Safety, pharmacokinetics and pharmacodynamics of obeticholic acid in subjects with fibrosis or cirrhosis from NASH.

BACKGROUND & AIMS: Fibrosis stage is a strong predictor of nonalcoholic steatohepatitis (NASH) outcomes. Two blinded studies evaluated the pharmacokinetics, pharmacodynamics and safety of obeticholic acid (OCA) in subjects with staged NASH fibrosis or cirrhosis. METHODS: Study 747-117 randomized 51 subjects with NASH (fibrosis stages F1-F4) to daily placebo, OCA 10 or OCA 25 mg (1:2:2) for 85 days. Study 747-118 randomized 24 subjects with NASH cirrhosis (F4; Child-Pugh [CP]-A) and normal liver control subjects matched for similar body weight to daily OCA 10 or OCA 25 mg (1:1) for 28 days. Individual and combined study data were analysed. RESULTS: No severe or serious adverse events (AEs) or AEs leading to discontinuation or death occurred. Pruritus was the most frequent AE. Plasma OCA exposure (dose-normalized area under the curve) increased with fibrosis stage but was a relatively poor predictor of hepatic OCA exposure (primary site of action), which remained constant across fibrosis stages F1-F3 and increased 1.8-fold compared with F1 in subjects with cirrhosis due to NASH. Both cohorts showed robust changes in farnesoid X receptor activation markers with OCA treatment and marked decreases in alanine transaminase, aspartate transaminase and gamma-glutamyltransferase. CONCLUSIONS: Despite higher drug exposures in subjects with NASH cirrhosis, short-term daily treatment with OCA 10 or 25 mg was generally safe and well tolerated in subjects with NASH fibrosis or NASH CP-A cirrhosis. Both cohorts experienced improvements in nonhistologic pharmacodynamic markers consistent with previously conducted OCA phase 2 and phase 3 studies in NASH fibrosis.

[Carrier screening model for Duchenne muscular dystrophy for women of reproductive age based on a pre-pregnancy birth defect control platform].

OBJECTIVE: To establish a screening model for females of reproductive age carrying Duchenne muscular dystrophy (DMD) variants based on a current community health examination platform. METHODS: A total of 61 870 participants were recruited between October 2017 and October 2019. Serum creatine kinase (CK) was measured with a Roche Cobasc 701/702 using an enzymatic rate method. Genetic testing was offered to those with a CK level of ≥ 200 U/L. For carriers of DMD variants, genetic counseling and follow up were provided. RESULTS: For the 61 870 females participating in the program, 1078 were found with raised serum CK (≥ 200 U/L), of which 618 (57.33%) accepted CK re-measurement after at least a two-week interval. One hundred and twenty cases were found with sustained serum CK elevation, of which 6 were confirmed to be definite DMD carriers regardless of family history. Genetic testing was provided to 33 females with a family history for DMD, and 13 were determined as definite carriers. An affected fetus was detected by prenatal diagnosis. After genetic counseling, the parents had opted induced abortion. CONCLUSION: Large-scale DMD carrier screening through a three-step approach based on the current community health examination platform is both feasible and cost effective.

Population-Wide Duchenne Muscular Dystrophy Carrier Detection by CK and Molecular Testing.

Carrier screening of Duchenne muscular dystrophy (DMD) has not been widely evaluated. To identify definite DMD female carriers prior to or in early pregnancy, we studied a large population of reproductive age females and provided informed reproductive options to DMD carriers. 37268 females were recruited from the Hangzhou Family Planning Publicity and Technology Guidance Station/Hangzhou Health Service Center for Children and Women, Hangzhou, China, between October 10, 2017, and December 16, 2018. CK activity was measured with follow-up serum DMD genetic testing in subjects with hyperCKemia, defined as CK > 200 U/L. The calculated upper reference limit (97.5th percentile) of serum creatine kinase (CK) for females aged 20-50 years in this study was near the reference limit recommended by the manufacturer (200 U/L), above which was defined as hyperCKemia. 427 females (1.2%) harbored initially elevated CK, among which 281 females (response rate of 65.8%) accepted CK retesting. DMD genetic testing was conducted on 62 subjects with sustained serum CK > 200 U/L and 16 females with a family history of DMD. Finally, 6 subjects were confirmed to be DMD definite carriers. The estimated DMD female carrier rate in this study was 1 : 4088 (adjusting for response rate), an underestimated rate, since only 50% to 70% of DMD female carriers manifest elevated serum CK, and carriers in this study may have been missed due to lack of follow-up or inability to detect all DMD pathogenic variants by current genetic testing.

Factors Associated With Histologic Response in Adult Patients With Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is a leading cause of liver transplantation, and many trials are underway to evaluate potential therapies. The farnesoid X receptor ligand obeticholic acid in the NASH treatment trial evaluated the effects of obeticholic acid vs placebo on histologic response (defined as decrease in nonalcoholic fatty liver disease activity score [NAS] by ≥2, with no worsening of fibrosis); 45% of patients had a histologic response to obeticholic acid (25 mg), and 21% had a response to placebo (P < .01). We performed a secondary analysis of data from this trial to identify clinical parameters associated with a histologic response. METHODS: We used a logistic regression model with a stepwise selection procedure to identify baseline and early on-treatment factors associated with a histologic response at 72 weeks. Baseline demographics, liver histology, medical history, concomitant medications, cardiometabolic parameters, and serum biochemistry, as well as the changes over the course of the trial (at weeks 12 and 24), were evaluated as potential predictors of a histologic response. The model was cross-validated by a jackknife method, and performance was evaluated with the area under the receiver operating characteristic curve. RESULTS: The logistic regression model found that obeticholic acid treatment, baseline NAS > 5, baseline triglyceride level ≤ 154 mg/dL, baseline international normalized ratio ≤ 1, baseline aspartate aminotransferase level ≤ 49 U/L, and a decrease in alanine aminotransferase level at week 24 by 17 U/L or more, to be significantly associated with histologic response (area under the receiver operating characteristic curve, 0.83; 95% confidence interval, 0.77-0.89; P < .0001). CONCLUSIONS: In a secondary analysis of data from a clinical trial of obeticholic acid in patients with NASH, we identified routine clinical and laboratory parameters during the first 24 weeks of treatment (such as baseline NAS, triglyceride levels, and a decrease in alanine aminotransferase level) to significantly associate with histologic markers of response.

[Clinical study on the effect of anti-gingivitis IgY toothpaste in control of gingivitis and dental plaque].

PURPOSE: To observe the effect of anti-gingivitis IgY toothpaste in control of gingivitis and plaque. METHODS: The study was a double-blind, randomized, parallel-controlled clinical trail with a total of 100 subjects who were divided into two groups, experimental group and control group. The subjects in experimental group used anti-gingivitis IgY toothpaste to brush twice daily for 3 minutes, and the subjects in control group used none anti-gingivitis IgY toothpaste. The examiner recorded GI, PI and BOP index of all subjects at the baseline, 6-weeks and 12-weeks. SPSS21.0 software package was used for statistical analysis. RESULTS: Twelve weeks later, there were significant differences in GI and BOP between the two groups. Yet no significant difference was found in PI. CONCLUSIONS: Anti-gingivitis IgY toothpaste is effective in control of gingivitis.

Effects of perfusion and dynamic loading on human neocartilage formation in alginate hydrogels.

Dynamic loading and perfusion culture environments alone are known to enhance cartilage extracellular matrix (ECM) production in dedifferentiated articular chondrocytes. In this study, we explored whether a combination of these factors would enhance these processes over a free-swelling (FS) condition using adult human articular chondrocytes embedded in 2% alginate. The alginate constructs were placed into a bioreactor for perfusion (P) only (100 µL/per minute) or perfusion and dynamic compressive loading (PL) culture (20% for 1 h, at 0.5 Hz), each day. Control FS alginate gels were maintained in six-well static culture. Gene expression analysis was conducted on days 7 and 14, while cell viability, immunostaining, and mechanical property testing were performed on day 14 only. Total glycosaminoglycan (GAG) content and GAG synthesis were assessed after 14 days. Col2a1 mRNA expression levels were significantly higher (at least threefold; p<0.05) in both bioreactor conditions compared with FS by days 7 and 14. For all gene studies, no significant differences were seen between P and PL treatments. Aggrecan mRNA levels were not significantly altered in any condition although both GAG/DNA and (35)S GAG incorporation studies indicated higher GAG retention and synthesis in the FS treatment. Collagen type II protein deposition was low in all samples, link protein distribution was more diffuse in FS condition, and aggrecan deposition was located in the outer regions of the alginate constructs in both bioreactor conditions, yet more uniformly in the FS condition. Catabolic gene expression (matrix metalloproteinase 3 [MMP3] and inducible nitric oxide synthase [iNOS]) was higher in bioreactor conditions compared with FS, although iNOS expression levels decreased to approximately fourfold less than the FS condition by day 14. Our data indicate that conditions created in the bioreactor enhanced both anabolic and catabolic responses, similar to other loading studies. Perfusion was sufficient alone to promote this dual response. PL increased the deposition of aggrecan surrounding cells compared with the other conditions; however, overall low GAG retention in the bioreactor system was likely due to both perfusion and catabolic conditions created. Optimal conditions, which permit appropriate anabolic and catabolic processes for accumulation of ECM and tissue remodeling for neocartilage development, specifically for humans, are needed.

Vimentin contributes to changes in chondrocyte stiffness in osteoarthritis.

Actin and tubulin cytoskeletal components are studied extensively in chondrocytes, but less is known about vimentin intermediate filaments. In other cell types, vimentin is a determinant of cell stiffness and disruption of vimentin networks weakens the mechanical integrity of cells. Changes in vimentin organization correlate with osteoarthritis progression, but the functional consequences of these changes remain undetermined in chondrocytes. The objective of this study was to compare the contribution of vimentin to the mechanical stiffness of primary human chondrocytes isolated from normal versus osteoarthritic cartilage. Chondrocytes were embedded in alginate and vimentin networks disrupted with acrylamide. Constructs were imaged while subjected to 20% nominal strain on a confocal microscope stage, and the aspect ratios of approximately 1,900 cells were measured. Cytosolic stiffness was estimated with a finite element model, and live-cell imaging of GFP-vimentin was used to further analyze the nature of vimentin disruption. Vimentin in normal chondrocytes formed an inner cage-like network that was substantially stiffer than the rest of the cytosol and contributed significantly to overall cellular stiffness. Disruption of vimentin reduced stiffness approximately 2.8-fold in normal chondrocytes. In contrast, osteoarthritic chondrocytes were less stiff and less affected by vimentin disruption. This 3D experimental system revealed contributions of vimentin to chondrocyte stiffness previously not apparent, and correlated changes in vimentin-based chondrocyte stiffness with osteoarthritis.

Rho kinase-dependent activation of SOX9 in chondrocytes.

OBJECTIVE: The transcription factor SOX9 directly regulates the expression of the major proteoglycans and collagens comprising the cartilage extracellular matrix. The DNA binding activity and cellular localization of SOX9 is controlled through posttranslational modifications, including phosphorylation. The activity of Rho kinase (ROCK) has profound effects on the actin cytoskeleton, and these effects are instrumental in determining the phenotype and differentiation of chondrocytes. However, the mechanisms linking ROCK to altered chondrocyte gene expression remain unknown. The purpose of the present study was to test for a direct interaction between ROCK and SOX9. METHODS: Human SW1353 chondrosarcoma cells were transfected with constructs coding for RhoA, ROCK, Lim kinase, and SOX9. The interaction between ROCK and SOX9 was tested on purified proteins, and was verified within a cellular context using induced overexpression and activation of the Rho pathway. The effects of SOX9 transcriptional activation were quantified with a luciferase reporter plasmid containing SOX9 binding sites from the COL2A1 enhancer element. RESULTS: SOX9 was found to contain a consensus phosphorylation site for ROCK. In vitro, ROCK directly phosphorylated SOX9 at Ser(181), and the overexpression of ROCK or the activation of the RhoA pathway in SW1353 chondrosarcoma cells increased SOX9(Ser181) phosphorylation. ROCK caused a dose-dependent increase in the transcription of a SOX9-luciferase reporter construct, and increased phosphorylation and nuclear accumulation of SOX9 protein in response to transforming growth factor beta treatment and mechanical compression. CONCLUSION: These results demonstrate a new interaction that directly links ROCK to increased cartilage matrix production via activation of SOX9 in response to mechanical and growth factor stimulation.

Characterization of the chondrocyte actin cytoskeleton in living three-dimensional culture: response to anabolic and catabolic stimuli.

The actin cytoskeleton is a dynamic network required for intracellular transport, signal transduction, movement, attachment to the extracellular matrix, cellular stiffness and cell shape. Cell shape and the actin cytoskeletal configuration are linked to chondrocyte phenotype with regard to gene expression and matrix synthesis. Historically, the chondrocyte actin cytoskeleton has been studied after formaldehyde fixation--precluding real-time measurements of actin dynamics, or in monolayer cultured cells. Here we characterize the actin cytoskeleton of living low-passage human chondrocytes grown in three-dimensional culture using a stably expressed actin-GFP construct. GFP-actin expression does not substantially alter the production of endogenous actin at the protein level. GFP-actin incorporates into all actin structures stained by fluorescent phalloidin, and does not affect the actin cytoskeleton as seen by fluorescence microscopy. GFP-actin expression does not significantly change the chondrocyte cytosolic stiffness. GFP-actin does not alter the gene expression response to cytokines and growth factors such as IL-1beta and TGF-beta. Finally, GFP-actin does not alter production of extracellular matrix as measured by radiosulfate incorporation. Having established that GFP-actin does not measurably affect the chondrocyte phenotype, we tested the hypothesis that IL-1beta and TGF-beta differentially alter the actin cytoskeleton using time-lapse microscopy. TGF-beta increases actin extensions, and lamellar ruffling indicative of Rac/CDC42 activation, while IL-1beta causes cellular contraction indicative of RhoA activation. The ability to visualize GFP-actin in living chondrocytes in 3D culture without disrupting the organization or function of the cytoskeleton is an advance in chondrocyte cell biology and provides a powerful tool for future studies in actin-dependent chondrocyte differentiation and mechanotransduction pathways.

[Analysis of hBMSCs spatial distribution and gene expression in biocoral scaffold with different seeding methods].

OBJECTIVE: To compare the effect of two different methods of cell seeding on spatial distribution and gene expression of hBMSCs in biocoral scaffold in vitro cultures. METHODS: The composite of hBMSCs and biocoral scaffold was prepared by traditional seeding (group A) and fibrin glue seeding (group B). The seeding efficiency was measured after 30 minutes of incubation in group B and after 3 hours in group A. At 2, 7, 14 and 21 days after culture, the samples were harvested and the serial longitudinal sections were cut for each embedded composite. The sections were stained with DAPI and were measured using fluorescence microscope with apostome under serial optical sections. The cell number in every 10 x objective field was automatically measured by AxioVision image analysis software and levels (from seeding surface to bottom L1-L5) or columns (from centre to margin) for comparing cell distribution were set up. The specific osteogenic genes [osteonectin (ON), core binding factor alpha1 (Cbfalpha1), osteocalcin (OC)] expression was measured by RT-PCR. RESULTS: The seeding efficiency was significantly higher in group B (88.32% +/- 4.2%) than in group A (66.51% +/- 12.33%, P < 0.01). At 2 days after culture, the cell number from L1 to L4 decreased gradully in two groups (P < 0.05); in the cell number of different columns, there was no significant difference in group A (P > 0.05) whereas significant difference in group B (P < 0.05); there was no significant difference in gene expression between two groups (P > 0.05). At 7 days after culture, the cell number was less than that at 2 days in group A and there was significant difference among levels (P < 0.05). The cell number and osteogenic gene expression increased sharply and there appeared uniform cell distribution in group B (P > 0.05). The gene expression of ON and Cbfalpha1 in group B was higher than that in group A (P < 0.05). At 14 days after culture, the cell number in levels or columns in group A decreased sharply and was less than that at 7 days (P < 0.05); whereas the cell number was similar to that at 7 days in group B (P > 0.05). The OC gene expression reached the highest level in group B at 14 days. The gene expression was higher in group B than in group A (P < 0.05). At 21 days after culture, there was significant difference in the cell number among levels and in the gene expression between group A and group B (P < 0.05); there was no significant difference in the cell number among columns in two groups (P > 0.05). In addition, the cell number of most levels and columns in group B was more than that in group A at 7, 14 and 21 days after culture (P < 0.05). CONCLUSION: More uniform cell distribution with rapid proliferation and osteogenic differentiation is available in different levels or columns of scaffold by fibrin glue seeding than by traditional seeding.

Rho kinase-dependent CCL20 induced by dynamic compression of human chondrocytes.

OBJECTIVE: Mechanical stimulation of cartilage affects tissue homeostasis and chondrocyte function. The chondrocyte phenotype is dependent on cell shape, which is largely determined by the actin cytoskeleton. Reorganization of the actin cytoskeleton results from Rho GTPase activation. The purpose of this study was to examine the roles of both actin and Rho in mechanotransduction in chondrocytes. METHODS: We embedded human articular chondrocytes in 2 x 6-mm agarose discs at 5 x 10(6) cells/ml and subjected the discs to unconfined dynamic compression at 0.5 Hz. By comparing samples with and without dynamic compression, we identified Rho activation according to the GTP-bound active RhoA measured in cell lysates. We identified rearrangements in filamentous actin structures using fluorescence-labeled phalloidin and confocal microscopy of fixed samples. We identified altered gene expression using TaqMan quantitative reverse transcription-polymerase chain reaction analysis. We tested for a requirement for Rho signaling by performing the dynamic compression in the presence of Rho kinase inhibitors. RESULTS: RhoA activation occurred within 5-10 minutes of dynamic compression. Rho kinase-dependent actin reorganization occurred within 20 minutes after application of dynamic compression and was apparent as "punctate" F-actin structures that were visible under confocal microscopy. We identified early-phase mechanoresponsive genes (CCL20 and inducible nitric oxide synthase) that were highly up-regulated within 1 hour of dynamic compression in a Rho kinase-dependent and actin-dependent manner. CONCLUSION: Together, these results are the first demonstration that the Rho-Rho kinase pathway and actin cytoskeletal reorganization are required for changes in the expression of genes involved in human chondrocyte mechanotransduction.