Serum Wisteria Floribunda Agglutinin-Positive Mac-2 Binding Protein Values Predict the Development of Hepatocellular Carcinoma among Patients with Chronic Hepatitis C after Sustained Virological Response

Measurement of Wisteria floribunda agglutinin-positive human Mac-2 binding protein (WFA⁺-M2BP) in serum was recently shown to be a noninvasive method to assess liver fibrosis. The aim of this study was to evaluate the utility of serum WFA⁺-M2BP values to predict the development of hepatocellular carcinoma (HCC) in patients who achieved a sustained virological response (SVR) by interferon treatment. For this purpose, we retrospectively analyzed 238 patients with SVR who were treated with interferon in our department. Serum WFA⁺-M2BP values were measured at pre-treatment (pre-Tx), post-treatment (24 weeks after completion of interferon; post-Tx), the time of HCC diagnosis, and the last clinical visit. Of 238 patients with SVR, HCC developed in 16 (6.8%) patients. The average follow-up period was 9.1 years. The cumulative incidence of HCC was 3.4% at 5 years and 7.5% at 10 years. The median pre-Tx and post-Tx WFA⁺-M2BP values were 1.69 (range: 0.28 to 12.04 cutoff index (COI)) and 0.80 (range: 0.17 to 5.29 COI), respectively. The WFA⁺-M2BP values decreased significantly after SVR (P < 0.001). The median post-Tx WFA⁺-M2BP value in patients who developed HCC was significantly higher than that in patients who did not (P < 0.01). Multivariate analysis disclosed that age (> 60 years), sex (male), pre-Tx platelet count (< 15.0×10³/μL), and post-Tx WFA⁺-M2BP (> 2.0 COI) were associated with the development of HCC after SVR.

Conclusion

Post-Tx WFA⁺-M2BP (> 2.0 COI) is associated with the risk for development of HCC among patients with SVR. The WFA⁺-M2BP values could be a new predictor for HCC after SVR.     

Monoclonal antibodies to three phospholipase C isozymes from bovine brain

Murine hybridoma cell lines secreting antibodies against the three bovine isozymes of phosphoinositide-specific phospholipase C (PLC) were established: 6, 23, and 12 lines were obtained for PLC-I (150 kDa), PLC-II (145 kDa), and PLC-III (85 kDa), respectively. The antibodies were purified from ascites fluid, and their properties were studied in detail. All the antibodies cross-reacted with their corresponding PLC enzymes, but not with the other two isozymes, suggesting that the three enzymes contain very different antigenic determinants. The six antibodies elicited by bovine PLC-I also cross-reacted with human and rat enzyme, whereas three each from anti-PLC-II antibodies and anti-PLC-III antibodies did not react with the enzymes from different species. Each antibody exerts different effects on the phosphatidylinositol-hydrolyzing activity of PLC. The most inhibitory antibody for either isozyme PLC-I or PLC-II exhibits 80% inhibition, whereas no more than 20% inhibition was observed for the anti-PLC-III antibodies. Purified PLC-I frequently contains catalytically active 140- and 100-kDa forms and an inactive 41-kDa protein in addition to the intact 150-kDa form, probably due to its high sensitivity to an unidentified endogenous protease. The five anti-PLC-I antibodies which bind to the denatured 150-kDa polypeptide also recognized the 140-kDa form, whereas only three cross-reacted with the 100-kDa form, and the remaining two bound to the 41-kDa protein. Competitive binding studies with intact PLC enzymes and Western blot experiments with proteolytic digests revealed that the 6 anti-PLC-I, 23 anti-PLC-II, and 12 anti-PLC-III antibodies bind at least five, six, and seven different epitopes on PLC-I, PLC-II, and PLC-III, respectively. The fact that these monoclonal antibodies bind to different epitopes on the same enzyme allowed one to develop a highly specific and sensitive tandem radioimmunoassay for quantitating PLC-I, PLC-II, and PLC-III. The principle of the assay is that binding of an 125I-labeled antibody to the antigen immobilized by another antibody at a distinctive binding site is proportional to the amount of antigen present. By using this method, PLC-I, PLC-II, and PLC-III could be measured quantitatively in the presence of other proteins, detergents, lipids, polyanions, and metal ions, all of which greatly affect the activity of PLC enzymes.     

Effects of cell-seeding methods of human osteoblast culture on biomechanical properties of porous bioceramic scaffold

Many studies have been performed to accelerate osteoinduction and osteoconduction into porous ceramic scaffolds by seeding them with cells. In this study, we compared available cell-seeding methods on a porous β-tricalcium phosphate (β-TCP) scaffold and evaluated the effects of cell-seeding on the mechanical properties of the porous β-TCP scaffold. Three types of porous bioceramic scaffolds were used: dry scaffold, scaffold wetted with media, and scaffold cultivated with normal human osteoblasts (NHOs). Cell-seeding into the porous β-TCP scaffolds was performed by conventional, centrifuge, high-density, and vacuum methods. After confirming cell proliferation with MTT assay and cell staining, a compressive test was performed after 2 and 4 weeks of cell culture. The vacuum method based on the high-density cell culture inserted effectively NHOs into the β-TCP scaffolds. The compressive elastic modulus of wetted β-TCP scaffolds decreased significantly (p < 0.05) about 20∼30% after 2 and 4 weeks of incubation in comparison with that of the dry scaffold. However, the compressive strength of the scaffolds cultivated with NHOs for 3 weeks was significantly (p < 0.05) higher than that of scaffolds without NHOs. The vacuum with the high-density of cell-seeding seems to be a suitable method for seeding cells into complex porous ceramic scaffolds. Cell proliferation and uniform distribution in the scaffolds can change the initial mechanical properties of porous ceramic scaffolds.     

Iron deposition and increased alveolar septal capillary density in nonfibrotic lung tissue are associated with pulmonary hypertension in idiopathic pulmonary fibrosis

Background

Early diagnosis of pulmonary hypertension (PH) in idiopathic pulmonary fibrosis (IPF) has potential prognostic and therapeutic implications but can be difficult due to the lack of specific clinical manifestations or accurate non-invasive tests. Histopathologic parameters correlating with PH in IPF are also not known. Remodeling of postcapillary pulmonary vessels has been reported in the nonfibrotic areas of explanted lungs from IPF patients. We hypothesized that iron deposition and increased alveolar capillaries, the findings often seen in postcapillary PH, might predict the presence of clinical PH, independent of the severity of fibrosis or ventilatory dysfunction in IPF patients. To test this hypothesis, we examined the association between these histologic parameters and the degree of PH, with consideration of the severity of disease in IPF.

Methods

Iron deposition and alveolar septal capillary density (ASCD) were evaluated on histologic sections with hematoxylin-eosin, iron, elastin and CD34 stainings. Percentage of predicted forced vital capacity (FVC%) was used for grading pulmonary function status. Fibrosis score assessed on high resolution computed tomography (HRCT) was used for evaluating overall degree of fibrosis in whole lungs. Right ventricular systolic pressure (RVSP) by transthoracic echocardiography was used for the estimation of PH. Univariate and multivariate regression analyses were performed.

Results

A cohort of 154 patients was studied who had the clinicopathological diagnosis of IPF with surgical lung biopsies or explants during the period of 1997 to 2006 at Mayo Clinic Rochester. In univariate analysis, RVSP in our IPF cases was associated with both iron deposition and ASCD (p < 0.001). In multivariate analysis with FVC% and HRCT fibrosis score included, iron deposition (p = 0.02), but not ASCD (p = 0.076), maintained statistically significant association with RVSP. FVC% was associated with RVSP on univariate analysis but not on multivariate analysis, while fibrosis score lacked any association with RVSP by either univariate or multivariate analyses.

Conclusions

Iron deposition and ASCD in non fibrotic lung tissue showed an association with RVSP, suggesting that these features are possible morphologic predictors of PH in IPF.     

Synthesis and phosphodiesterase 5 inhibitory activity of novel phenyl ring modified sildenafil analogues.

New sildenafil analogues containing an ether ring fused into the phenyl moiety, 6a--d and 7a--d, were efficiently synthesized from the readily available starting materials, 1a--d and 2, in five steps. Ab initio calculations indicated that introduction of a cyclic ether to the phenyl group might enhance the co-planarity of the molecule. The torsional angles were calculated to be 2--3 degrees for the 5-membered cyclic ether derivatives, 6a, 6c, 7a, and 7c, and 12--16 degrees for the 6-membered ones, 6b, 6d, 7b, and 7d. On the other hand, sildenafil showed the least co-planarity with the torsional angle of 23 degrees compared with the target compounds, 6a--d and 7a--d. In the enzyme assay, however, the in vitro PDE 5 inhibitory activity was found out to be inversely related to the degree of co-planarity. In other words, the least planar sildenafil showed the highest activity, and the most planar 5-membered cyclic ether derivatives were least active by 100--200-fold compared with sildenafil. Our study clearly demonstrated that the open chain 2'-alkoxy group of the phenyl ring, although less effective for inducing the co-planarity, seemed to act as a much better lipophilic requirement than the cyclic alkoxy moiety.     

A parametric study on ethanol production from xylose byPichia stipitis

Characteristics of ethanol production by a xylose-fermenting yeast,Pichia stipitis Y-7124, were studied. The sugar consumption rate and specific growth rate were higher in the glucose-containing medium than in the xylose-containing medium. Specific activities of xylose reductase and xylitol dehydrogenase were higher in the medium with xylose than glucose, suggesting their induction by xylose. Maximum specific growth rate and ethanol yield were achieved at 30 g xylose/L concentration without formation of by-products such as xylitol and acetic acid whereas a maximum ethanol concentration was obtained at 130 g/L xylose. Adding a respiratory inhibitor, rotenone, increased a maximum ethanol concentration by 10% compared with the control experiment. In order to evaluate the pattern of ethanol inhibition on specific growth rate, a kinetic model based on Luong’s equations was applied. The relationship between ethanol concentration and specific growth rate was hyperbolic for glucose and parabolic for xylose. A maximum ethanol concentration at which cells did not grow was 33.6 g/L for glucose and 44.7 g/L for xylose.     

Isoegomaketone induces apoptosis through caspase-dependent and caspase-independent pathways in human DLD1 cells

Isoegomaketone (IK) is an essential oil component of Perilla frutescens (L.), but the mechanism by which IK induces apoptosis has never been studied. The purpose of this study was to elucidate the IK-induced apoptotic pathway in DLD1 human colon cancer cells. We observed that IK treatment over 24 h significantly inhibited cell viability in a dose-dependent manner. We also found that IK triggered cleavage of PARP. Moreover, IK treatment resulted in cleavage of caspase-8, -9, and -3 in a dose- and time-dependent manner. IK treatment also resulted in cleavage of Bid and translocation of Bax, and triggered the release of cytochrome c from the mitochondria to the cytoplasm. Furthermore, it resulted in the translocation of apoptosis inducing factor (AIF), a caspase-independent mitochondrial apoptosis factor, from the mitochondria into the nucleus. Overall, these results suggest that IK induces apoptosis through caspase-dependent and capase-independent pathways in DLD1 cells.     

Local kallikrein�Ckinin system is involved in podocyte apoptosis under diabetic conditions

The kallikrein-kinin system (KKS) serves as the physiologic counterbalance to the renin-angiotensin system. This study was conducted to examine the changes in the expression of KKS components in podocytes under diabetic conditions and to elucidate the functional role of bradykinin (BK) in diabetes-associated podocyte apoptosis. Thirty-two rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with BK infusion for 6 weeks. Immortalized mouse podocytes were cultured in media containing 5.6 mmol/l glucose (NG), NG + 10(-7) mol/l AII (AII), or 30 mmol/l glucose (HG) with or without 10(-8) mol/l BK. Urinary albumin excretion was significantly higher in DM rats, and this increase was ameliorated by BK. Not only kininogen, kallikrein, and BK B1- and B2-receptor expression but also BK levels were significantly decreased in DM glomeruli and in cultured podocytes exposed to HG. The changes in the expressions of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG- and AII-stimulated podocytes were significantly abrogated by BK. The suppressed KSS within podocytes under diabetic condition was associated with podocyte apoptosis, suggesting that BK may be beneficial in preventing podocyte loss in diabetic nephropathy.