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.

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.

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.

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.

Prevalence of precore-defective mutant of hepatitis B virus in HBV carriers.

Two hundred and seventy-three serum specimens from hepatitis B virus (HBV) carriers were examined for the presence of a characteristic one point mutation at nucleotide (nt) 1896 from the EcoRI site of the HBV genome in the precore region (the preC mutant) using restriction fragment length polymorphism (RFLP) analysis. This assay approach could detect preC mutants or wild-type sequences when either form constituted more than 10% of the total sample. Overall, 65.5% (76/116) of HBeAg-positive carriers had only the preC wild-type. All HBeAg-positive asymptomatic carriers (n = 14) had only the preC wild-type. In patients with chronic hepatitis B and in anti-HBe-positive asymptomatic carriers, increased prevalence of the preC mutant was associated with the development of anti-HBe antibodies and normalization of the serum alanine aminotransferase concentration. Furthermore, 27 (29.0%) of 93 HBeAg-negative carriers had unexpectedly preC wild-type sequences only. Direct sequencing of the HBV precore region of HBV specimens from 24 patients revealed no mutation at nt 1896, supporting the specificity of the RFLP analysis. These results suggest that RFLP analysis was accurate for the detection of the preC mutation and that the absence of serum HBeAg cannot be explained solely by the dominance of the preC mutant.

Changes of serum hepatitis C virus levels in patients with chronic hepatitis C treated with two courses of interferon administration.

To assess the efficacy of repeated interferon (IFN) administration in patients with chronic hepatitis C unresponsive to initial therapy, serum hepatitis C virus (HCV)-RNA levels were measured in 12 patients who had failed prior IFN therapy. Serum HCV-RNA was assayed by measuring DNA complementary to HCV-RNA using a quantitative reverse transcription-polymerase chain reaction assay. The mean total dose of IFN was 227.8 mega-units for first treatment and 270.7 mega-units for second treatment. Five responders with a normal alanine aminotransferase (ALT) concentration (less than 40 IU/liter) at the end of the first treatment also had a normal ALT concentration at the end of the second treatment. By contrast, all nonresponders with an elevated ALT concentration during the first treatment likewise had an elevated ALT concentration at the end of the second treatment. HCV-RNA levels before the first treatment varied from 10(6) to 10(8) copies/microliters. The serum HCV-RNA levels fell in 9 out of 10 patients after the first treatment and in 11 out of 12 patients after the second treatment. One patient had unchanged normal serum ALT levels after two courses of IFN treatment. These results suggested that the outcome of a second course of IFN treatment was similar biochemically and virologically to a first course, and that patients who did not respond initially seldom respond to additional IFN therapy. Therefore, readministration of IFN should be restricted to patients who respond biochemically and virologically to initial treatment. The optimal second dose shall be determined with further studies.

Two cases of porphyria cutanea tarda associated with chronic hepatitis positive for the antibody against hepatitis C virus.

We report two cases of porphyria cutanea tarda (PCT) positive for the antibody against hepatitis C virus (anti-HCV). The serological and histological examinations revealed that they were persistently infected with HCV and were suffering from liver disease compatible with chronic viral hepatitis. It is suggested that one of the factors which contribute to liver damage of patients with PCT may be HCV infection. It now may be advisable to examine anti-HCV in PCT patients with liver disease.

Relationship between molecular state of serum HBV-DNA and clinical features of hepatitis B virus carriers.

Molecular species of serum hepatitis B virus (HBV)-DNA in HBV carriers were classified by Southern blot hybridization into three types; type I with two bands of 4.0 kb and 3.2 kb, type II with the two bands of type I plus the smear between 4.0 kb and 3.2 kb, and type III with a broad band below 4.0 kb. A total of 51 HBV carriers were classified into three groups (group I, n = 19 with type I; group II, n = 12 with type II; and group III, n = 20 with type III). Serum aminotransferase levels of group I were significantly lower than those of groups II and III. Liver pathology revealed that 18 of the 19 (94.7%) group I patients showed nonspecific reactive hepatitis (NSRH), while 11 of the 12 (91.7%) group II patients and 19 of the 20 (95.0%) group III patients showed chronic persistent hepatitis (CPH) or chronic active hepatitis (CAH). Immunohistochemical study showed that hepatitis B core antigen (HBcAg) was localized in the nucleus of hepatocytes in most of patients with type I while it was localized in both the nucleus and cytoplasm in those with types II and III. Since the smear between 4.0 kb and 3.2 kb is specifically found in groups II and III, HBV-DNA with this smear may be related to active hepatitis.

Demonstration of hepatitis C virus genome in saliva and urine of patients with type C hepatitis: usefulness of the single round polymerase chain reaction method for detection of the HCV genome.

The possible transmission routes of hepatitis C virus (HCV) in patients without overt parenteral exposure (sporadic or community acquired form) were examined. Saliva and urine specimens obtained from type C hepatitis patients, whose sera were positive for the HCV genome, were examined by reverse transcription and polymerase chain reaction (RT-PCR). By analyzing the factors that influenced the detection of the HCV genome by PCR, we developed a single round method which enabled semiquantitative detection with higher sensitivity than that obtained with nested PCR. Single round PCR revealed that 34.8% (8 of 23) of saliva and 56.5% (13 of 23) of urine specimens from patients with type C hepatitis contained the HCV genome. The amounts of HCV genome in saliva and urine specimens correlated with those in serum. The relative amounts of HCV genome in serum, saliva, and urine from a chronic type C hepatitis patient were determined by comparing the reciprocal of the smallest volume of the specimens in which the PCR products were visualized in agarose gels (PCR units/ml), and the values were 1 x 10(5), 5 x 10(1), and 3 x 10(1) PCR units/ml for serum, saliva, and urine specimens, respectively.

Three distinct Southern blot hybridization patterns of HBV-DNA in the sera of HBV carriers.

The molecular species of hepatitis B virus (HBV)-DNA of 44 sera taken from 25 HBV carriers were examined by Southern blot hybridization with a biotin-labeled HBV-DNA probe and classified into three patterns. Type I consisted of two distinct bands of 4.0 kb and 3.2 kb. Type II consisted of the 4.0 kb and 3.2 kb bands and a smear between these two bands. Type III showed a broad band below 4.0 kb. With endogenous HBV-DNA polymerase reaction, the smear disappeared and 4.0 kb and 3.2 kb bands appeared. After EcoRI digestion, the 4.0 kb band disappeared to form a single band of 3.2 kb. These results suggest that the 4.0 kb and 3.2 kb forms are a relaxed circular, fully double-stranded DNA and a linear, fully double-stranded DNA, respectively, and that the smear between 4.0 kb and 3.2 kb is formed from the sum of relaxed circular, partially double-stranded DNAs with various length of plus strands. Comparison between histological diagnosis and Southern blot hybridization patterns of 25 HBV carriers indicates that these three patterns are closely related to the degree of hepatitis.

Helicobacter pylori infection and chronic atrophic gastritis among Japanese blood donors: a cross-sectional study.

To evaluate an association between Helicobacter pylori (H. pylori) infection and chronic atrophic gastritis (CAG), an established precursor of gastric cancer, we performed a cross-sectional study using IgG antibody against H. pylori and pepsinogens of blood donors in four prefectures in Japan. Although a geographic correlation between the age-adjusted prevalence rates for H. pylori infection and those for CAG was not seen, the age-adjusted odds ratios (OR) of H. pylori infection for CAG were high in each area (around five for men and from four to 12.6 for women). The association between them weakened with advancing age; the ORs in the youngest age group (16-29 yrs) and in the oldest age group (50-64 yrs) were 12.5 and 2.8 for men, and 11.5 and 5.2 for women, respectively. These findings suggest that H. pylori infection is strongly associated with CAG, while there are some other factors interacting in the development of CAG. A prospective cohort study in which CAG and H. pylori infection are taken into account will be necessary to assess the risks of gastric cancer.

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.

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.

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.

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.

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.

Virological significance of HBeAg subtypes (HBeAg/1 and HBeAg/2) in patients with type B hepatitis.

In order to establish the virological significance of HBeAg subtypes (HBeAg/1 and HBeAg/2) during hepatitis B virus infection, HBsAg, HBeAg and hepatitis B virus DNA in serum and HBcAg in liver were determined quantitatively in relation to the detection of HBeAg subtypes in agar gel diffusion. Thirty-eight chronic HBsAg carriers with HBeAg, including 16 non-specific reactive hepatitis, 8 chronic persistent hepatitis, 11 chronic active hepatitis and 3 liver cirrhosis, who were seen at Tohoku University Hospital from 1983 to 1985, were examined. Significantly larger amounts of HBsAg, HBeAg and hepatitis B virus DNA in serum and HBcAg in liver were found in patients positive for both HBeAg/1 and HBeAg/2 in serum than in those positive for only HBeAg/1 or negative for both subtypes. These results suggest that the presence of HBeAg/2 in serum may reflect the occurrence of active viral replication. When the detection pattern of HBeAg subtypes was examined during serial follow-up for at least 1 year, three groups of patients were classified with respect to the presence of HBeAg/2, i.e., Type I, consistently positive for HBeAg/2; Type II, consistently negative for HBeAg/2, and Type III, intermittently positive for HBeAg/2. More than 80% of Type I patients were histologically diagnosed having as nonspecific reactive hepatitis, while more than 80% of Type II and III patients had more progressive liver diseases such as chronic persistent hepatitis, chronic active hepatitis and liver cirrhosis. These results suggest that the serial examination of HBeAg subtypes in serum may be important for more detailed evaluations of type B hepatitis.

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.

Establishment of passive hemagglutination assay (PHA) system for anti-HBc in plasma.

A sensitive and specific assay system for anti-HBc, the passive hemagglutination (PHA) method, has been established. The reactivity of PHA cells prepared by conjugating purified recombinant HBcAg-particles with fixed sheep blood cells (SRBC) was highly specific to monoclonal- and polyclonal anti-HBc IgGs. The sensitivity of PHA method was higher than that of radioimmunoassay (RIA). However, uncertainty for the positivity of anti-HBc still remained in plasma with the PHA titers lower than 2(5). A relatively high ratio (19%, 37 of 196) of anti-HBc-positive plasma, which had been confirmed to be HBsAg negative, was demonstrated in blood donors whose bloods have been considered suitable for transfusion. The hazards of anti-HBc-positive bloods and the importance of anti-HBc detection in plasma are discussed in this paper.