Second-Line Treatment after Failure of Immune Checkpoint Inhibitors in Hepatocellular Carcinoma: Tyrosine Kinase Inhibitor, Retrial of Immunotherapy, or Locoregional Therapy?

Background: Immune checkpoint inhibitor (ICI)-based therapy such as atezolizumab plus bevacizumab or durvalumab plus tremelimumab became mainstream first-line systemic treatment in advanced hepatocellular carcinoma (HCC) patients since remarkably superior efficacy of ICI-based therapy compared to tyrosine kinase inhibitors (TKIs) was reported in two recent randomized controlled trials (RCTs) (IMbrave150, HIMALAYA). However, the optimal second-line therapy after treatment failure of first-line ICI-based therapy remains unknown as no RCT has examined this issue. Summary: Therefore, at present, most clinicians are empirically treating patients with TKIs or retrial of ICI or locoregional treatment (LRT) modality such as transarterial therapy, radiofrequency ablation, and radiation therapy in this clinical setting without solid evidence. In this review, we will discuss current optimal strategies for second-line treatment after the failure of first-line ICI-based therapy by reviewing published studies and ongoing prospective trials. Key Messages: Clinicians should consider carefully whether to treat the patients with TKI, other ICI-based therapy, or LRT in this situation by considering several factors including liver function reserve, performance status, adverse events of previous therapy, and presence of lesion that can consider LRT such as oligoprogression and vascular invasion. In the meantime, we await the results of ongoing prospective trials to elucidate the best management options.

Outcome of Atezolizumab Plus Bevacizumab Combination Therapy in High-Risk Patients with Advanced Hepatocellular Carcinoma.

Real-world data regarding treatment with atezolizumab plus bevacizumab in high-risk patients with advanced HCC are lacking. In this multicenter retrospective cohort study, a total of 215 patients with advanced HCC received atezolizumab plus bevacizumab treatment at four tertiary hospitals. High-risk patients were those with grade Vp4 portal vein thrombus, bile duct invasion, or more than 50% liver infiltration. In total, 98 (45.6%) were the high-risk population, 186 (86.5%) were considered to be Child-Pugh class A, and 128 (59.5%) had previously received neoadjuvant or concomitant radiation treatment. Median overall survival (OS) was 11.25 months (95% CI, 9.50-13.10), and the median progression-free survival (PFS) was 8.00 months (95% CI, 6.82-9.18). In the high-risk population, the median OS was 10 months (95% CI, 8.19-11.82) and the median PFS was 6.50 months (95% CI, 3.93-9.08). In the high-risk population, multivariate analysis indicated that radiation therapy and lower ALBI grade were associated with better OS and PFS. A total of 177 (82.3%) patients experienced adverse events of any grade, the most common being proteinuria (23.7%). Atezolizumab plus bevacizumab treatment showed consistent efficacy and tolerability in both the total and high-risk population. Radiation therapy combined with atezolizumab plus bevacizumab treatment might be helpful to improve PFS and OS in high-risk populations.

Oxidative Mechanisms and Cardiovascular Abnormalities of Cirrhosis and Portal Hypertension.

In patients with portal hypertension, there are many complications including cardiovascular abnormalities, hepatorenal syndrome, ascites, variceal bleeding, and hepatic encephalopathy. The underlying mechanisms are not yet completely clarified. It is well known that portal hypertension causes mesenteric congestion which produces reactive oxygen species (ROS). ROS has been associated with intestinal mucosal injury, increased intestinal permeability, enhanced gut bacterial overgrowth, and translocation; all these changes result in increased endotoxin and inflammation. Portal hypertension also results in the development of collateral circulation and reduces liver mass resulting in an overall increase in endotoxin/bacteria bypassing detoxication and immune clearance in the liver. Endotoxemia can in turn aggravate oxidative stress and inflammation, leading to a cycle of gut barrier dysfunction → endotoxemia → organ injury. The phenotype of cardiovascular abnormalities includes hyperdynamic circulation and cirrhotic cardiomyopathy. Oxidative stress is often accompanied by inflammation; thus, blocking oxidative stress can minimize the systemic inflammatory response and alleviate the severity of cardiovascular diseases. The present review aims to elucidate the role of oxidative stress in cirrhosis-associated cardiovascular abnormalities and discusses possible therapeutic effects of antioxidants on cardiovascular complications of cirrhosis including hyperdynamic circulation, cirrhotic cardiomyopathy, and hepatorenal syndrome.

Dysregulated Calcium Handling in Cirrhotic Cardiomyopathy.

Cirrhotic cardiomyopathy is a syndrome of blunted cardiac systolic and diastolic function in patients with cirrhosis. However, the mechanisms remain incompletely known. Since contractility and relaxation depend on cardiomyocyte calcium transients, any factors that impact cardiac contractile and relaxation functions act eventually through calcium transients. In addition, calcium transients play an important role in cardiac arrhythmias. The present review summarizes the calcium handling system and its role in cardiac function in cirrhotic cardiomyopathy and its mechanisms. The calcium handling system includes calcium channels on the sarcolemmal plasma membrane of cardiomyocytes, the intracellular calcium-regulatory apparatus, and pertinent proteins in the cytosol. L-type calcium channels, the main calcium channel in the plasma membrane of cardiomyocytes, are decreased in the cirrhotic heart, and the calcium current is decreased during the action potential both at baseline and under stimulation of beta-adrenergic receptors, which reduces the signal to calcium-induced calcium release. The study of sarcomere length fluctuations and calcium transients demonstrated that calcium leakage exists in cirrhotic cardiomyocytes, which decreases the amount of calcium storage in the sarcoplasmic reticulum (SR). The decreased storage of calcium in the SR underlies the reduced calcium released from the SR, which results in decreased cardiac contractility. Based on studies of heart failure with non-cirrhotic cardiomyopathy, it is believed that the calcium leakage is due to the destabilization of interdomain interactions (dispersion) of ryanodine receptors (RyRs). A similar dispersion of RyRs may also play an important role in reduced contractility. Multiple defects in calcium handling thus contribute to the pathogenesis of cirrhotic cardiomyopathy.

Role of Galectin in Cardiovascular Conditions including Cirrhotic Cardiomyopathy.

Abnormal cardiac function in the setting of cirrhosis and in the absence of a primary cardiac disease is known as cirrhotic cardiomyopathy. The pathogenesis of cirrhotic cardiomyopathy is multifactorial but broadly is comprised of two pathways. The first is due to cirrhosis and synthetic liver failure with abnormal structure and function of many substances, including proteins, lipids, hormones, and carbohydrates such as lectins. The second is due to portal hypertension which invariably accompanies cirrhosis. Portal hypertension leads to a leaky, congested gut with resultant endotoxemia and systemic inflammation. This inflammatory phenotype comprises oxidative stress, cellular apoptosis, and inflammatory cell infiltration. Galectins exert all these pro-inflammatory mechanisms across many different tissues and organs, including the heart. Effective therapies for improving cardiac function in patients with cirrhosis are not available. Conventional strategies for other noncirrhotic heart diseases, including vasodilators, are not feasible because of the significant baseline vasodilation in cirrhotic patients. Therefore, exploring new treatment modalities for cirrhotic cardiomyopathy is of great importance. Galectin-3 inhibitors such as modified citrus pectin, N-acetyllactosamine, TD139 and GB0139 exert anti-apoptotic, anti-oxidative and anti-inflammatory effects and thus have potential therapeutic interest. This review briefly summarizes the physiological and pathophysiological role of galectin and specifically examines its role in cardiac disease processes. We present a more detailed discussion of galectin in cardiovascular complications of cirrhosis, particularly cirrhotic cardiomyopathy. Finally, therapeutic studies of galectin-3 inhibitors in cirrhotic cardiomyopathy are reviewed.

A case of nearly complete response in hepatocellular carcinoma with disseminated lung metastasis by combination therapy of nivolumab and ipilimumab after treatment failure of atezolizumab plus bevacizumab.

Recently, the efficacy of immuno-oncologic agents for advanced hepatocellular carcinoma (HCC) has been proven in several trials. In particular, atezolizumab with bevacizumab (AteBeva), as a first-line therapy for advanced HCC, has shown tremendous advances in the IMBrave150 study. However, second or third-line therapy after treatment failure with AteBeva has not been firmly established. Moreover, clinicians have continued their attempts at multidisciplinary treatment that includes other systemic therapy and radiotherapy (RT). Here, we report a case that showed a near complete response (CR) of lung metastasis to nivolumab with ipilimumab therapy after achieving a near CR of intrahepatic tumor using sorafenib and RT in a patient with advanced HCC who had experienced treatment failure of AteBeva.

Inverse Propensity Score-Weighted Analysis of Entecavir and Tenofovir Disoproxil Fumarate in Patients with Chronic Hepatitis B: A Large-Scale Multicenter Study.

Tenofovir disoproxil fumarate (TDF) is reportedly superior or at least comparable to entecavir (ETV) in preventing hepatocellular carcinoma (HCC) among chronic hepatitis B (CHB) patients; however, it remains controversial. This study aimed to conduct comprehensive comparisons between the two antivirals. CHB patients initially treated with ETV or TDF between 2012 and 2015 at 20 referral centers in Korea were included. The primary outcome was the cumulative incidence of HCC. The secondary outcomes included death or liver transplantation, liver-related outcome, extrahepatic malignancy, development of cirrhosis, decompensation events, complete virologic response (CVR), seroconversion rate, and safety. Baseline characteristics were balanced using the inverse probability of treatment weighting (IPTW). Overall, 4210 patients were enrolled: 1019 received ETV and 3191 received TDF. During the median follow-ups of 5.6 and 5.5 years, 86 and 232 cases of HCC were confirmed in the ETV and TDF groups, respectively. There was no difference in HCC incidence between the groups both before (p = 0.36) and after IPTW was applied (p = 0.81). Although the incidence of extrahepatic malignancy was significantly higher in the ETV group than in the TDF group before weighting (p = 0.02), no difference was confirmed after IPTW (p = 0.29). The cumulative incidence rates of death or liver transplantation, liver-related outcome, new cirrhosis development, and decompensation events were also comparable in the crude population (p = 0.24-0.91) and in the IPTW-adjusted population (p = 0.39-0.80). Both groups exhibited similar rates of CVR (ETV vs. TDF: 95.1% vs. 95.8%, p = 0.38), and negative conversion of hepatitis B e antigen (41.6% vs. 37.2%, p = 0.09) or surface antigen (2.8% vs. 1.9%, p = 0.10). Compared to the ETV group, more patients in the TDF group changed initial antivirals due to side effects, including decreased kidney function (n = 17), hypophosphatemia (n = 20), and osteoporosis (n = 18). In this large-scale multicenter study, ETV and TDF demonstrated comparable effectiveness across a broad range of outcomes in patients with treatment-naïve CHB during similar follow-up periods.

Activation of crosslinked lipases in mesoporous silica via lid opening for recyclable biodiesel production.

Lipases catalyze a wide range of industrially important reactions, including the transesterification of triglycerides with alcohols for biodiesel production, and the stabilization of lipases are critical to achieve their recycled uses. Here, nanoscale enzyme reactor (NER) of lipase from Rhizopus oryzae (LP) was prepared via a simple two-step process, comprising of enzyme adsorption into magnetically-separable mesoporous silica and follow-up crosslinking of adsorbed enzymes. In aqueous phase, the specific hydrolysis activity of NER-LP was 4.7 times lower than that of free LP. On the other hand, however, the specific transesterification activity of NER-LP (130.4 µmol/min/mg LP) in organic phase for biodiesel production was 50 times higher than that of free LP (2.6 µmol/min/mg LP). These results reveal that the enzyme crosslinking for the preparation of NER does not interfere with the interfacial activation of LP molecules, opening the lid of LP active site under an optimal hydrophobic environment provided by the combination of organic solvent and mesoporous silica. Magnetic separation and optimized washing protocol facilitated the recycled uses of NER-LP. Highly stable and active NER-LP in magnetically-separable mesoporous silica has demonstrated its great potentials as an environmentally-friendly nanobiocatalyst for various lipase applications, including plasticizers, biosurfactants, functional fatty acids, as well as recyclable biodiesel production.

Complete response in hepatocellular carcinoma with lymph node metastasis by combination therapy of atezolizumab and bevacizumab: a case report.

Sorafenib is the oldest first line systemic treatment in patients with advanced hepatocellular carcinoma (HCC) and has been used exclusively for nearly 10 years. The superiority of administering a combination of atezolizumab plus bevacizumab (AteBeva) compared to sorafenib as first line systemic treatment for unresectable HCC was recently proven during the IMbrave150 Phase III randomized trial. While clinicians can expect improved responses and treatment outcomes due to the good results of the IMbrave 150 trial, they must also consider that atezolizumab can cause various immune-related adverse events (IrAEs). Based on the above suggestions, we herein present a case of HCC with lymph node metastasis who achieved complete remission following treatment with AteBeva and developed an IrAE (adrenal insufficiency). Further study of real-life data regarding combination therapy with AteBeva is needed to manage patients with advanced HCC.

Real-world systemic sequential therapy with sorafenib and regorafenib for advanced hepatocellular carcinoma: a multicenter retrospective study in Korea.

Background/Aims Regorafenib has been approved as a second-line systemic therapy for hepatocellular carcinoma (HCC) patients after the phase III RESORCE trial. This study analyzed real-world data to assess the clinical effectiveness and safety of regorafenib compared to the RESORCE trial. Methods This multicenter cohort study included HCC patients treated with regorafenib after sorafenib (n = 133). We evaluated the time to progression (TTP), progression-free survival (PFS), overall survival (OS), and safety in patients receiving regorafenib along with the predictors of prognosis. Results The median age was 60 years and 81.2% patients were men. Hepatitis B virus infection (68.4%) was the commonest etiology. Most patients were classified as Child-Pugh A (98.5%) and had extrahepatic metastasis (84%) and vascular invasion (45.1%). This study demonstrated similar characteristics apart from more frequent hepatitis B etiology and more vascular or extrahepatic involvement compared with the RESORCE trial. An objective response rate of 12.5% was obtained for response assessment (n = 112); the disease control rate was 34.8%. Thirty-eight patients died during follow-up. With regorafenib, the median OS, PFS, and TTP were 10.0, 2.7, and 2.6 months, respectively. In the exploratory analysis after sorafenib administration, the median OS was 25.8 months. The rate of response and survival were comparable to those in the RESORCE trial. Child-Pugh score > 5, alpha-fetoprotein > 400 ng/ml, and TTP for sorafenib ≥ median were independently associated with OS. Conclusions This real-word regorafenib study showed comparable effectiveness and safety to the RESORCE trial. Regorafenib improves the prognosis of patients with prolonged TTP during previous sorafenib therapy.

Imaging-based analysis of liposome internalization to macrophage cells: Effects of liposome size and surface modification with PEG moiety.

Liposome is one of the frequently used carriers for active targeting systems in vivo. Such parameters as its size, surface charge, and surface modifiers are known to influence the liposome uptake by macrophage cells. In this study, we investigated the effects of liposome size and polyethylene glycol (PEG) surface modifier on the liposomal internalization to murine macrophage (RAW-264.7), by using an imaging analysis technique. Three different sized liposomes (100, 200, and 400 nm in nominal diameter) labeled with rhodamine fluorescence were used. Liposome internalization appeared to reach a pseudo-steady plateau in about 5h incubation, and most of the internalized liposomes were seen to accumulate in the cytosol including cellular extensions. The maximum fluorescent density from the internalized liposomes was similar between 100 nm and 200 nm liposomes. However, that of the larger 400 nm liposome was approximately 1.7 times higher than the others, confirming the previous report that the larger the liposomes are the higher the degree of internalization is. When the outside of the 200 nm liposomes was modified with biocompatible anchor molecule (BAM) consisting of PEG (ca. 2kD molecular weight) moiety, the endocytosis was indeed reduced by about 2.1-fold, despite the increase of the hydrodynamic size due to BAM conjugation. This fluorescence-based cellular imaging analysis can be used to quantitatively monitor and optimize cellular internalization systems.

Reduction of oxidative stress attenuates lipoapoptosis exacerbated by hypoxia in human hepatocytes.

Chronic intermittent hypoxia, a characteristic of obstructive sleep apnea (OSA), is associated with the progression of simple hepatic steatosis to necroinflammatory hepatitis. We determined whether inhibition of a hypoxia-induced signaling pathway could attenuate hypoxia-exacerbated lipoapoptosis in human hepatocytes. The human hepatocellular carcinoma cell line (HepG2) was used in this study. Palmitic acid (PA)-treated groups were used for two environmental conditions: Hypoxia (1% O2) and normoxia (20% O2). Following the treatment, the cell viability was determined by the 3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay, and the mechanism of lipoapoptosis was evaluated by Western blotting. Hypoxia exacerbated the suppression of hepatocyte growth induced by palmitic acid via activation of mitochondrial apoptotic pathways as a result of endoplasmic reticulum (ER) and oxidative stresses. Ammonium pyrrolidine dithiocarbamate, a scavenger of reactive oxygen species, attenuated the hypoxia-exacerbated lipoapoptosis in hepatocytes, whereas glycerol, which reduces ER stress, did not. This may have been because inhibition of oxidative stress decreases the expression of pro-apoptotic proteins, such as caspase 9 and cytochrome c. These results suggested that modulation of apoptotic signaling pathways activated by oxidative stress can aid in identifying novel therapeutic strategies for the treatment of nonalcoholic steatohepatitis (NASH) with OSA. Further in vivo studies are necessary to understand the pathophysiologic mechanism of NASH with OSA and to prove the therapeutic effect of the modulation of the signaling pathways.

Emodin attenuates radioresistance induced by hypoxia in HepG2 cells via the enhancement of PARP1 cleavage and inhibition of JMJD2B.

Radioresistance in the tumor and radiotoxicity in the non­tumorous liver significantly restrict efficient radiotherapy of hepatocellular carcinoma (HCC). It is therefore important to study the radioresistance mechanism and development of radiosensitization to optimize the effect of irradiation on cancer cells. Emodin (1, 3, 8­trihydroxy­6­methylanthraquinone) is a plant­derived polyphenol, possessing anticancer properties. It is known to act as a radiosensitizer in human HCC cell lines. The aim of this study was to evaluate the role of emodin in radioresistance of human HCC cell lines as well as the underlying radiosensitization mechanism. The human HCC cell line (HepG2) was used in this study. Four different treatment groups, i.e., no treatment (control), irradiation (10 Gy, one fraction), emodin (10 µM), and a combination of irradiation and emodin (10 Gy+10 µM) were used for two environmental conditions: hypoxia (1% O2) and normoxia (20% O2). The cells were exposed to the respective treatments for 24 and 72 h. Following the treatment, the cell viability was determined by the 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) assay, and the radiosensitization mechanism was evaluated by western blotting. The proliferation of HepG2 cells was significantly suppressed in the treatment groups under hypoxic and normoxic conditions in the following order: combination of irradiation and emodin>irradiation only >emodin only. The combination of irradiation and emodin induced apoptotic signaling activities such as cleavage of poly (ADP­ribose) polymerase (PARP)­1 as well as the downregulation of epigenetic signaling such as JMJD1A and JMJD2B. Emodin attenuated radioresistance in the HepG2 cells via upregulation of the apoptotic signals and down-regulation of the proliferative signals. These results suggested that emodin is a potential candidate for the radiosensitization of HCC cells and can aid in identifying novel therapeutic strategies for HCC radiotherapy.

Extramedullary plasmacytoma of the pancreas diagnosed using endoscopic ultrasonography-guided fine needle aspiration.

Extramedullary plasmacytoma involves organs outside the bone marrow; however, involvement of the pancreas is rare. We recently experienced a case of extramedullary plasmacytoma of the pancreas that was diagnosed by endoscopic ultrasonography-guided fine needle aspiration (EUS-FNA). EUS-FNA, which has a high diagnostic accuracy and an excellent safety profile, is the modality of choice for establishing tissue diagnosis. We report a case of extramedullary plasmacytoma of the pancreas diagnosed using EUS-FNA.

A highly sensitive immunoassay using antibody-conjugated spherical mesoporous silica with immobilized enzymes.

A highly sensitive immunoassay was developed by using antibody-conjugated spherical mesoporous silica with immobilized enzymes. The higher ratio of enzyme/antibody than conventional ELISA improved both the sensitivity and dynamic range. Especially, the use of spherical mesoporous silica could achieve a limit of detection (LOD) with a sensitivity that is 20 times more than that of ELISA using amorphous silica.

Thermal stability and starch degradation profile of α-amylase from Streptomyces avermitilis.

Amylases from Streptomyces are useful in the production of maltooligosaccharides, but they have weak thermal stability at temperatures higher than 40 °C. In this study, α-amylase (SAV5981 gene of Streptomyces avermitilis) was expressed from Streptomyces lividans 1326 and purified by ammonium sulfate fractionation followed by anionic chromatography (Q-HP sepharose). The properties of the purified SAV5981 amylase were determined by the starch-iodine method. The effect of metal ions on amylase activity was investigated. The optimal temperature shifted from 25 to 50 °C with the addition of the Ca(2+) ion. The thermal stability of SAV5981 was also dramatically enhanced by the addition of 10 mM CaCl2. Improvement of the thermal stability of SAV5981 was examined by CD spectra in the presence and the absence of the Ca(2+) ion. Thin-layer chromatography (TLC) analysis and HPLC analysis of starch degradation revealed that SAV5981 mainly produced maltose and maltotriose, not glucose. The maltoorigosaccharide-producing amylase examined in this study has the potential in the industrial application of oligosaccharide production.

Effects of operating parameters on the efficiency of liposomal encapsulation of enzymes.

Encapsulation of active proteins in the hydrophilic core of vesicular liposomes is important for developing a therapeutic protein carrier system. The efficiency of liposomal encapsulation of proteins is generally low. A better understanding of the fundamental mechanisms of encapsulation is needed to increase this efficiency. In this study we investigated the effects of operating parameters such as phospholipid concentration, buffer pH and ionic strength, protein size and surface charge, and liposome size on the enzyme encapsulation yield. Four model enzymes of different molecular weights and isoelectric points (trypsin, horseradish peroxidase, enterokinase and hyaluronidase) were encapsulated into three different sized liposomes (125 nm, 194 nm, and 314 nm in mean diameter). Relatively inert and neutral DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) was used as the main phospholipid in the liposomes. Size exclusion chromatography was used to separate the enzyme-encapsulated liposomes from the free enzyme, and the encapsulation yield was determined from the peak area. The encapsulation yield was generally low ranging from ca. 5% to 20%, and did not depend much on the molecular weight of the enzyme encapsulated. Larger liposomes had higher encapsulation yields. The electrostatic interaction between the phospholipid and enzyme was the most significant parameter in determining the encapsulation yield. Thus adjusting buffer pH and ionic strength and adding charged phospholipids to the liposome preparation to impart electric charge to the lipid bilayer could significantly improve the yield. This approach can be used to optimize the liposomal encapsulation of clinically significant proteins.

Simple scoring system predicting genotypic resistance during rescue therapy for Lamivudine-resistant chronic hepatitis B.

GOAL: In this study, we aimed to devise a simple scoring system predicting the risk of genotypic resistance (GR) to current rescue therapies for patients with lamivudine (LAM)-resistant chronic hepatitis B. BACKGROUND: LAM and adefovir (ADV) combination therapy should be recommended for an initial rescue therapy against LAM-resistant hepatitis B virus (HBV). However, there still are many LAM-resistant patients being treated with entecavir (ETV) or ADV monotherapy. STUDY: This retrospective cohort study included consecutive patients treated for LAM-resistant chronic hepatitis B with ETV or ADV monotherapy, or LAM/ADV combination therapy. The cumulative probabilities of GR and virological responses and breakthrough according to clinical variables were analyzed by survival analyses and derived an index for future GR. RESULTS: A total of 224 patients were included (median treatment duration=117.9 wk). Using risk factors indentified on multivariable analyses, a simple index for future GR (Antiviral Resistance Prediction Index, ARPI) was developed with 3 clinical variables: the rescue therapy regimens (+0, ADV; +1, ETV; +2, LAM/ADV), HBV DNA reduction at 12 weeks (+0, <3 log10 copies/mL; +1, >3 log10 copies/mL), and the initial HBV DNA level (+0, >10 copies/mL; +1, <10 copies/mL). No patient with ARPI ≥2 exhibited GR, whereas 47% of the patients with an ARPI <2 developed GR by week 144 (P=0.005). CONCLUSIONS: The results of this study suggest that the ARPI is a simple and early index that can be used to determine the risk for subsequent GR during rescue therapy for LAM-resistant chronic hepatitis B.

Cellular imaging assay for early evaluation of an apoptosis inducer.

The objective of this study was to propose a feasibility of a cellular imaging assay as an alternative to the conventional cytotoxicity assay, such as MTS assay, for apoptosis monitoring. As an apoptosis monitoring parameter, affinity interaction between phosphatidylserine (PS) and annexin V was chosen. First, the specific binding affinity between annexin V and PS in phospholipid bilayers consisting of various molar (0-15%) composition of PS was measured using a surface plasmon resonance biosensor. As PS composition increased, the binding level of annexin V increased proportionally. Second, various concentrations (0.1-10 µM) of staurosporine were used as to induce apoptosis and introduced to MCF-7 breast carcinoma cells. The cellular fluorescence images from annexin V-FITC conjugate were obtained by confocal microscopy, and their fluorescence intensities were quantified by image scanning. Dose-apoptosis (or cell death) relationships were very similar to those from MTS and FACS assays. In summary, our cellular imaging method could serve as a quicker and simpler alternative to MTS (end point assay) and FACS (flow cytometry) to screen potential apoptosis inducers.

Enzyme precipitate coatings of lipase on polymer nanofibers.

Lipase (LP) was immobilized on electrospun and ethanol-dispersed polystyrene-poly(styrene-co-maleic anhydride) (PS-PSMA) nanofibers (EtOH-NF) in the form of enzyme precipitate coatings (EPCs). LP precipitate coatings (EPCs-LP) were prepared in a three-step process, consisting of covalent attachment, LP precipitation, and crosslinking of precipitated LPs onto the covalently attached LPs via glutaraldehyde treatment. The LP precipitation was performed by adding various concentrations of ammonium sulfate (20-50%, w/v). EPCs-LP improved the LP activity and stability when compared to covalently attached LPs (CA-LP) and the enzyme coatings of LPs (EC-LP) without the LP precipitation. For example, the use of 40% (w/v) ammonium sulfate resulted in EPC40-LP with the highest activity, which was 4.0 and 3.6 times higher than those of CA-LP and EC-LP, respectively. After 165-day incubation under rigorous shaking at 200 rpm, the residual activities of EPC50-LP were 0.5 µM/min mg of EtOH-NF, representing 113 and 75 times higher than those of CA-LP and EC-LP, respectively. When LP was partially purified via a simple ammonium sulfate precipitation and dialysis, both activities and stabilities of EC-LP and EPC-LP could be marginally improved. It is anticipated that the improved LP activity and stability in the form of EPCs would allow for their potential applications in various bioconversion processes such as biodiesel production and ibuprofen resolution.