Fibrinogen γ-chain peptide-coated, ADP-encapsulated liposomes rescue thrombocytopenic rabbits from non-compressible liver hemorrhage.

BACKGROUND: We developed a fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV [H12])-coated, ADP-encapsulated liposome (H12-[ADP]-liposome) that accumulates at bleeding sites via interaction with activated platelets via glycoprotein IIb-IIIa and augments platelet aggregation by releasing ADP. OBJECTIVE: To evaluate the efficacy of H12-(ADP)-liposomes for treating liver hemorrhage in rabbits with acute thrombocytopenia. METHODS: Thrombocytopenia (platelets < 50 000 µL(-1)) was induced in rabbits by repeated blood withdrawal (100 mL kg(-1) in total) and isovolemic transfusion of autologous washed red blood cells. H12-(ADP)-liposomes with platelet-poor plasma (PPP), platelet-rich plasma (PRP), PPP, ADP liposomes with PPP or H12-(PBS)-liposomes/PPP, were administered to the thrombocytopenic rabbits, and liver hemorrhage was induced by penetrating liver injury. RESULTS: Administration of H12-(ADP)-liposomes and of PRP rescued all thrombocytopenic rabbits from liver hemorrhage as a result of potent hemostasis at the liver bleeding site, although rabbits receiving PPP or ADP liposomes showed 20% survival in the first 24 h. Administration of H12-(ADP)-liposomes and of PRP suppressed both bleeding volume and time from the site of liver injury. H12-(phosphate-buffered saline)-liposomes lacking ADP also improved rabbit survival after liver hemorrhage, although their hemostatic effect was weaker. In rabbits with severe thrombocytopenia (25 000 platelets µL(-1)), the hemostatic effects of H12-(ADP)-liposomes tended to be attenuated as compared with those of PRP treatment. Histologic examination revealed that H12-(ADP)-liposomes accumulated at the bleeding site in the liver. Notably, neither macrothombi nor microthrombi were detected in the lung, kidney or liver in rabbits treated with H12-(ADP)-liposomes. CONCLUSIONS: H12-(ADP)-liposomes appear to be a safe and effective therapeutic tool for acute thrombocytopenic trauma patients with massive bleeding.

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.

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.

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.

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.

Successful combined resection of carcinoma of the esophagus and adrenal metastasis: report of a case.

We report herein the case of a 56-year-old man who underwent successful combined resection of carcinoma of the esophagus and an adrenal metastasis. The patient presented with carcinoma of the thoracic esophagus, and an adrenal tumor was incidentally detected by computed tomography(CT). Complete removal of the carcinoma was accomplished along with a combined resection of the thoracic esophagus and left adrenal gland. Surgery was followed by the administration of anticancer chemotherapeutic agents. He is currently doing well with a grade 1 performance status and no signs of recurrence 22 months after his operation. To our knowledge, no previous report of the successful simultaneous resection of esophageal carcinoma and an adrenal metastasis has been documented in the literature.

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.

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.

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.

A neutral sugar is responsible for serovar specificity of the antigenic determinant of Leptospira interrogans serovar canicola.

To provide information on the chemical structures of antigenic determinants of leptospira, glycolipids of Leptospira interrogans serovar canicola strain Hond Utrecht IV (Ut-IV) and its antigenic variant selected in the presence of a serovar-specific monoclonal antibody were compared physicochemically. Gas-liquid chromatography-mass spectrometry analysis revealed that the glycolipid of Ut-IV contained 6 neutral sugar species; rhamnose, mannose, galactose, glucose, and unknown sugars III and IV, in addition to unknown sugars I and II that had been previously reported. On the other hand, the glycolipid of the variant lacked unknown sugar III, suggesting that this sugar is responsible for the serovar-specific antigenic determinant.

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.

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.

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.

Chemotaxis of leptospires to hemoglobin in relation to virulence.

A guinea pig-lethal line of Leptospira interrogans serovar copenhageni strain Shibaura, but not an avirulent line of the same strain, moved in larger numbers toward hemoglobin than toward distilled water (control) in a U-shaped polypropylene tube. L. interrogans serovar lai strains 017 and KH-1, which were also guinea pig lethal, showed a similar move to hemoglobin. No such move toward hemoglobin was shown by 14 avirulent strains of L. interrogans (with one exception) or any of the 8 strains of L. biflexa tested.

Selective medium containing fosfomycin, nalidixic acid, and culture supernatant of Rhodococcus equi for isolation of Corynebacterium pseudotuberculosis.

FNR medium containing fosfomycin, nalidixic acid, bovine blood and culture supernatant of Rhodococcus equi was prepared by the present authors, and the medium did not inhibit growth of Corynebacterium pseudotuberculosis but completely hampered the growth of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. The culture supernatant of R. equi facilitated detection of suspected colonies of C. pseudotuberculosis due to synergistic hemolysis. Rate of isolation of the organisms (from the trachea, larynx and nasal cavity of 16 slaughtered sheep with caseous abscess in the lung) was higher with FNR, the selective medium, than with nonselective medium. The selective medium was thus found to be useful for isolation of C. pseudotuberculosis from sheep.

Typing of Rhodococcus equi isolated from submaxillary lymph nodes of pigs in Japan.

Isolation of Rhodococcus equi from the submaxillary lymph nodes of pigs, with or without caseous lymphadenitis, and typing of the isolates by two serological methods were carried out. The rate of isolation of the organisms from the lymph nodes of pigs was 5 times higher in the lymph nodes with caseous lymphadenitis than in those without the lesion. Of 219 isolates, 146 (66.7%) were typable by the method of Prescott, while all the 219 isolates (100%) were typable by the method of Nakazawa et al. The most frequently isolated were serotype 2 of Prescott (identical to serogroup 16 of Nakazawa et al.), and serogroup 3 of Nakazawa et al., which did not correspond with any serotypes of Prescott. Serotypes/serogroups of R. equi from pigs were thus first clarified in Japan.

Phase variation of pili of Corynebacterium pilosum.

Whether or not (?) phase variation occurs in the pili of Corynebacterium pilosum was biologically examined using the colony enzyme-linked immunosorbent assay (ELISA) blot test with anti-pili immune serum. From the densely piliated clone (35P+) of C. pilosum 35, non-piliated variants were isolated at a frequency of 2.45 x 10(-3). From one of the non-piliated variants (designated as P(-11)/35P+), a piliated variant was isolated at a frequency of 4.68 x 10(-4), one log less frequently than the non-piliated variant. From this piliated variant as designated (P+4/P(-11)/35P+), a non-piliated variant was isolated at a frequency of 3.86 x 10(-3). C. pilosum was thus alternated between piliated and non-piliated at a fairly high frequency, suggesting that the pili may undergo phase variation. This is the first finding of phase variation of the pili of Gram-positive bacteria.

A basic study of chemotaxis of leptospiras.

Chemotaxis of leptospiras was studied using a U-tube made of a commercially available straw-shaped polypropylene tube with an uneven flexible part. The U-tube was made by folding the polypropylene tube double and binding the vertical parts by a piece of tape, thus making the uneven flexible part the bottom. Semi-solid Korthof's medium was poured into the U-tube. Preliminary experiments showed that the medium containing 0.5% agar and measuring 2 cm in in length was appropriate. Leptospiras inoculated on the top of the medium of one side of the U-tube moved toward the top of the medium of the other side. The leptospiral move toward the uninoculated top was significantly faster when 6 micrograms of hemoglobin were added to the uninoculated top. Chemotaxis of leptospiras to hemoglobin was thus shown, which is the first evidence of chemotaxis of leptospiras.