Efficient Selective Adsorption of SARS-CoV-2 via the Recognition of Spike Proteins Using an Affinity Spongy Monolith.

Since the outbreak of COVID-19, SARS-CoV-2, the infection has been spreading to date. The rate of false-negative result on a polymerase chain reaction (PCR) test considered the gold standard is roughly 20%. Therefore, its accuracy poses a question as well as needs improvement in the test. This study reports fabrication of a substrate of an anti-spike protein (AS)-immobilized porous material having selective adsorption toward a spike protein protruding from the surface of SARS-CoV-2. We have employed an organic polymer substrate called spongy monolith (SPM). The SPM has through-pores of about 10 µm and is adequate for flowing liquid containing virus particles. It also involves an epoxy group on the surface, enabling arbitrary proteins such as antibodies to immobilize. When antibodies of the spike protein toward receptor binding domain were immobilized, selective adsorption of the spike protein was observed. At the same time, when mixed analytes of spike proteins, lysozymes and amylases, were flowed into an AS-SPM, selective adsorption toward the spike proteins was observed. Then, SARS-CoV-2 was flowed into the BSA-SPM or AS-SPM, amounts of SARS-CoV-2 adsorption toward the AS-SPM were much larger compared to the ones toward the BSA-SPM. Furthermore, rotavirus was not adsorbed to the AS-SPM at all. These results show that the AS-SPM recognizes selectively the spike proteins of SARS-CoV-2 and may be possible applications for the purification and concentration of SARS-CoV-2.

Rational Supramolecular Strategy via Halogen Bonding for Effective Halogen Recognition in Molecular Imprinting.

Halogen bonding is a highly directional interaction and a potential tool in functional material design through self-assembly. Herein, we describe two fundamental supramolecular strategies to synthesize molecularly imprinted polymers (MIPs) with halogen bonding-based molecular recognition sites. In the first method, the size of the σ-hole was increased by aromatic fluorine substitution of the template molecule, enhancing the halogen bonding in the supramolecule. The second method involved sandwiching hydrogen atoms of a template molecule between iodo substituents, which suppressed competing hydrogen bonding and enabled multiple recognition patterns, improving the selectivity. The interaction mode between the functional monomer and the templates was elucidated by 1H NMR, 13C NMR, X-ray absorption spectroscopy, and computational simulation. Finally, we succeeded in the effective chromatographic separation of diiodobenzene isomers on the uniformly sized MIPs prepared by multi-step swelling and polymerization. The MIPs selectively recognized halogenated thyroid hormones via halogen bonding and could be applied to screening endocrine disruptors.

Rapid and Highly Efficient Purification of Extracellular Vesicles Enabled by a TiO2 Hybridized Spongy-like Polymer.

We developed a novel purification medium of extracellular vesicles (EVs) by constructing a spongy-like monolithic polymer kneaded with TiO2 microparticles (TiO2-hybridized spongy monolith, TiO2-SPM). TiO2-SPM was applied in a solid-phase extraction format and enabled simple, rapid, and highly efficient purification of EVs. This is due to the high permeability caused by the continuous large flow-through pores of the monolithic skeleton (median pore size; 5.21 µm) and the specific interaction of embedded TiO2 with phospholipids of the lipid bilayers. Our method also excels in efficiency and comprehensiveness, collecting small EVs (SEVs) from the same volume of a cell culture medium 130.7 times more than typical ultracentrifugation and 4.3 times more than affinity purification targeting surface phosphatidylserine by magnetic beads. The purification method was completed within 1 h with simple operations and was directly applied to serum SEVs. Finally, we demonstrated flexibility toward the shape and size of our method by depleting EVs from fetal bovine serum (FBS), which is a necessary process to prevent contamination of culture cell-derived EVs with exogenous FBS-derived EVs. Our method will eliminate the tedious and difficult purification processes of EVs, providing a universal purification platform for EV-based drug discovery and pathological diagnosis.

Erk5 in Bone Marrow Mesenchymal Stem Cells Regulates Bone Homeostasis by Preventing Osteogenesis in Adulthood.

Extracellular signal-regulated kinase 5 (Erk5) belongs to the mitogen-activated protein kinase (MAPK) family. Previously, we demonstrated that Erk5 directly phosphorylates Smad-specific E3 ubiquitin protein ligase 2 (Smurf2) at Thr249 (Smurf2Thr249) to activate its E3 ubiquitin ligase activity. Although we have clarified the importance of Erk5 in embryonic mesenchymal stem cells (MSCs) on skeletogenesis, its role in adult bone marrow (BM)-MSCs on bone homeostasis remains unknown. Leptin receptor-positive (LepR+) BM-MSCs represent a major source of bone in adult bone marrow and are critical regulators of postnatal bone homeostasis. Here, we identified Erk5 in BM-MSCs as an important regulator of bone homeostasis in adulthood. Bone marrow tissue was progressively osteosclerotic in mice lacking Erk5 in LepR+ BM-MSCs with age, accompanied by increased bone formation and normal bone resorption in vivo. Erk5 deficiency increased the osteogenic differentiation of BM-MSCs along with a higher expression of Runx2 and Osterix, essential transcription factors for osteogenic differentiation, without affecting their stemness in vitro. Erk5 deficiency decreased Smurf2Thr249 phosphorylation and subsequently increased Smad1/5/8-dependent signaling in BM-MSCs. The genetic introduction of the Smurf2T249E mutant (a phosphomimetic mutant) suppressed the osteosclerotic phenotype in Erk5-deficient mice. These findings suggest that the Erk5-Smurf2Thr249 axis in BM-MSCs plays a critical role in the maintenance of proper bone homeostasis by preventing excessive osteogenesis in adult bone marrow.

Amelioration of Osteoarthritis Development by Daily Oral Supplementation of Royal Jelly.

Royal jelly (RJ), an essential food for the queen honeybee, has a variety of biological activities. Although RJ exerts preventive effects on various lifestyle-related diseases, such as osteoporosis and obesity, no study evaluated the effect of RJ on the development of osteoarthritis (OA), the most common degenerative joint disease. Here, we showed that daily oral administration of raw RJ significantly prevented OA development in vivo following surgically-induced knee joint instability in mice. Furthermore, in vitro experiments using chondrocytes, revealed that raw RJ significantly reduced the expression of inflammatory cytokines and enzymes critical for the degradation of the extracellular matrix (ECM). Similar results were observed after treatment with 10-hydroxy-2-decenoic acid, the most abundant and unique fatty acid in raw RJ. Our results suggest that oral supplementation with RJ would benefit the maintenance of joint health and prophylaxis against OA, possibly by suppressing the activity of inflammatory cytokines and ECM-degrading enzymes.

Conditional inactivation of the L-type amino acid transporter LAT1 in chondrocytes models idiopathic scoliosis in mice.

Scoliosis, usually diagnosed in childhood and early adolescence, is an abnormal lateral curvature of the spine. L-type amino acid transporter 1 (LAT1), encoded by solute carrier transporter 7a5 (Slc7a5), plays a crucial role in amino acid sensing and signaling in specific cell types. We previously demonstrated the pivotal role of LAT1 on bone homeostasis in mice, and the expression of LAT1/SLC7A5 in vertebral cartilage of pediatric scoliosis patients; however, its role in chondrocytes on spinal homeostasis and implications regarding the underlying mechanisms during the onset and progression of scoliosis, remain unknown. Here, we identified LAT1 in mouse chondrocytes as an important regulator of postnatal spinal homeostasis. Conditional inactivation of LAT1 in chondrocytes resulted in a postnatal-onset severe thoracic scoliosis at the early adolescent stage with normal embryonic spinal development. Histological analyses revealed that Slc7a5 deletion in chondrocytes led to general disorganization of chondrocytes in the vertebral growth plate, along with an increase in apoptosis and a decrease in cell proliferation. Furthermore, loss of Slc7a5 in chondrocytes activated the general amino acid control (GAAC) pathway but inactivated the mechanistic target of rapamycin complex 1 (mTORC1) pathway in the vertebrae. The spinal deformity in Slc7a5-deficient mice was corrected by genetic inactivation of the GAAC pathway, but not by genetic activation of the mTORC1 pathway. These findings suggest that the LAT1-GAAC pathway in chondrocytes plays a critical role in the maintenance of proper spinal homeostasis by modulating cell proliferation and survivability.

Separation of Glycoproteins Based on Sugar Chains Using Novel Stationary Phases Modified with Poly(ethylene glycol)-Conjugated Boronic-Acid Derivatives.

Boronic acid (BA) reversibly complexes with the diol structure. BA derivatives separate glycoproteins based on the differences in the sugar chains. Separation typically occurs under basic conditions, which does not guarantee the structural stability of glycoproteins. Here, 5-boronopicolinic acid (BPA) is used to prepare silica-gel based columns with poly(ethylene glycol) (PEG) as a linker to suppress nonselective adsorption and poly(ethylene imine) (PEI) as a scaffold to increase the BPA moiety density. High-performance liquid chromatography (HPLC) using only aqueous buffer solutions without organic solvents demonstrates the selective retention ability of the BPA columns for glycoproteins. BPA interacts with the diols in the sugar chains but not the proteins. In an evaluation for N-glycans, the BPA columns show a higher retention ability toward high mannose type and a lower affinity to N-acetylneuraminic acid (Neu5Ac). Finally, a pair of glycoproteins, fetuin and asialofetuin, are selectively separated due to the presence of Neu5Ac on the nonreducing end.

Classification of Extracellular Vesicles Based on Surface Glycan Structures by Spongy-like Separation Media.

Extracellular vesicles (EVs) are lipid bilayer vesicles that enclose various biomolecules. EVs hold promise as sensitive biomarkers to detect and monitor various diseases. However, they have heterogeneous molecular compositions. The compositions of EVs from identical donor cells obtained using the same purification methods may differ, which is a significant obstacle for elucidating objective biological functions. Herein, the potential of a novel lectin-based affinity chromatography (LAC) method to classify EVs based on their glycan structures is demonstrated. The proposed method utilizes a spongy-like monolithic polymer (spongy monolith, SPM), which consists of poly(ethylene-co-glycidyl methacrylate) with continuous micropores and allows an efficient in situ protein reaction with epoxy groups. Two distinct lectins with different specificities, Sambucus sieboldiana agglutinin and concanavalin A, are effectively immobilized on SPM without impacting the binding activity. Moreover, high recovery rates of liposomal nanoparticles as a model of EVs are achieved due to the large flow-through pores (>10 µm) of SPM compared to a typical agarose gel. Finally, lectin-immobilized SPMs are employed to classify EVs based on the surface glycan structures and demonstrate different subpopulations by proteome profiling. This is the first approach to clarify the variation of protein contents in EVs by the difference of surface glycans via lectin immobilized media.

Identification by Liquid Chromatography-Tandem Mass Spectrometry and Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry of the Contributor to the Thyroid Hormone Receptor Agonist Activity in Effluents from Sewage Treatment Plants.

3,3',5-Triiodothyroacetic acid (TRIAC) was identified as a major contributor to the activity of thyroid hormone receptor (TR) agonists in environmental water. TRIAC contributed 60-148% of the TR-agonist activity in effluents from sewage treatment plants (STPs). Meanwhile, the contributions of 3,5,3'-triiodothyronine (T3), 3,3',5,5'-tetraiodothyronine (T4), and analogues were <1%. TRIAC concentrations in the range of 0.30-4.2 ng/L are likely enough to cause disruption of the thyroid system in living aquatic organisms. The origin of TRIAC in the STP effluents was investigated by analyzing both STP influents and effluents. Relatively high concentrations of T3 and T4 (2.5 and 6.3 ng/L, respectively) were found only in the influents. TRIAC was identified only in the effluents. These findings suggested that T3 and T4 in STP influents were potentially converted into TRIAC during activated sludge treatment or by other means. The evaluation of TRIAC at relevant environmental concentrations by in vivo assays and an appropriate treatment to reduce the TR activity in sewage are needed.

Development of transient trapping micellar electrokinetic chromatography coupled with mass spectrometry for steroids analysis.

An on-line sample preconcentration technique based on transient trapping (tr-trapping) in micellar electrokinetic chromatography (MEKC) was applied for steroid detection with UV (tr-trapping-UV) and electrospray ionization mass spectrometry detection (tr-trapping-ESI-MS). ESI-MS was used to improve the sensitivity in MEKC. The MEKC separation was carried out using volatile ammonium formate as a background solution to facilitate the coupling with ESI-MS. The partial introduction of a sodium dodecyl sulfate (SDS) micellar solution before the introduction of a sample solution to the capillary provided the effective preconcentration of analytes. At the same time, the SDS micelle would not enter the ESI-MS system, so its interference in ESI-MS detection was suppressed under the optimal condition, then five steroids can be separated by the developed method. In tr-trapping-ESI-MS, an acidic condition of pH 3.5 was employed to suppress the electroosmotic flow, which can avoid micellar solution migrating to the MS instrument. The developed method showed that the micellar solution requires a twofold slower time than the sample to migrate along the column, which can prohibit the cause of the problem with the MS instrument and interference signal of SDS in the steroid's detection. The tr-trapping-ESI-MS protocol showed up to 540-fold enhancements of the peak intensity and 50-fold improvement of the limit of detection compared with capillary zone electrophoresis using androsterone as a model sample.

Selective Recovery of Estrogenic Endocrine Disruptors from 48 Environmental Samples Using a Substrate for Activity-Specific Concentration.

hER-MIP is a molecularly imprinted polymer (MIP) that has been shown to selectively collect human estrogen receptor (hER) binding active substances. However, environmental samples contain various chemicals depending on the location and regional differences, and the hER binding activity depends on the sample type. Thus, the general applicability of hER-MIP to actual environmental samples must be elucidated. In this study, 48 environmental samples were collected and screened with hER-MIP, and a yeast assay was performed to evaluate the adsorption characteristics of the samples according to the adsorption and elution fractions. The results showed that hER-MIP collects hER binding active substances almost selectively but does not collect constitutive androstane receptor (CAR) binding active substances selectively. CAR binding activity was detected in the adsorbed fraction because several hER binding active substances also demonstrate CAR binding activity.

LPIAT1/MBOAT7 depletion increases triglyceride synthesis fueled by high phosphatidylinositol turnover.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a common prelude to cirrhosis and hepatocellular carcinoma. The genetic rs641738 C>T variant in the lysophosphatidylinositol acyltransferase 1 (LPIAT1)/membrane bound O-acyltransferase domain-containing 7, which incorporates arachidonic acid into phosphatidylinositol (PI), is associated with the entire spectrum of NAFLD. In this study, we investigated the mechanism underlying this association in mice and cultured human hepatocytes. DESIGN: We generated the hepatocyte-specific Lpiat1 knockout mice to investigate the function of Lpiat1 in vivo. We also depleted LPIAT1 in cultured human hepatic cells using CRISPR-Cas9 systems or siRNA. The effect of LPIAT1-depletion on liver fibrosis was examined in mice fed high fat diet and in liver spheroids. Lipid species were measured using liquid chromatography-electrospray ionisation mass spectrometry. Lipid metabolism was analysed using radiolabeled glycerol or fatty acids. RESULTS: The hepatocyte-specific Lpiat1 knockout mice developed hepatic steatosis spontaneously, and hepatic fibrosis on high fat diet feeding. Depletion of LPIAT1 in cultured hepatic cells and in spheroids caused triglyceride accumulation and collagen deposition. The increase in hepatocyte fat content was due to a higher triglyceride synthesis fueled by a non-canonical pathway. Indeed, reduction in the PI acyl chain remodelling caused a high PI turnover, by stimulating at the same time PI synthesis and breakdown. The degradation of PI was mediated by a phospholipase C, which produces diacylglycerol, a precursor of triglyceride. CONCLUSION: We found a novel pathway fueling triglyceride synthesis in hepatocytes, by a direct metabolic flow of PI into triglycerides. Our findings provide an insight into the pathogenesis and therapeutics of NAFLD.

Recognition of Polymer Terminus by Metal-Organic Frameworks Enabling Chromatographic Separation of Polymers.

Unlike commonly used molecular recognition techniques, recognition of polymer structures requires an additional aspect of extremely high recognition ability, by which marginal structural differences can be identified in a large polymer chain. Herein we show that metal-organic frameworks (MOFs) can recognize polymer terminal structures, thus enabling the first reported chromatographic separation of polymers. End-functionalized polyethylene glycols (PEGs) are selectively inserted into the MOF channel, the insertion kinetics being dependent on the projection size of the PEG terminus. This size-selective insertion mechanism facilitates precise discrimination of end-functionalized PEGs using liquid chromatography (LC). An MOF-packed column thus provides an efficient and easily accessible method for the separation of such end-functionalized polymers using conventional LC systems.

Tunable Liquid Chromatographic Separation of H/D Isotopologues Enabled by Aromatic π Interactions.

We report hydrogen/deuterium (H/D) isotope effects based on weak intermolecular interactions with polar functional groups and aromatic rings in liquid chromatography (LC). Various LC experiments with different aromatic analytes, separation media, and nonpolar mobile phases were conducted under normal phase LC conditions, where the hydrophobic interaction was completely suppressed. The separation media that had polar functional groups, such as silanol groups, allowed for higher separation efficiencies for the pairs of aromatic H/D isotopologues. In comparing the 13C NMR spectra of protiated and deuterated aromatic analytes, the electron density of the deuterated analyte was found to be slightly higher than that of the protiated analytes. In the case of silanol functional groups, aromatic rings of the analyte acted as donors through the OH-π interaction to hydrogen atoms in the silanol groups. Thus, the deuterated analytes were able to be greatly retained by the stronger OH-π interactions. Furthermore, a C70-fullerene bonded monolithic column (C70 column), which effectively provides CH-π interactions, allowed the opposite isotope effect. Briefly, an electrostatic attraction based on the dipole-(induced) dipole interaction dominated in the CH-π interactions, according to a van't Hoff analysis. Hence, the bonding lengths of the C-H or D bonds were sensitively affected, such that we were able to conclude that the CH-π interaction depended on the geometric effect. Applying these opposing H/D isotope effects, we were able to finally demonstrate effective H/D isotopologue separations by utilizing the complementary action of the OH-π and CH-π interactions.

Effectiveness of Lusutrombopag in Patients with Mild to Moderate Thrombocytopenia.

AIMS: Thrombocytopenia is a common complication among patients with chronic liver disease (CLD). To increase platelet counts, lusutrombopag, a small-molecule, second-generation thrombopoietin receptor agonist, was developed in September 2015. Lusutrombopag is mainly used in patients with platelet counts <50,000/µL. However, its usefulness in patients with platelet counts ≥50,000/µL remains unknown. We studied the effectiveness of lusutrombopag administration in patients with platelet counts of ≥50,000/µL. METHODS: We evaluated 36 patients who received lusutrombopag for CLD. Changes in platelet counts were evaluated. A treatment response was defined as an increasing platelet count ≥20,000/µL from baseline after drug administration. The differences related to these changes between platelet counts ≥50,000 and <50,000/µL were evaluated. RESULTS: Of the patients, 25 had platelet counts ≥50,000/µL. The increase in platelet count and the date in which it reached a maximum did not significantly differ between the groups. The effectiveness of lusutrombopag did not significantly differ between the groups. In both groups, no adverse reaction was observed during lusutrombopag administration. CONCLUSION: In this study, we showed the effectiveness of lusutrombopag, which had no complications. This study is the first to report that the effectiveness of lusutrombopag was the same for patients with platelet counts ≥50,000/µL and <50,000/µL.

Effect of three or more treatments with lusutrombopag in patients with cirrhotic thrombocytopenia: A retrospective single-center study.

AIMS: Thrombocytopenia is a common complication among patients with chronic liver disease (CLD). Lusutrombopag, an oral thrombopoietin receptor agonist, is used to reduce the risk of hemorrhage in patients with thrombocytopenia who are undergoing invasive procedures. Platelet transfusion was the standard treatment for thrombocytopenia; however, multiple platelet transfusions lead to the production of antiplatelet antibody. The effect of giving lusutrombopag three times or more has not been previously reported. In this study, we investigated the effect of lusutrombopag readministration in patients with thrombocytopenia. METHODS: This study included 14 patients (total, 24 readministrations) who received lusutrombopag two times or more. Changes in platelet counts were evaluated. Treatment response was defined as an increased platelet count of ≥20 000/µL after lusutrombopag treatment. RESULTS: Lusutrombopag was given twice in nine patients, three times in three patients, five times in one patient, and six times in one patient. An elevated platelet count of <20 000/µL was noted in only one of the 24 readministrations. There were no postoperative hemorrhagic complications, and no patient had an increased platelet count of >200 000/µL. One patient had a portal venous mural thrombus; however, he was asymptomatic, and the thrombus resolved after anticoagulant treatment, without recurrence. The comparison between the first, second, and third or more treatments showed there was no significant difference in platelet increase. CONCLUSION: Repeated treatment of lusutrombopag is effective for CLD patients with thrombocytopenia. Moreover, three or more treatments with lusutrombopag showed equal effect compared with one and two treatments with the medication.

Effect of furosemide on muscle cramps in patients with liver cirrhosis.

BACKGROUND AND AIM: Patients with cirrhosis usually experience muscle cramps of varying severity. Although diuretics have been reported to cause muscle cramps, clinical evidence is limited. Also, it has been pointed out that the use of diuretics is associated with the progression of sarcopenia in patients with cirrhosis. We conducted a questionnaire survey to clarify the effects of diuretics and skeletal muscle loss on muscle cramps. METHODS: Overall, we enrolled 152 adults with cirrhosis in this study. Cramp questionnaires were obtained after informed consent. Study variables (demographics, physical findings, serum metabolic panel, and drugs taken that affect muscle cramps) were extracted from medical records. Body composition, including muscle volume, was analyzed using a bioelectrical impedance analysis method, and muscle strength (handgrip) was evaluated at enrollment. Cross-sectional skeletal muscle area was evaluated on computed tomography imaging at the L3 vertebral level to investigate the relationship between muscle cramps and sarcopenia. RESULTS: The proportion of furosemide administration was higher in patients with cramping compared with those without. On a multivariate logistic regression analysis, furosemide use was a significant factor in the presence of muscle cramps. Furthermore, regarding factors contributing to muscle cramp severity, furosemide use was extracted by multivariate logistic regression analysis. In the presence or severity of muscle cramps, skeletal muscles did not show any significant difference. CONCLUSIONS: Furosemide use for patients with cirrhosis was considered a risk factor for occurrence and severity of muscle cramps. On the other hand, skeletal muscle mass loss was not associated with muscle cramps.

Differentiating π Interactions by Constructing Concave/Convex Surfaces Using a Bucky Bowl Molecule, Corannulene in Liquid Chromatography.

Convex-concave π conjugated surfaces in hemispherical bucky bowl such as corannulene (Crn) have shown increasing utility in constructing self-assembled new functional materials owing to its unique π electrons and strong dipole. Here, we investigate these specific molecular recognitions on Crn by developing new silica-monolithic capillary columns modified with Crn and evaluating their performance in the separation of different aromatic compounds by liquid chromatography (LC). We synthesized two Crn derivatives and conjugated them onto the surface of a silica monolith. The first Crn derivative was edge functionalized, which can undergo free inversion of a convex-concave surface. The second Crn derivative was synthesized by modifying the spoke of Crn, which suppresses the convex-concave inversion. Results of LC suggest that each surface showed different shape recognition based on π interaction. Furthermore, the concave surface of Crn showed strong CH-π interaction with a planar molecule, coronene, demonstrated by the shifts of the 1H NMR signals of both Crn and coronene resulting from the multiple interactions between Crn and π electrons in coronene. These results clearly demonstrated the presence of CH-π interactions at multiple points, and the role of shape recognition.

Combining probiotics and an angiotensin-II type 1 receptor blocker has beneficial effects on hepatic fibrogenesis in a rat model of non-alcoholic steatohepatitis.

AIM: Intestinal endotoxin is important for the progression of non-alcoholic steatohepatitis (NASH). Circulating endotoxin levels are elevated in most animal models of diet-induced non-alcoholic fatty liver disease (NAFLD) and NASH. Furthermore, plasma endotoxin levels are significantly higher in NAFLD patients, which is associated with small intestinal bacterial overgrowth and increased intestinal permeability. By improving the gut microbiota environment and restoring gut-barrier functions, probiotics are effective for NASH treatment in animal models. It is also widely known that hepatic fibrosis and suppression of activated hepatic stellate cells (Ac-HSCs) can be attenuated using an angiotensin-II type 1 receptor blocker (ARB). We thus evaluated the effect of combination probiotics and ARB treatment on liver fibrosis using a rat model of NASH. METHODS: Fisher 344 rats were fed a choline-deficient/L-amino acid-defined (CDAA) diet for 8 weeks to generate the NASH model. Animals were divided into ARB, probiotics, and ARB plus probiotics groups. Therapeutic efficacy was assessed by evaluating liver fibrosis, the lipopolysaccharide Toll-like receptor (TLR)4 regulatory cascade, and intestinal barrier function. RESULTS: Both probiotics and ARB inhibited liver fibrosis, with concomitant HSC activation and suppression of liver-specific transforming growth factor-ß and TLR4 expression. Probiotics reduced intestinal permeability by rescuing zonula occludens-1 disruption induced by the CDAA diet. Angiotensin-II type 1 receptor blocker was found to directly suppress Ac-HSCs. CONCLUSIONS: Probiotics and ARB are effective in suppressing liver fibrosis through different mechanisms. Currently both drugs are in clinical use; therefore, the combination of probiotics and ARB is a promising new therapy for NASH.

Identification of clinical risk factors for histological progression of primary biliary cholangitis.

AIM: To identify laboratory predictors of histological progression (HP) of primary biliary cholangitis (PBC). METHODS: Sequential biopsies were carried out on 35 (11.4%) of 308 patients with PBC treated with ursodeoxycholic acid (UDCA). Patients were divided into high γ-glutamyl transpeptidase (GGT) (n = 18) and low GGT (n = 17) groups, based on the median value of GGT at baseline. Patients were then categorized as showing HP (progressive group, PG) or lacking HP (non-progressive group, NPG) according to the Scheuer and Nakanuma classifications, with the latter grading liver fibrosis (fibrosis score) and bile duct loss (BDL score). RESULTS: According to the Scheuer definition, 12 patients had HP and 23 did not. According to the Nakanuma definition, 8 and 27 patients were in the PG and NPG groups, respectively. The fibrosis and BDL scores progressed in 13 and 8 patients, respectively, whereas 22 and 25 patients did not show HP, respectively. Fisher's exact probability test analysis revealed that the rate of HP using the Nakanuma fibrosis score was significantly higher in the high GGT group compared to the low GGT group (P < 0.05). However, no significant correlation was found between the HP of PBC and the biochemical response to UDCA therapy. Both univariate and multivariate logistic regression analyses indicated that the serum GGT level at baseline is an independent risk factor for an increased Nakanuma fibrosis score. CONCLUSIONS: The level of serum GGT at baseline is significantly associated with liver fibrosis progression in PBC, and therefore could help to predict the HP of PBC.