S-1 and irinotecan plus bevacizumab versus mFOLFOX6 or CapeOX plus bevacizumab as first-line treatment in patients with metastatic colorectal cancer (TRICOLORE): a randomized, open-label, phase III, noninferiority trial.

Background: Combination therapy with oral fluoropyrimidine and irinotecan has not yet been established as first-line treatment of metastatic colorectal cancer (mCRC). We carried out a randomized, open-label, phase III trial to determine whether S-1 and irinotecan plus bevacizumab is noninferior to mFOLFOX6 or CapeOX plus bevacizumab in terms of progression-free survival (PFS). Patients and methods: Patients from 53 institutions who had previously untreated mCRC were randomly assigned (1 : 1) to receive either mFOLFOX6 or CapeOX plus bevacizumab (control group) or S-1 and irinotecan plus bevacizumab (experimental group; a 3-week regimen: intravenous infusions of irinotecan 150 mg/m2 and bevacizumab 7.5 mg/kg on day 1, oral S-1 80 mg/m2 twice daily for 2 weeks, followed by a 1-week rest; or a 4-week regimen: irinotecan 100 mg/m2 and bevacizumab 5 mg/kg on days 1 and 15, S-1 80 mg/m2 twice daily for 2 weeks, followed by a 2-week rest). The primary end point was PFS. The noninferiority margin was 1.25; noninferiority would be established if the upper limit of the 95% confidence interval (CI) for the hazard ratio (HR) of the control group versus the experimental group was less than this margin. Result: Between June 2012 and September 2014, 487 patients underwent randomization. Two hundred and forty-three patients assigned to the control group and 241 assigned to the experimental group were included in the primary analysis. Median PFS was 10.8 months (95% CI 9.6-11.6) in the control group and 14.0 months (95% CI 12.4-15.5) in the experimental group (HR 0.84, 95% CI 0.70-1.02; P < 0.0001 for noninferiority, P = 0.0815 for superiority). One hundred and fifty-seven patients (64.9%) in the control group and 140 (58.6%) in the experimental group had adverse events of grade 3 or higher. Conclusion: S-1 and irinotecan plus bevacizumab is noninferior to mFOLFOX6 or CapeOX plus bevacizumab with respect to PFS as first-line treatment of mCRC and could be a new standard treatment. Clinical trials number: UMIN000007834.

Circular dichroism and biochemical properties of the hepatitis B virus core antigen.

The hepatitis B virus (HBV) core antigen was purified by mild procedures, including hydroxyapatite column chromatography, with care taken to avoid the degradation of the particles. Circular dichroism (CD) of the HBV core particles in saline showed low intensities of negative ellipticities in the region dominated by amide bond absorption. Acid treatment of the particles induced a remarkable change in the CD spectrum, with the appearance of a positive extremum at about 208 nm. The amino acid composition and the COOH-terminal residue of the isolated core polypeptide (Mr 21,000-21,500) were shown to be essentially the same as those of the polypeptide deduced from the nucleotide sequences which had been proposed for the HBV core antigen by other laboratories. We failed to detect any NH2-terminal dansyl-derivatives from the core polypeptide by the dansyl-Edman method. We also showed by the method of fluorescein polarization that the core polypeptide conjugated with fluorescein isothiocyanate has an affinity for serum albumin. This may indicate a state of disassembled or non-assembled core polypeptide in sera.

Intra- and extracellular distribution and immunochemical characterization of hepatitis B virus nucleocapsid proteins produced by a human hepatoma cell line transfected with cloned viral DNA.

Hepatitis B virus (HBV)-related antigens produced by the human hepatoma cell line (HB611 cell), which had been transfected with a cloned HBV DNA and established as a stable producer of HBV (T. Tsurimoto, A. Fujiyama, and K. Matsubara, 1987, Proc. Natl. Acad. Sci. USA 84, 444-448), were investigated immunochemically and morphologically. All HBV-related antigens, HBV surface (HBsAg), e (HBeAg), and core (HBcAg), were semiquantitatively examined by the respective reversed passive hemagglutination assay (RPHA). RPHAs for HBcAg and for HBeAg were characterized as reacting only to the core particles and to the free form of nucleocapsid proteins, respectively. The amounts of HBsAg and nucleocapsid protein in culture medium were roughly related to the number of viable cells. The amount of core particles was, instead, proportional to the number of dead cells. Relative amounts of HBsAg, core particles, and nucleocapsid proteins in culture medium, cell surface, and cell lysate were determined and it was found that HBsAg and nucleocapsid proteins were effectively secreted into culture medium but core particles were not. Molecular species of nucleocapsid proteins were identified to be p17 and p18 (HBeAg polypeptides) in the culture medium and HBeAg polypeptides and p21.5 (HBcAg polypeptide) in the cytosol fraction. The p21.5 was preferentially found in the nuclear fraction.

Immunochemical characteristics of hepatitis B e antigen subspecificities, HBeAg/1 and HBeAg/2.

The molecular interrelation between hepatitis B e Ag 1 (HBeAg/1) and HBeAg/2 was examined immunochemically. Major polypeptides with m.w. around 17,000 (P17) and one minor polypeptide with m.w. 18,000 (P18) that had HBeAg activity were consistently detected in 12 different serum samples by immunoblotting analysis. To examine the polypeptide composition of HBeAg/1 and HBeAg/2, precipitin lines of HBeAg/1-anti-HBeAg/1 and HBeAg/2-anti-HBeAg/2 were separately cut from the agarose gel and analyzed by immunoblotting. HBeAg/1 and HBeAg/2 showed similar P17 and P18 compositions. Monomeric forms of P17 and P18 in a solution of 0.1% SDS showed HBeAg/1 activity, whereas polymeric forms of these polypeptides showed HBeAg/2 activity. The stability of HBeAg subspecificities to SDS and 2-ME was examined. When HBeAg was incubated in a buffer containing 0.1% SDS and 0.1% 2-ME, only the HBeAg/2 precipitin line disappeared in agarose gel concurrently with the conversion of HBeAg molecules to a monomeric from a polymeric form. In contrast, neither monomerization nor the disappearance of HBeAg/2 was seen when SDS or 2-ME was used by itself. These results indicate that hydrophobic forces and disulfide bonds may be involved in the association of P17 and P18 in serum and that HBeAg/2 activity depends upon association of HBeAg-polypeptides but that HBeAg/1 activity does not. The possibility that the epitopes of HBeAg/2 are specific for the conformation of polymerized molecules is discussed.

DNA-binding activity of hepatitis B e antigen polypeptide lacking the protaminelike sequence of nucleocapsid protein of human hepatitis B virus.

The characteristics of binding of hepatitis B core antigen (HBcAg) and hepatitis B e antigen (HBeAg) polypeptides to hepatitis B virus (HBV) DNA were analyzed. HBcAg polypeptide from recombinant HBV core particles and HBeAg polypeptide from partially purified serum HBeAg were prepared and verified to have molecular weights of 21,500 (P21.5) and of 17,000 (P17) and 18,000 (P18), respectively, by immunoblot analysis. By reaction of these proteins on a nitrocellulose membrane with cloned 32P-HBV DNA, it was revealed that the HBeAg polypeptide, which lacks the C-terminal 34 amino acids of P21.5, as well as the HBcAg polypeptide, bound to the DNA. The secondary structures of nucleocapsid proteins of HBV, woodchuck hepatitis virus, and ground squirrel hepatitis virus were predicted by the Garnier algorithm. Amino acid sequences which, in addition to those of the C-terminal regions, may contribute to binding were proposed to be the 21-amino-acid residues located at amino acids 100 to 120 of the nucleocapsid proteins of these hepadnaviruses.

Physicochemical heterogeneity of hepatitis B e antigen detected in asymptomatic carriers and carriers in a hemodialysis unit.

Physicochemical studies of hepatitis B e antigen (HBeAg) revealed a clear cut difference between e1 and e2 antigen. The e1 antigen was found to have a MW of Ca 150,000 and a pI of 6.4-7.2, whereas both the MW and pI of the e2 antigen were heterogeneous depending upon the source of serum. Sera obtained from asymptomatic carriers were characterized by low titers of HBs antigen, HBc antigen and DNA polymerase and contained e2 antigen of larger molecular weight (200,000-300,000) with a narrow distribution range and a pI of 4.8 to 5.2 (type 1). On the other hand, the sera from patients in a hemodialysis unit who were HBs antigen carriers and had high titers of HBs antigen, HBc antigen and DNA polymerase contained e2 antigen of heterogeneous distribution in MW (from 300,000 to 70,000) and pI (type 2 and 3). The e2 antigen obtained from the higher MW type 3 serum had lower isoelectric points (pI 4.5 to 5.2) as was the case with e2 antigen obtained from asymptomatic carriers whereas relatively wide range of isoelectric points (pI 5.1 to 8.2) was found with the lower molecular weight e2 antigen.

Collagen binding of Bifidobacterium adolescentis.

In this study, 13 bifidobacterial strains were tested for their ability to adhere to immobilized extracellular matrix (ECM) proteins. Only two Bifidobacterium adolescentis strains adhered to immobilized type I and type V collagens, but not to laminin, fibronectin, and type III and IV collagens. The adhesion of B. adolescentis BB-119 to type V collagen was inhibited by type I and V collagens and gelatin, and was diminished after protease treatment of the cells. Periodate treatment of immobilized collagen and the presence of galactose inhibited the adhesion of strain BB-119 to type V collagen. Two cell surface proteins with molecular masses of 36 kDa and 52 kDa from strain BB-119 were found to bind to horseradish peroxidase-conjugated type V collagen by ligand blotting. We concluded that B. adolescentis BB-119 binds to type V collagen at galactose chains as target via these two cell surface proteins by their lectin-like activity.

Demonstration of hepatitis B e antigen (HBeAg) in association with intact Dane particles.

Mild detergent treatment (0.1% Sarkosyl-0.1% beta-mercaptoethanol) of Dane particle-rich fraction from human serum resulted in the release of core particles together with HBe antigen activity when examined by the reversed passive haemagglutination method. Furthermore, when the core particles isolated by the above procedure were exposed to stronger detergent (1% Sarkosyl-0.1% beta-mercaptoethanol), additional HBe antigen activity was released only from intact core particles with DNA polymerase activity and not from empty core particles.

Molecular heterogeneity of hepatitis B virus e antigen in liver and serum.

Hepatitis B virus e antigen (HBeAg) derived from liver at autopsy or from the serum of asymptomatic carriers has been characterized. The liver-derived HBeAg consisted of two different molecules, one with a mol. wt. of 30 000 (monomer) and the other with a mol. wt. of 90 000 (trimer), in a ratio of 3:1. Both were free of IgG. The serum-derived HBeAgs were heterogeneous with mol. wt. of 30 000, 90 000, 240 000, 400 000 and 540 000. Among them, the so-called IgG-free HBeAgs consisted almost exclusively of the 30 000 and 90 000 molecular species, in a ratio of 1:9. The serum HBeAg of mol. wt. 90 000 was further differentiated into two molecular species, one trimer and the other associated with albumin. The large mol. wt. HBeAgs (240 000, 400 000 and 540 000) were associated with IgG in ratios of one molecule of HBeAg to one, two or three molecules of IgG respectively. The complete dissociation of the IgG molecule was not achieved by 5 M-urea treatment of such HBeAgs, suggesting that it was bound in an immune complex. A hypothetical model is proposed which describes the heterogeneity of the HBeAgs derived from both the liver and serum, and containing HBeAgs either in a free form or associated with serum IgG.

Immunochemical characterization of hepatitis B e antigen subtypes, HBeAg/1 and HBeAg/2, in sera of hepatitis B virus carriers.

The interrelation between HBeAg subtypes, HBeAg/1 and HBeAg/2, in sera was examined immunochemically. The detection of HBeAg subtypes in immunodiffusion (ID) depended upon the amount of HBeAg determined by reversed passive haemagglutination (RPHA), ie, the titres of HBeAg in sera positive for both HBeAg/1 and HBeAg/2, positive for only HBeAg/1 and negative for both HBeAg/1 and HBeAg/2 were 2(9.8) +/- 1.5, 2(7.0) +/- 1.6, and 2(5.6) +/- 1.3, respectively. When the sera belonging to the latter two groups were concentrated up to 2(10) RPHA titre, the precipitin line corresponding to that of HBeAg/2-anti-HBeAg/2 was visualized in ID. Monoclonal anti-HBeAg antibody that absorbed only the precipitin line of HBeAg/1-anti-HBeAg/1 in ID was prepared for the characterization of HBeAg subtypes. A linear correlation (r = 0.91) between titres of HBeAg determined by the RPHA cells prepared with monoclonal and polyclonal antibodies was found in almost all HBeAg-positive sera. The reactivities of this monoclonal anti-HBeAg antibody to both HBeAg/1 and HBeAg/2 were demonstrated in affinity chromatography experiments using a Sepharose 4B column conjugated with this antibody. These results suggest that both HBeAg/1 and HBeAg/2 are constantly present in HBeAg-positive sera and that they are closely associated. Based upon these results, a hypothetical model for the elucidation of the immunological relationship between HBeAg/1 and HBeAg/2 is proposed.

Haemagglutination and glycolipid-binding activities of Lactobacillus reuteri.

The carbohydrate-binding activity of Lactobacillus reuteri was studied by haemagglutination (HA), HA inhibition and thin layer chromatography (TLC) overlay assays. Three of the six Lact. reuteri strains examined showed HA activity. Two strains (JCM1081 and JCM1112T) agglutinated neuraminidase-treated, but not untreated, erythrocytes. Strain JCM2762 agglutinated both treated and untreated erythrocytes. The HA activity of JCM 1081 was inhibited by galactose, lactose, methyl beta-galactoside and asialoglycophorin A. Among 12 glycosphingolipids, TLC overlay assay showed that JCM1081 strongly bound to asialo-GM1. These results indicated that JCM1081 bound to the beta-galactosyl residues of the non-reducing terminal of sugar chains of glycoconjugates. The carbohydrate-binding ability of JCM1081 may be responsible for the adhesion of this strain to the mucosal surface of the intestine.

Depletion of dietary sulphamonomethoxine and sulphadimethoxine from various tissues of laying hens.

Sulphamonomethoxine (SMM) or sulphadimethoxine (SDM) was fed to laying hens at 400 mg/kg diet for 5 successive days. After withdrawal of the drugs, contents (mg/kg) of SMM and SDM in the blood, kidney, liver, ovary, muscle and adipose tissue were determined by HPLC. 2. The disappearance of dietary SMM and SDM from the tissues of laying hens was rapid and, except for the liver, was very similar in all tissues. 3. A common biological half-life (t1/2) of SMM in the above 6 tissues was estimated to be 5.2 h. The t1/2 of SDM in the liver was 6.9 h, significantly longer than that of 4.4 h in the other 5 tissues. The values were much shorter than 51/2 (reported elsewhere) for other drugs. 4. Comparing the data found in this study with those obtained from previous papers, the depletion velocities of SMM and SDM from the hen's body were much faster than those from albumen in egg. The reason for this is probably related to the longer time period over which albumen formation occurs.

Electron microscopy of human hepatitis B virus cores by negative staining-carbon film technique.

The ultrastructure of the nucleocapsid component of human hepatitis B virus (core particle) was studied by negative staining-carbon film technique. Using this method an improved image of core particles was obtained in respect of resolution and contrast. Two-dimensional crystalline arrays of core particles were formed in vitro. Under these arrays the distance between the particle centers was 28.3 nm, corresponding to the capsid diameter, when analyzed through optical diffraction patterns. Positively stained images of these arrays revealed that core particles contain an electron-dense center of nucleoid-like area about 21 nm in diameter. The capsid surface rarely exhibited small capsomeres, ie, small spheres or ring-like structures measuring 4.0-4.2 nm. From the dimension of these structures and the analysis by Markham's rotational technique, it was suggested that each of these capsomeres is an individual subunit (monomer) and 180 of these subunits build up the core particle capsid according to the icosahedral symmetry (T = 3), but not clustering into distinct morphological features.

Prevalence of Williams e1 antigen in comparison with e2 antigen in hepatitis B antigen carriers and patients in hemodialysis unit.

The prevalence of both e1 and e2 antigens in 1,158 sera of asymptomatic HBsAg carriers, carriers in hemodialysis units, and HBsAg-negative blood donors was examined. The detection rate of e1 antigen was as high as 80% in asymptomatic carriers, 95% in hemodialysis patients, and even 13.1% in HBsAg-negative donors. All of the e1 antigen-positive specimens in such HBsAg-negative sera were found to have both or either anti-HBs and anti-HBc, suggesting the past history of Hepatitis B virus (HBV) infection of the donors. In the HBsAg-positive serum, the detection rate of e2 antigen (17%) was lower than that of e1 (80%), and all sera having e2 antigen were positive for e1 antigen. The titers of HBsAg, HBcAg, and anti-HBc in e2 antigen-positive sera were higher than that of sera detecting only e1 antigen. The appearance of e1 antigen and e2 antigen in the course of post-transfusion hepatitis B was studied with five cases. Retrospective study showed that three of them each received one unit of HBsAg-positive blood, and the other two received HBsAg-negative blood but with high-titered anti-HBc. In four cases out of five, in which e2 antigen was detected during the course of infection, the initial detection of e2 antigen occurred at or just before the elevation of liver enzyme levels. On the other hand, e1 antigen was detected relatively early after transfusion, and the time of onset. Moreover, the detection period of e1 antigen persisted longer, even after the disappearance of HBsAg antigenemia. These two separate studies suggest that not only e2 antigen but also e1 antigen are associated with the infection of HBV, but they are distinct from each other; the e2 antigen may have the properties of a signal of the viral activity in the patient as suggested by many others, but e1 antigen does not seem to bear such diagnostic values.

Evidence for the multiplication of hepatitis B virus in "oval cell" culture originated from human embryonic liver.

Growth conditions fof human oval cells (immature hepatocytes),evidence of hepatitis B (HB) antigen synthesis in oval cells as revealed by immunofluorescent staining and successful passage of such an agent in the culture fluid up to the 4th passage are described. The results have been proved to be readily reproducible with different inocula. The oval cells used in these experiments were defined as small round cells, with scant cytoplasm, vesicular nuclei and small nucleoli, vitally stained with indocyanine green and synthesizing alpha-foetoprotein but no albumin.

Relationship between the replication of hepatitis B virus and the localization of virus nucleocapsid antigen (HBcAg) in hepatocytes.

According to the localization of hepatitis B virus (HBV) core antigen (HBcAg), detected by the avidin-biotin complex method, infected hepatocytes were classified into three types, i.e. those having nuclear (type I), nuclear and cytoplasmic (type II) or only cytoplasmic (type III) antigen. HBcAg-positive hepatocytes of all specimens (three) from non-specific reactive hepatitis and of most (five of seven) from chronic persistent hepatitis (CPH) patients were only type I; the other two CPH samples and all (seven) chronic active hepatitis samples were composed of a mixture of types I, II and III. Linear correlations between the frequency of type I, as well as that of of all types (I, II and III) of the HBcAg-positive hepatocytes, and the amount of HBV DNA in serum were found. The relative HBV production of HBcAg-positive hepatocytes (serum HBV DNA amount/frequency of HBcAg-positive cells) was 0.11 in type I and 0.07 in all hepatocytes including types I, II and III. HBV core particles and complete HBV particles were found in type I hepatocytes. On the other hand, these particles were not found in a predominantly type III liver specimen. These results suggest that type I hepatocytes are more involved in the propagation of HBV than types II and III.

Severe acute exacerbation in chronic hepatitis B virus infection in Sendai, Japan.

In order to examine the clinical features of severe acute exacerbation in chronic hepatitis B virus (HBV) infection, 297 hepatitis B surface antigen (HBsAg) carriers were followed for 35 +/- 22 months (mean +/- S.D.) in Tohoku University Hospital from 1976 to 1987. Of these, 10 experienced severe acute exacerbation with hepatic decompensation. All of these patients had intense subjective symptoms related to the hepatitis. They were all icteric and 8 had ascites. Three developed to fulminant hepatic failure, eventually died. Histology after the exacerbation showed severe hepatic damage such as massive hepatic necrosis and bridging hepatic necrosis in a half of them. Six cases were suspected to result from spontaneous reactivation and 2 from drug-induced reactivation of chronic HBV infection, and the other 2 from superinfection with non-A, non-B hepatitis agent (s). These results suggest that the reactivation of chronic HBV infection is an important factor of severe acute exacerbations in chronic HBV infection in Japan.