Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray: identification of genes involved in viral carcinogenesis and tumor progression.

To disclose detailed genetic mechanisms in hepatocellular carcinoma (HCC) with a view toward development of novel therapeutic targets, we analyzed expression profiles of 20 primary HCCs and their corresponding noncancerous tissues by means of cDNA microarrays consisting of 23,040 genes. Up-regulation of mitosis-promoting genes was observed in the majority of the tumors examined. Some genes showed expression patterns in hepatitis B virus-positive HCCs that were different from those in hepatitis C virus-positive HCCs; most of them encoded enzymes that metabolize carcinogens and/or anticancer agents. Furthermore, we identified a number of genes associated with malignant histological type or invasive phenotype. Accumulation of such data will make it possible to define the nature of individual tumors, to provide clues for identifying new therapeutic targets, and ultimately to optimize treatment of each patient.

Alterations of gene expression during colorectal carcinogenesis revealed by cDNA microarrays after laser-capture microdissection of tumor tissues and normal epithelia.

To identify a set of genes involved in the development of colorectal carcinogenesis, we compared expression profiles of colorectal cancer cells from eight tumors with corresponding noncancerous colonic epithelia using a DNA microarray consisting of 9216 human genes. These cell populations had been rendered homogeneous by laser-capture microdissection. Expression change in more than half of the tumors was observed for 235 genes, i.e., 44 up-regulated and 191 down-regulated genes. The differentially expressed genes include those associated with signal transduction, metabolizing enzymes, production of reactive oxygen species, cell cycle, transcription, mitosis, and apoptosis. Subsequent examination of 10 genes (five up-regulated and five down-regulated) by semiquantitative reverse transcription-PCR using the eight tumors together with an additional 12 samples substantiated the reliability of our analysis. The extensive list of genes identified in these experiments provides a large body of potentially valuable information of colorectal carcinogenesis and represents a source of novel targets for cancer therapy.

Prediction of sensitivity of esophageal tumors to adjuvant chemotherapy by cDNA microarray analysis of gene-expression profiles.

We applied cDNA microarray analyses of 9216 genes to establish a genetic method for predicting the outcome of adjuvant chemotherapy to esophageal cancers. We analyzed expression profiles of 20 esophageal cancer tissues from patients who were treated with the same adjuvant chemotherapy after removal of tumor by operation, and we attempted to find genes associated with the duration of survival after surgery. By comparing expression profiles of those cancer tissues, we identified by statistical analysis 52 genes that were likely to be correlated with prognosis and possibly with sensitivity/resistance to the anticancer drugs. We also developed a drug response score based on the differential expression of these genes, and we found a significant correlation between the drug response score and individual patients' prognoses. Our results indicated that this scoring system, based on microarray analysis of selected genes, is likely to have great potential for predicting the prognosis of individual cancer patients with the adjuvant chemotherapy.

Genome-wide screening of genes showing altered expression in liver metastases of human colorectal cancers by cDNA microarray.

In spite of intensive and increasingly successful attempts to determine the multiple steps involved in colorectal carcinogenesis, the mechanisms responsible for metastasis of colorectal tumors to the liver remain to be clarified. To identify genes that are candidates for involvement in the metastatic process, we analyzed genome-wide expression profiles of 10 primary colorectal cancers and their corresponding metastatic lesions by means of a cDNA microarray consisting of 9121 human genes. This analysis identified 40 genes whose expression was commonly upregulated in metastatic lesions, and 7 that were commonly downregulated. The upregulated genes encoded proteins involved in cell adhesion, or remodeling of the actin cytoskeleton. Investigation of the functions of more of the altered genes should improve our understanding of metastasis and may identify diagnostic markers and/or novel molecular targets for prevention or therapy of metastatic lesions.

Spin-labeling of influenza virus hemagglutinin permits analysis of the conformational change at low pH and its inhibition by antibody.

To study the conformational changes in the hemagglutinin (HA) molecule of A/seal/Mass/1/80 (H7N7) (Seal) influenza virus at low pH, a spin-labeling method was used. This method also permits study of antibody interaction with the HA. A synthetic nitroxide compound was used for spin-labeling of tyrosine residues of the isolated HA molecule. Electron spin resonance (ESR) spectra of the spin-labeled HA at various pH values indicated that a conformational transition occurred under acidic conditions, and around pH 5.8 the HA molecule has maximal flexibility. Since virus-induced hemolysis occurs optimally at pH 5.8-5.9, the HA molecule in the maximally flexible conformation is considered to mediate membrane fusion. The ESR spectra of the antibody-bound HA at various pH values revealed that monoclonal antibodies to different regions on the molecule may inhibit the conformational change by different modes. One antibody inhibited the changes in the HA that resulted in flexibility, while the other did not. These results support the assumption that monoclonal antibodies, which failed to inhibit hemagglutination of the virus yet neutralized viral infectivity, inhibited the fusion step in the viral replication process by interfering with a low pH-induced conformational change in the HA molecule (Kida, H., Webster, R.G. and Yanagawa, R. (1983) Arch. Virol. 76, 91-99).

Leptospiral attachment to extracellular matrix of mouse fibroblast (L929) cells.

The attachment of leptospires to the extracellular matrix (ECM) remaining after mouse fibroblast (L929) cells on coverslips had been solubilized with Triton X-100 was examined. Each highly virulent line of Leptospira interrogans serovar copenhageni, canicola and pomona attached to ECM more effectively than intermediately virulent and avirulent lines of the same strains, suggesting a correlation between virulence and attachment to ECM. Inhibition of the attachment of highly virulent copenhageni to ECM was found in the presence of the homologous immunoglobulin G Fab fragment.

Opsonic effect of monoclonal antibodies against Leptospira interrogans serovar copenhageni.

The opsonic effect of monoclonal antibodies (McAbs) against Leptospira interrogans serovar copenhageni strain Shibaura was examined in vitro using radiolabeled organisms and mouse peritoneal exudate macrophages. Four IgG McAbs (all IgG3) and 2 IgM McAbs were used, all showed different reactivities and were bound to homologous lipopolysaccharide (LPS). IgG3 McAbs at the sub-agglutinating concentration were opsonic, but the degree of the opsonic effect varied among IgG3 McAbs. Opsonization with IgG3 McAbs at the concentration showing the same level of ELISA binding showed that the highest opsonic effect was exerted by Sw-6 antibody. The other IgG3 McAbs showed a similar but lower opsonic effect. IgM McAbs, which were not opsonic at the sub-agglutinating concentration even in the presence of complement, showed opsonic effect at the agglutinating concentration.

Adhesion of Corynebacterium renale and Corynebacterium pilosum to the epithelial cells of various parts of the bovine urinary tract from the renal pelvis to vulva.

The various parts of the bovine urinary tract, the renal pelvis, the ureter, the urinary bladder, the urethra, the vaginal vestibule and the vulva, were examined for the capacity of the epithelial cells to bind Corynebacterium renale and C. pilosum. C. renale adhered best to the epithelial cells of the vulva, and then to those of the ureter and renal pelvis. C. pilosum also adhered best to the epithelial cells of the vulva, followed by those of the vaginal vestibule. The results indicate that the most important target tissue for these bacteria may be the vulva, and the results correlate with the fact that C. renale frequently causes pyelonephritis and ureteritis, while C. pilosum causes the same diseases less frequently and behaves like normal flora of the vaginal vestibule.

Survival of Corynebacterium renale, Corynebacterium pilosum and Corynebacterium cystitidis in soil.

Survival of the causative agents of bovine pyelonephritis, Corynebacterium renale, C. pilosum and C. cystitidis, was examined at 30 degrees C in autoclaved soil. In the soil from a paddock, C. renale and C. cystitidis survived for 56 and 63 days, respectively, and C. pilosum for a longer period of at least 210 days. In soil from a pasture, sand from an athletic field and sea sand, the survival of these bacteria was of shorter duration.

Comparison of surface hydrophobicity of piliated and non-piliated clones of Corynebacterium renale and Corynebacterium pilosum.

Piliated (P+) and non-piliated (P-) clones of Corynebacterium renale and C. pilosum were similar in hydrophobicity as measured by hydrophobic interaction chromatography, bacterial adherence to hydrocarbons and the salt aggregation test. Therefore, the previously reported adherence of P+ clone to various cells, which is more effective than that of P- clone, may be uncorrelated with the degree of hydrophobicity of both clones of these bacteria. Hydrophobicity of P+ and P- clones was found to be high when measured by hydrophobic interaction chromatography and bacterial adherence to hydrocarbons, but low when measured by the salt aggregation test.

Isolation and characterization of monoclonal antibodies against pili of Corynebacterium renale and Corynebacterium pilosum.

Five monoclonal antibodies against pili of Corynebacterium renale 115 P+ (piliated clone) and two monoclonal antibodies against pili of C. pilosum 92 P+ (piliated clone) were produced. These antibodies bound to pili of the homologous strain in in enzyme-linked immunosorbent assay (ELISA) and agglutinated P+ but not P- (non-piliated clone) of each homologous strain. The five monoclonal antibodies against C. renale 115 P+ pili were divided into 2 groups, comprising 16/5, 160/1 and 32/6 and 13/4 and B20/3, based on the results of a competitive binding assay. The results may indicate the presence of at least 2 distinct antigenic areas on the pilus of C. renale 115 P+. The monoclonal antibodies of the first group inhibited adhesion of C. renale 115 P+ bacteria to the epithelial cells of bovine vulva, while the second group did not. Two monoclonal antibodies against C. pilosum 92 P+ pili recognized the same area on the pilus of C. pilosum 92 P+, and inhibited the adhesion of C. pilosum 92 P+ bacteria to the epithelial cells of bovine vulva. The adhesion of these bacteria was inhibited by the monoclonal antibodies in the form of IgG as well as by the Fab fragment. The strains of C. renale and C. pilosum which reacted with each of the anti-C. renale 115 P+ pili and anti-C. pilosum 92 P+ pili monoclonal antibodies were small in number and of restricted distribution.

Human influenza virus infection in mink: serological evidence of infection in summer and autumn.

During the period from July to November 1981, 42 out of 128 young mink of a flock were found to possess antibodies against the viruses A/Bangkok/1/79 (H3N2) and A/Kumamoto/37/79 (H1N1), which were currently prevailing human influenza viruses. Seroconversion against A/Bangkok/1/79 was found in 12 mink from August to November. HI antibody titers of greater than 1: 128 were found in 8 out of 42 mink at the first examination in July and August. These findings suggest that infection with these human influenza viruses was present in this flock during the period from birth (the beginning of May) to autumn, the non-prevalent season in man. Attempts at virus isolation were unsuccessful.

Plasmid profiles of Klebsiella pneumoniae isolated from bovine mastitis.

Plasmids of Klebsiella pneumoniae strains isolated from mastitic milk of cows were detected, and the epidemiological significance of their profiles was investigated. Of 47 K. pneumoniae isolates, 41 (87.2%) possessed plasmid(s). Although the molecular size of the plasmids ranged from 1.9 to 140 megadaltons (Md), the 125 Md plasmid was predominant (31/41, 75.6%). There was a great diversity in the plasmid profiles, however, no correlation was found between the plasmid profiles and capsular types of Klebsiella or the source of strains (dairy farms), except for one farm. All isolates obtained from cows on that farm possessed the 125 Md plasmid and their capsular type was 9.

Isolation of Corynebacterium pseudotuberculosis from the cervical canal of clinically normal sows.

Two isolates from the cervical canal of clinically normal sows were indistinguishable from Corynebacterium pseudotuberculosis in their biochemical properties and genetic homology by DNA-DNA hybridisation. Furthermore, three sheep inoculated with one of the isolates developed typical lesions of caseous lymphadenitis and gave antibody responses specifically to C pseudotuberculosis.

Detection of leptospiral plasmid and comparison of plasmid profiles between virulent and avirulent leptospires.

Detection of plasmid from leptospires and a comparison of the plasmid profiles between virulent and avirulent strains were performed to investigate whether leptospires contained plasmid(s) associated with virulence. Virulent strains of Leptospira interrogans serovars copenhageni, lai, canicola and pomona, which were virulent for the guinea pig and/or hamster and which showed chemotaxis toward hemoglobin, contained approximately 370 kilobases (kb) plasmid. Avirulent strains of L. interrogans also contained identical plasmid. Similar plasmid profiles in virulent and avirulent strains of L. interrogans were observed. These data showed that no plasmids associated with virulence or chemotaxis were detected. Strains of saprophytic leptospires, L. biflexa and Leptonema illini, did not possess any plasmid.

Protection from caseous lymphadenitis in sheep by spraying iodine tincture on shearing wounds.

The effect of spraying shearing wounds with iodine tincture on Corynebacterium pseudotuberculosis infection in lambs was examined. The ELISA-negative lambs which had received some visible wounds during their first shearing were randomly divided into two groups: one was sprayed with iodine tincture on wounds after shearing, and the other was not. Anti-C. pseudotuberculosis toxin titers were measured by ELISA. The seroconversion ratio of the group with iodine tincture treatment 3 months after shearing was smaller than that of the untreated group (P < 0.05). These results suggest that treatment of shearing wounds with iodine tincture is effective in the protection of C. pseudotuberculosis infection in lambs.

Assignment of the bacterial agent of urinary calculus in young rats by the comparative sequence analysis of the 16S rRNA genes of corynebacteria.

Comparative 16S rRNA gene sequencing was used to assign four isolates of spontaneous urinary calculus in young laboratory rats. The phylogenetic relationships among the rat isolates and selected species of corynebacteria were also inferred. Based on the homology and evolutionary distance analysis, the 16S rRNA genes of the rat isolates were almost identical with that of Corynebacterium renale ATCC 19412. Also the results of the phylogenetic analysis showed a close relationship among the isolates and C. renale, but they were clearly different from C. pilosum, C. cystitidis, C. kutscheri and Rhodococcus equi. The results of the present study and previously published biochemical data demonstrate that the organism involving urinary infections in young rats is identified to be C. renale.

Seroepidemiological evidence that shearing wounds are mainly responsible for Corynebacterium pseudotuberculosis infection in sheep.

Anti-Corynebacterium pseudotuberculosis toxin titers were examined by an enzyme-linked immunosorbent assay (ELISA) at 3 to 5 month intervals in sheep of three age groups (lambs, 1- and 2-year-old sheep). A significant increase in the ELISA-positive ratio was found only in July, 3 months after shearing, in 1- and 2-year-old sheep. A decrease in the positive ratio was found in the same month in lambs, possibly due to a decrease in maternal antibodies. Since the 1- and 2-year-old sheep had been sheared in April, it was considered that the increase in the positive ratio in July might be closely related to the shearing in April.

Pili-mediated attachment of Corynebacterium renale to mucous membrane of urinary bladder of mice.

Corynebacterium renal strain 115 with numerous pili became attached in vivo to the mucous membrane of the urinary bladder of mice 10 to 30 times more frequently than did that of C renale American Type Culture Collection 19412, which showed few pili. Antipili serum-treated C renale strain 115 was not recovered from the membrane in as large amounts as was untreated bacteria. Antisomatic serum-treated strain 115, on the other hand, was recovered from the membrane in amounts similar to untreated bacteria. Untreated organisms became attached to the mucous membrane of the urinary bladder more effectively than did the antipili serum-treated bacteria, as seen on scanning electron micrographs. It may be concluded that C renale strain 115 attaches itself to the mucous membrane of the urinary bladder om mice by the pili.

Attachment of Corynebacterium renale to tissue culture cells by the pili.

One or more cells of Corynebacterium renale strains (serologic types, I, II and III), which possessed numerous pili, frequently were attached to BHK-21 cells, primary dog kidney cells, and primary rabbit kidney cells. The percentage of the cultured cells to which C renal cells were attached was about 70%. The percentage was less with cells of C renale possessing fewer pili, around 30%. After C renale was treated with the homologous anti-pili serum, the percentage of BHK-21 cells to which bacterial cells were attached was even less (22%). In electron micrographs, the pili of C renale were observed to attach themselves to the membranes of BHK-21 cells. The adhesive property of the pili of C renale to tissue culture cells was thus demonstrated.