weight. The 120 kDa protein plays an important role in the association of FSG constituent proteins (258 and 237 kDa) through disulfide bonds.

Effects of uroguanylin and guanylin against antigen-induced bronchoconstriction and airway microvascular leakage in sensitized guinea-pigs.

Uroguanylin and guanylin are isolated mainly from the gastrointestinal tract and are activators of guanylyl cyclase C receptor (GC-C), which mediates the production of intracellular cyclic guanosine 3',5'-monophosphate (cyclic GMP). The bronchodilator effects of agents that raise cyclic GMP levels, such as atrial natriuretic peptide, have been reported, and uroguanylin mRNA has recently been detected in extra-gastrointestinal tissues, including the lung, suggesting their role in pulmonary activity. In the first step of this study, we examined the relaxant effects of uroguanylin and guanylin on isolated tracheal smooth muscle of guinea-pigs, and measured tissue cyclic GMP levels by means of enzymeimmunoassay. Uroguanylin produced concentration-dependent relaxant effects on resting tone and significant elevated cyclic GMP levels. Guanylin produced the same, but less potent, effects. In this study, we first investigated the effects of uroguanylin and guanylin on antigen-induced bronchoconstriction and airway microvascular leakage in actively sensitized guinea-pigs. Anesthetized male guinea-pigs, ventilated via a tracheal cannula, were placed in a plethysmograph to measure pulmonary mechanics for 10 min after challenging with 1 mg/kg of ovalbumin. Evans blue dye was then extravasated into their airway tissues to measure microvascular leakage. Intravenous pretreatment with uroguanylin significantly inhibited ovalbumin-induced bronchoconstriction and microvascular leakage in a dose-dependent manner. These inhibitory effects were mimicked by 8-bromoguanosine 3', 5'-cyclic monophosphate. This study is the first to show that uroguanylin not only had a potent bronchodilatory effect but also inhibited microvascular leakage. These results encouraged us to continue the above experimental and clinical studies in bronchial asthma.

Neutral endopeptidase 3.4.24.11 inhibition potentiates the inhibitory effects of type-C natriuretic peptide on leukotriene D4-induced airway changes.

1. Microvascular leakage, a primary feature of inflammation, is well known for worsening the asthmatic condition. Gene expression of and a specific receptor for type-C natriuretic peptide (CNP), initially considered a neuropeptide, have been detected in the human vascular wall and secretion of CNP from vascular endothelial cells has recently been demonstrated. These facts suggest the presence of a vascular natriuretic peptide system and led us to expect that CNP may act beneficially on airway microvascular leakage in asthma. In the present study, we investigated the effects of CNP against leukotriene (LT) D4-induced airway microvascular leakage and bronchoconstriction and how these effects were potentiated by thiorphan, a potent neutral endopeptidase 3.4.24.11 (NEP) inhibitor. 2. Anaesthetized male guinea-pigs, ventilated via a tracheal cannula, were placed into a plethysmograph for 10 min, in order to measure pulmonary mechanics and mean blood pressure, after challenge with 2 micrograms/kg LTD4 and then the extravasation of 20 mg/kg Evans blue dye into airway tissue was investigated to indicate and evaluate microvascular leakage. 3. Intravenous administration of CNP (100, 300 and 1000 micrograms/kg) significantly inhibited the LTD4-induced microvascular leakage and bronchoconstriction in a dose-dependent manner. These inhibitory effects were enhanced by pretreatment with 20 mg/kg thiorphan, suggesting the important role of NEP in the pulmonary metabolism of CNP. 4. We believe that these results are encouraging for the further investigation of the therapeutic applications of exogenous CNP in asthma.

Enhancing effect of wortmannin on muscarinic stimulation of phospholipase D in rat pheochromocytoma PC12 cells.

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK), enhanced carbachol-induced formation of [3H]phosphatidylethanol ([3H]PEt), a marker of phospholipase D (PLD) activity, in [3H]palmitic acid-labeled PC12 cells. The apparent EC50 value was 1.5 microM, and the effect was maximal at 3 microM and slightly attenuated at higher concentration. Wortmannin alone had no significant effect on [3H]PEt formation. The enhancing effect of wortmannin was observed at the initial increasing phase of [3H]PEt formation but not at the subsequent plateau phase. Wortmannin enhanced also phorbol ester-induced PLD activation. Although the precise mechanism remains to be clarified, these results suggest that MLCK may be involved in PLD regulation in PC12 cells.

Secretory immunoglobulin A carries oligosaccharide receptors for Escherichia coli type 1 fimbrial lectin.

Type 1 fimbriae with mannose-specific lectins are widely distributed among members of the family Enterobacteriaceae and confer the ability to attach to a range of host cells, including colonic epithelial cells. The mucosal surfaces are protected by secretory immunoglobulin A (IgA), which agglutinates microorganisms and prevents their attachment to host epithelial cells. This action has been attributed to a specificity of the antigen-combining site of mucosal immunoglobulins for bacterial and viral surface components. Here, we report a novel mechanism for the antibacterial effect of secretory IgA. Secretory IgA and IgA myeloma proteins, especially those of the IgA2 subclass, were shown to possess carbohydrate receptors for the mannose-specific lectin of type 1-fimbriated Escherichia coli. The presence of the high-mannose oligosaccharide chain Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc correlated with binding activity. The interaction between bacterial mannose-specific lectins and IgA receptor oligosaccharide resulted in agglutination of the bacteria and in inhibition of bacterial attachment to colonic epithelial cells. Thus, this interaction could form the basis for a broad antibacterial function of secretory IgA against enterobacteria regardless of the specificity of antibody molecules.

Novel methods to determine the epitopes on the asparagine-linked oligosaccharides of glycoproteins.

Oligosaccharides obtained from glycoproteins by hydrazinolysis followed by N-acetylation were covalently coupled to an amino-bonded thin-layer plate or to a poly-L-lysine coating on the microtiter plate. Bound oligosaccharides can be directly detected by immunostaining or by enzyme-linked immunosorbent assay (ELISA) using a mouse monoclonal antibody. These methods are simple and require small amounts of oligosaccharides and antibodies. The methods were successfully applied to determine the epitope of F48-60, a monoclonal antibody which reacts with the N-linked sugar chains of nonspecific cross-reacting antigen 2, but not with those of carcinoembryonic antigen. The results revealed that the antibody recognizes the Gal beta 1----3GlcNAc beta 1----group.

Structural studies of the asparagine-linked sugar chains of two immunoglobulin M's purified from a patient with Waldenström's macroglobulinemia.

The structures of the sugar chains present in two human monoclonal IgM molecules purified from the serum of a patient with Waldenström's macroglobulinemia have been determined. The asparagine-linked sugar chains were liberated as oligosaccharides by hydrazinolysis and labeled by reduction with NaB3H4 after N-acetylation. Their structures were studied by serial lectin column chromatography and sequential exoglycosidase digestion in combination with methylation analysis. These two IgM's were shown to contain almost the same sugar chains. The sugar chains were a mixture of a series of high-mannose-type and biantennary complex-type oligosaccharides. The complex-type oligosaccharides contain Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAc as their core and GlcNAc beta 1----, Gal beta 1----4GlcNAc beta 1---- and Neu5Ac alpha 2----6Gal beta 1----4GlcNAc beta 1---- groups in their outer chain moieties.

Structural study on the carbohydrate moiety of human placental alkaline phosphatase.

Alkaline phosphatase purified from human placenta contains a single asparagine-linked sugar chain in one molecule. The sugar chain was quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction, and separated by paper electrophoresis into one neutral and two acidic fractions. By a combination of sequential exoglycosidase digestion and methylation analysis, the structures of oligosaccharides in the neutral fraction were confirmed to be as follows: Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of the neutral fraction. All the sialic acid residues of the sugar chains occur as the NeuAc alpha 2----3Gal group. In the case of monosialyl derivatives, the N-acetylneuraminic acid was exclusively linked to the Man alpha 1----3 arm.