Serum Osteopontin Predicts Degree of Hepatic Fibrosis and Serves as a Biomarker in Patients with Hepatitis C Virus Infection

Background & Aims

Osteopontin (OPN) is a matricellular protein that upregulates during pathogenesis of hepatic fibrosis. The present study was aimed to evaluate whether serum OPN could be used as a biomarker to assess the degree of hepatic fibrosis in patients with hepatitis C virus (HCV) infection.

Methods

Needle biopsy was performed on HCV patients and scored as zero fibrosis (F0), mild fibrosis (F1), moderate fibrosis (F2), severe fibrosis (F3) and liver cirrhosis (F4) based on Masson’s trichrome and α-smooth muscle actin (α-SMA) staining. Serum OPN levels were measured using ELISA and correlated with the degree of fibrosis. Furthermore, the OPN values were correlated and evaluated with platelets count, serum hyaluronic acid (HA), and collagen type IV and subjected to receiver operating characteristic (ROC) curve analysis.

Results

Serum OPN levels were remarkably increased from F0 through F4 in a progressive manner and the differences were significant (P < 0.001) between each group. The data were highly correlated with the degree of hepatic fibrosis. The ROC curve analysis depicted that serum OPN is an independent risk factor and an excellent biomarker and a prognostic index in HCV patients.

Conclusions

The results of the present study indicate that serum OPN levels reflect the degree of hepatic fibrosis and could be used as a biomarker to assess the stage of fibrosis in HCV patients which would help to reduce the number of liver biopsies. Furthermore, serum OPN serves as a prognostic index towards the progression of hepatic fibrosis to cirrhosis and hepatocellular carcinoma.     

Improved analytical method for the hexuronic acids at the reducing terminals of glycosaminoglycans

When endo-uronidases act on glycosaminoglycans, the reaction products have hexuronic acid residues at the reducing terminals. An analytical method for hexuronic acids at the reducing terminals was devised for hyaluronate oligosaccharides having hexuronic acid residues at the reducing terminals.The procedure is as follows: Hexuronic acid residues at the reducing terminals of hyaluronate oligosaccharides were tritiated with reduction using NaB[³H]₄ and the products were hydrolyzed with trifluoroacetic acid and nitrous acid. As a result, the tritiated and reduced hexuronic acid residues, that is aldonic acids, were liberated from the reducing terminals. After passing them through anion and cation ion-exchange resins, the aldonic acids were lactonized. The lactones were developed on paper chromatography, and their radioactivities determined on the paper.The method is also useful for discrimination between glucoronic acid and iduronic acid at the reducing terminals of glycosaminoglycans.     

Heterogeneity of reducing terminals of urinary chondroitin sulfates

Human urinary chondroitin sulfates were isolated by precipitation with cetylpyridinium chloride of the non-dialyzable fraction of pooled urine, followed by ethanol fractionation and successive enzymic digestions with neuraminidase and mucopolysaccharides. Further purification was achieved by Dowex-1 chromatography with stepwise elution by increasing the concentration of NaCl at intervals of 0.25 M from 0.75 M to 1.5 M. The chondroitin sulfates thus obtained were characterized by the analysis and quantification on of carbohydrate, amino acid and sulfate, and by electrophoresis on cellulose acetate membrane. Then reducing terminals were identified by gas liquid chromatographic analyses of the acetyl and butaneboronate derivatives of hydrolysates, after reduction of the reducing terminals with sodium borohydride. About 22.8% of the urinary chondroitin sulfate in the 1.5 M fraction was peptide-bound, and the remainder was peptide-free, with xylose (29.8%), galactose (23.6%) and glucuronic acid (18.7%) at the reducing terminal. The amount of peptide-free chondroitin sulfate with xylose and galactose at its reducing terminals in the 0.75 M-, 1.0 M-, 1.25 M- and 1.5 M-fractions increased in the order described in parallel with the increase of sulfation and the decrease of peptide content. It was thus suggested that the endo-types of beta-xylosidase, beta-galactosidase and beta-glucuronidase acted on the carbohydrate-peptide linkage region of proteo-chondroitin sulfate in the tissues and produced various types of urinary chondroitin sulfate with heterogeneity at reducing terminals.     

Monoclonal antibodies to human recombinant interleukin 1 (IL 1)beta: quantitation of IL 1 beta and inhibition of biological activity

Monoclonal antibodies (McAb) were developed to the Mr 17,500 form of human recombinant interleukin 1, IL 1 beta. Four McAb have been identified that inhibit the biological activity of IL 1 beta. McAb H34 and H67, at 1 microgram/ml (6 X 10(-9) M), completely inhibit the capacity of 1 ng/ml (6 X 10(-11) M) recombinant IL 1 beta to stimulate the proliferation of murine thymocytes or human fibroblasts in vitro. McAb H6 and H21 are approximately 10-fold less potent, and completely inhibit IL 1 beta activity at 10 micrograms/ml (6 X 10(-8) M) in both assays. The McAb do not have a significant effect on the biological activity of human recombinant IL 1 alpha in either assay. These McAb block the binding of recombinant [125I]IL 1 beta to IL 1 receptors on mouse 3T3 fibroblasts and have affinity constants for IL 1 beta in the range of 10(9) to 10(10) liters/mol. Competition studies suggest that two nonoverlapping epitopes on the IL 1 beta molecule are recognized by the McAb. H6 and H34 recognize one epitope, and H21 and H67 another. McAb H6 and H67 have been used together in a two-site ELISA to detect IL 1 beta. The sensitivity of the ELISA, which is 15 pg/ml (0.86 pM), approaches the limit of sensitivity of the thymocyte proliferation assay. The ELISA and thymocyte proliferation assay were used to quantitate IL 1 beta in E. coli LPS-stimulated human monocyte culture supernatants (HMCS). The level of IL 1 beta detected by ELISA in culture supernatants from eight donors ranged from 1.7 to 5.6 ng/ml, with a mean value of approximately 3 ng/ml. By comparison, the thymocyte proliferation assay gave levels of IL 1 in HMCS that were eight fold higher when quantitated by using recombinant IL 1 beta as a standard. This discrepancy with the bioassay used was reflected by the three fold higher maximum stimulation of thymocyte proliferation by HMCS as compared with recombinant IL 1 alpha or IL 1 beta, and only 45% inhibition of HMCS IL 1 activity by McAb. Thus, factors other than IL 1 beta account for the IL 1-like activity in monocyte culture supernatant as measured by the bioassay. The ILB1 McAb and ELISA allow for the first time-sensitive, accurate, and convenient quantitation of IL 1 beta levels in biological fluids or specimens.     

Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus.

Tyrosinase (monophenol, L-DOPA:oxygen oxidoreductase) was isolated from the ink of the squid, Illex argentinus. Squid tyrosinase, termed ST94, was found to occur as a covalently linked homodimeric protein with a molecular mass of 140.2 kDa containing two copper atoms per a subunit. The tyrosinase activity of ST94 was enhanced by proteolysis with trypsin to form a protein, termed ST94t, with a molecular mass of 127.6 kDa. The amino acid sequence of the subunit was deduced from N-terminal amino acid sequencing and cDNA cloning, indicating that the subunit of ST94 is synthesized as a premature protein with 625 amino acid residues and an 18-residue signal sequence region is eliminated to form the mature subunit comprised of 607 amino acid residues with a deduced molecular mass of 68,993 Da. ST94 was revealed to contain two putative copper-binding sites per a subunit, that showed sequence similarities with those of hemocyanins from mollusks, tyrosinases from microorganisms and vertebrates and the hypothetical tyrosinase-related protein of Caenorhabditis elegans. The squid tyrosinase was shown to catalyze the oxidation of monophenols as well as o-diphenols and to exhibit temperature-dependency of o-diphenolase activity like a psychrophilic enzyme.     

Negative dielectrophoretic patterning with different cell types

In this paper, a novel method for patterning different cell types based on negative dielectrophoresis (n-DEP), without any special pretreatment of a culture slide, has been described. An interdigitated array (IDA) electrode with four independent microelectrode subunits was fabricated with indium-tin-oxide (ITO) and used as a template to form cellular micropatterns. A suspension of C2C12 cells was introduced into the patterning device between the upper slide and the bottom IDA. In the present system, the n-DEP force is induced by applying an ac voltage (typically 12V(pp), 1MHz) to direct cells toward a weaker region of electric field strength. The cells aligned above one of the bands of IDA within 1min since the aligned areas on the slide were regions with the lower electric field. The application of an ac voltage for 5min allows the cells to adsorb onto the cell culture slide. After removing excess cells, the second cell type was patterned in lines using the same method as with the first set of cells. Periodic and alternate cell lines incorporating two cell types were also fabricated by changing the ac voltage mode. A second cell type was introduced into the device and guided to other areas to form a different pattern. The described system enables two cell types to be patterned in 15min. The patterning method provides a novel tool for use in fundamentals studies of cell biology based on cell-cell interactions between different cell types.     

The involvement of Cry1 and Cry2 genes in the regulation of the circadian body temperature rhythm in mice

The criptochrome genes (Cry1 and Cry2) are involved in the molecular mechanism that controls the circadian clock, and mice lacking these genes (Cry1(-/-)/Cry2(-/-)) are behaviorally arrhythmic. It has been speculated that the circadian clock modulates the characteristics of thermoregulation, resulting in body temperature (T(b)) rhythm. However, there is no direct evidence proving this speculation. We show here that T(b) and heat production in Cry1(-/-)/Cry2(-/-) mice are arrhythmic under constant darkness. In contrast, both rhythms occur under a light-dark cycle and/or periodical food restriction linked with spontaneous activity and/or eating, although they are not robust as those in wild-type mice. The relationship between heat production and T(b) in Cry1(-/-)/Cry2(-/-) mice is linear and identical under any conditions, indicating that their T(b) rhythm is determined by heat production rhythm associated with activity and eating. However, T(b) in wild-type mice is maintained at a relatively higher level in the active phase than the inactive phase regardless of the heat production level. These results indicate that the thermoregulatory responses are modulated according to the circadian phase, and the Cry genes are involved in this mechanism.     

Characteristics of bovine hemoglobin as a potential source of hemoglobin-vesicles for an artificial oxygen carrier

Hemoglobin-vesicles (HbV) have been developed for use as artificial O(2) carriers in which a purified Hb solution is encapsulated within a phospholipid bilayer membrane. In this study, bovine Hb (BHb) was tested as a source of HbV instead of human Hb (HHb). We compared the preparation process and characteristics of BHbV with those of HHbV. The purification of BHb was effectively performed simply with an ultrafiltration system including a process for removing virus and scrapie reagent. The removal ratio of the phospholipid components of bovine red blood cells was over 99.99%, and the protein purity was over 99.9%. The deoxygenated and carbonylated BHb showed denaturation transition temperatures at 83 and 87 degrees C, respectively, which are higher than those of HHb (80 and 78 degrees C, respectively), and resistant to pasteurization (60 degrees C, 10 h). The purified BHb was concentrated to over 40 g/dl, and encapsulated in a phospholipid bilayer membrane to form BHbV with a diameter of about 280 nm. The O(2) affinity (P(50)) of the BHbV was regulated by coencapsulation of an appropriate amount of Cl(-) (as NaCl), which binds to BHb as an allosteric effector, in the range 16-28 Torr, comparable to human blood (P(50) = 28 Torr). This is quite simple in comparison with HHb which requires phosphate derivatives such as pyridoxal 5'-phosphate as a replacement for 2,3-diphoshoglyceric acid. The viscosity and colloid osmotic pressure of the BHbV when suspended in 5% human serum albumin are 3.5 cP and 20 Torr, respectively, comparable to those of human blood. In conclusion, BHb can be used as a source for the production of HbV, not only because of its abundance in the cattle industry, but also because of the physicochemical advantages of the purification process, thermal stability, and regulation of O(2) affinity in comparison with HHb.     

Fluorescent labeling of pectic oligosaccharides with 2-aminobenzamide and enzyme assay for pectin

Oligogalacturonides [oligomers composed of (1-->4)-linked alpha-D-galactosyluronic acid residues] with degrees of polymerization (DP) from 1 to 10, and a tri-, penta-, and heptasaccharide generated from the backbone of rhamnogalacturonan I (RG-I) were labeled at their reducing ends using aqueous 2-aminobenzamide (2AB) in the presence of sodium cyanoborohydride in over 90% yield. These derivatives were analyzed by high-performance anion-exchange chromatography (HPAEC) and structurally characterized by electrospray-ionization mass spectrometry (ESIMS) and by 1H and 13C NMR spectroscopy. The 2AB-labeled oligogalacturonides and RG-I oligomers are fragmented by endo- and exo-polygalacturonase and by Driselase, respectively. 2AB-labeled oligogalacturonide is an exogenous acceptor for galacturonosyltransferase of transferring galacturonic acid from UDP-GalA. Thus, the 2AB-labeled oligogalacturonides and RG-I oligomers are useful for studying enzymes involved in pectin degradation and biosynthesis and may be of value in determining the biological functions of pectic fragments in plants.     

Characteristics of specific bindings of nitrendipine and PN200-110 to various crude membranes: induction of irreversible bindings by UV irradiation

The characteristics of the specific bindings of [3H]nitrendipine (Nit) and [3H](+)PN200-110 (PN) to crude membranes from rat skeletal, cardiac, and uterine muscle and whole brain were investigated, with special interest in the effect of UV irradiation on these bindings. The specific bindings of [3H]Nit and [3H](+)PN to these crude membranes were saturable and reversible. The specific bindings of [3H]Nit to all these membranes except crude skeletal membranes was maximum in the presence of 0.15 M NaCl plus 1 mM CaCl2 and minimal in the absence of these ions, but the specific bindings of [3H](+)PN to these crude membranes was not affected significantly by these ions. A calcium agonist and antagonists inhibited the specific bindings of [3H]Nit and [3H](+)PN to these crude membranes, the order of their inhibitory effects on specific [3H]Nit bindings being roughly Nit greater than or equal to (+)PN greater than or equal to (-)PN much greater than Bay K 8644 (Bay) greater than verapamil (Ver) greater than diltiazem (Dil). In crude skeletal membranes only, PN caused significant stereospecific inhibition. The order of inhibitions of specific [3H](+)PN bindings to these crude membranes was generally (+)PN greater than Nit greater than or equal to (-)PN greater than Bay much greater than Ver greater than or equal to Dil. In all these crude membranes, UV irradiation completely prevented decrease in the amount of specific binding of [3H](+)PN binding on addition of excess unlabeled (+)PN. These findings suggested that [3H]Nit and [3H](+)PN bind to voltage-sensitive calcium channels in crude membranes from rat skeletal, cardiac, and uterine muscle and whole brain, and that UV irradiation changes the specific bindings of [3H]Nit and [3H](+)PN from reversible to irreversible bindings.