Composite Remineralization of Bone-Collagen Matrices by Low-Temperature Ceramics and Serum Albumin: A New Approach to the Creation of Highly Effective Osteoplastic Materials.

This study examined the effectiveness of coating demineralized bone matrix (DBM) with amorphous calcium phosphate (DBM + CaP), as well as a composite of DBM, calcium phosphate, and serum albumin (DBM + CaP + BSA). The intact structure of DBM promotes the transformation of amorphous calcium phosphate (CaP) into dicalcium phosphate dihydrate (DCPD) with a characteristic plate shape and particle size of 5-35 µm. The inclusion of BSA in the coating resulted in a better and more uniform distribution of CaP on the surface of DBM trabeculae. MG63 cells showed that both the obtained forms of CaP and its complex with BSA did not exhibit cytotoxicity up to a concentration of 10 mg/mL in vitro. Ectopic (subcutaneous) implantation in rats revealed pronounced biocompatibility, as well as strong osteoconductive, osteoinductive, and osteogenic effects for both DBM + CaP and DBM + CaP + BSA, but more pronounced effects for DBM + CaP + BSA. In addition, for the DBM + CaP + BSA samples, there was a pronounced full physiological intrafibrillar biomineralization and proangiogenic effect with the formation of bone-morrow-like niches, accompanied by pronounced processes of intramedullary hematopoiesis, indicating a powerful osteogenic effect of this composite.

Low-Temperature Calcium Phosphate Ceramics Can Modulate Monocytes and Macrophages Inflammatory Response In Vitro.

Creating bioactive materials for bone tissue regeneration and augmentation remains a pertinent challenge. One of the most promising and rapidly advancing approaches involves the use of low-temperature ceramics that closely mimic the natural composition of the extracellular matrix of native bone tissue, such as Hydroxyapatite (HAp) and its phase precursors (Dicalcium Phosphate Dihydrate-DCPD, Octacalcium Phosphate-OCP, etc.). However, despite significant scientific interest, the current knowledge and understanding remain limited regarding the impact of these ceramics not only on reparative histogenesis processes but also on the immunostimulation and initiation of local aseptic inflammation leading to material rejection. Using the stable cell models of monocyte-like (THP-1ATRA) and macrophage-like (THP-1PMA) cells under the conditions of LPS-induced model inflammation in vitro, the influence of DCPD, OCP, and HAp on cell viability, ROS and intracellular NO production, phagocytosis, and the secretion of pro-inflammatory cytokines was assessed. The results demonstrate that all investigated ceramic particles exhibit biological activity toward human macrophage and monocyte cells in vitro, potentially providing conditions necessary for bone tissue restoration/regeneration in the peri-implant environment in vivo. Among the studied ceramics, DCPD appears to be the most preferable for implantation in patients with latent inflammation or unpredictable immune status, as this ceramic had the most favorable overall impact on the investigated cellular models.

Gender versus sex in predicting outcomes of traumatic brain injury: a cohort study utilizing large administrative databases.

Understanding the factors associated with elevated risks and adverse consequences of traumatic brain injury (TBI) is an integral part of developing preventive measures for TBI. Brain injury outcomes differ based on one's sex (biological characteristics) and gender (social characteristics reflecting norms and relationships), however, whether it is sex or gender that drives differences in early (30-day) mortality and discharge location post-TBI is not well understood. In the absence of a gender variable in existing data, we developed a method for "measuring gender" in 276,812 residents of Ontario, Canada who entered the emergency department and acute care hospitals with a TBI diagnostic code between April 1st, 2002, and March 31st, 2020. We applied logistic regression to analyse differences in diagnostic codes between the sexes and to derive a gender score that reflected social dimensions. We used the derived gender score along with a sex variable to demonstrate how it can be used to separate the relationship between sex, gender and TBI outcomes after severe TBI. Sex had a significant effect on early mortality after severe TBI with a rate ratio (95% confidence interval (CI)) of 1.54 (1.24-1.91). Gender had a more significant effect than sex on discharge location. A person expressing more "woman-like" characteristics had lower odds of being discharged to rehabilitation versus home with odds ratio (95% CI) of 0.54 (0.32-0.88). The method we propose offers an opportunity to measure a gender effect independently of sex on TBI outcomes.

A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling.

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.

Transcriptomic and proteomic assessment of tocilizumab response in a randomized controlled trial of patients hospitalized with COVID-19.

High interleukin (IL)-6 levels are associated with greater COVID-19 severity. IL-6 receptor blockade by tocilizumab (anti-IL6R; Actemra) is used globally for the treatment of severe COVID-19, yet a molecular understanding of the therapeutic benefit remains unclear. We characterized the immune profile and identified cellular and molecular pathways modified by tocilizumab in peripheral blood samples from patients enrolled in the COVACTA study, a phase 3, randomized, double-blind, placebo-controlled trial of the efficacy and safety of tocilizumab in hospitalized patients with severe COVID-19. We identified markers of inflammation, lymphopenia, myeloid dysregulation, and organ injury that predict disease severity and clinical outcomes. Proteomic analysis confirmed a pharmacodynamic effect for tocilizumab and identified novel pharmacodynamic biomarkers. Transcriptomic analysis revealed that tocilizumab treatment leads to faster resolution of lymphopenia and myeloid dysregulation associated with severe COVID-19, indicating greater anti-inflammatory activity relative to placebo and potentially leading to faster recovery in patients hospitalized with COVID-19.

Genomic diversity landscapes in outcrossing and selfing Caenorhabditis nematodes.

Caenorhabditis nematodes form an excellent model for studying how the mode of reproduction affects genetic diversity, as some species reproduce via outcrossing whereas others can self-fertilize. Currently, chromosome-level patterns of diversity and recombination are only available for self-reproducing Caenorhabditis, making the generality of genomic patterns across the genus unclear given the profound potential influence of reproductive mode. Here we present a whole-genome diversity landscape, coupled with a new genetic map, for the outcrossing nematode C. remanei. We demonstrate that the genomic distribution of recombination in C. remanei, like the model nematode C. elegans, shows high recombination rates on chromosome arms and low rates toward the central regions. Patterns of genetic variation across the genome are also similar between these species, but differ dramatically in scale, being tenfold greater for C. remanei. Historical reconstructions of variation in effective population size over the past million generations echo this difference in polymorphism. Evolutionary simulations demonstrate how selection, recombination, mutation, and selfing shape variation along the genome, and that multiple drivers can produce patterns similar to those observed in natural populations. The results illustrate how genome organization and selection play a crucial role in shaping the genomic pattern of diversity whereas demographic processes scale the level of diversity across the genome as a whole.

Expanding the stdpopsim species catalog, and lessons learned for realistic genome simulations.

Simulation is a key tool in population genetics for both methods development and empirical research, but producing simulations that recapitulate the main features of genomic datasets remains a major obstacle. Today, more realistic simulations are possible thanks to large increases in the quantity and quality of available genetic data, and the sophistication of inference and simulation software. However, implementing these simulations still requires substantial time and specialized knowledge. These challenges are especially pronounced for simulating genomes for species that are not well-studied, since it is not always clear what information is required to produce simulations with a level of realism sufficient to confidently answer a given question. The community-developed framework stdpopsim seeks to lower this barrier by facilitating the simulation of complex population genetic models using up-to-date information. The initial version of stdpopsim focused on establishing this framework using six well-characterized model species (Adrion et al., 2020). Here, we report on major improvements made in the new release of stdpopsim (version 0.2), which includes a significant expansion of the species catalog and substantial additions to simulation capabilities. Features added to improve the realism of the simulated genomes include non-crossover recombination and provision of species-specific genomic annotations. Through community-driven efforts, we expanded the number of species in the catalog more than threefold and broadened coverage across the tree of life. During the process of expanding the catalog, we have identified common sticking points and developed the best practices for setting up genome-scale simulations. We describe the input data required for generating a realistic simulation, suggest good practices for obtaining the relevant information from the literature, and discuss common pitfalls and major considerations. These improvements to stdpopsim aim to further promote the use of realistic whole-genome population genetic simulations, especially in non-model organisms, making them available, transparent, and accessible to everyone.

Gender versus sex in predicting outcomes of traumatic brain injury: A cohort study utilizing large administrative databases.

Understanding the factors associated with elevated risks and adverse consequences of traumatic brain injury (TBI) is an integral part of developing preventive measures for TBI. Brain injury outcomes differ based on one's sex (biological characteristics) and gender (social characteristics reflecting norms and relationships), however, whether it is sex or gender that drives differences in early (30-day) mortality and discharge location post-TBI event are unknown. In the absence of gender variable in existing data, we developed a method for "measuring gender" in 276,812 residents of Ontario, Canada who entered the emergency department and acute care hospitals with a TBI diagnostic code between April 1st, 2002 and March 31st, 2020. We analysed differences in diagnostic codes between the sexes to derive gender score that reflected social dimensions. Sex had a significant effect on early mortality after severe TBI with a rate ratio (95% confidence interval (CI)) of 1.54 (1.24-1.91). Gender had a more significant effect than sex on discharge location. A person expressing more female-like characteristics have lower odds of being discharged to rehabilitation versus home with odds ratio (95% CI) of 0.54 (0.32-0.88). The method we propose offers an opportunity to measure gender effect independently of sex on TBI outcomes.

Biomimetic Remineralized Three-Dimensional Collagen Bone Matrices with an Enhanced Osteostimulating Effect.

Bone grafts with a high potential for osseointegration, capable of providing a complete and effective regeneration of bone tissue, remain an urgent and unresolved issue. The presented work proposes an approach to develop composite biomimetic bone material for reconstructive surgery by deposition (remineralization) on the surface of high-purity, demineralized bone collagen matrix calcium phosphate layers. Histological and elemental analysis have shown reproduction of the bone tissue matrix architectonics, and a high-purity degree of the obtained collagen scaffolds; the cell culture and confocal microscopy have demonstrated a high biocompatibility of the materials obtained. Adsorption spectroscopy, scanning electron microscopy, microcomputed tomography (microCT) and infrared spectroscopy, and X-ray diffraction have proven the efficiency of the deposition of calcium phosphates on the surface of bone collagen scaffolds. Cell culture and confocal microscopy methods have shown high biocompatibility of both demineralized and remineralized bone matrices. In the model of heterotopic implantation in rats, at the term of seven weeks, an intensive intratrabecular infiltration of calcium phosphate precipitates, and a pronounced synthetic activity of osteoblast remodeling and rebuilding implanted materials, were revealed in remineralized bone collagen matrices in contrast to demineralized ones. Thus, remineralization of highly purified demineralized bone matrices significantly enhanced their osteostimulating ability. The data obtained are of interest for the creation of new highly effective osteoplastic materials for bone tissue regeneration and augmentation.

Prognostic value of severe acute respiratory syndrome coronavirus-2 viral load and antibodies in patients hospitalized with COVID-19.

Observational studies have identified the potential prognostic value for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral load and anti-SARS-CoV-2 antibodies in coronavirus disease 2019 (COVID-19). However, viral load in nasopharyngeal (NP) swabs produced inconsistent results in prognostic analyses, and the prognostic value of viral load or antibodies has not been confirmed in large clinical trials. COVACTA and REMDACTA were double-blind, randomized, controlled trials with a combined enrollment of 1078 patients hospitalized with COVID-19 treated with tocilizumab or placebo in COVACTA or tocilizumab plus remdesivir or placebo plus remdesivir in REMDACTA. We assessed the potential prognostic value of NP and serum SARS-CoV-2 viral load and serum anti-SARS-CoV-2 antibodies at baseline as biomarkers for clinical outcomes in patients enrolled in these trials. In adjusted Cox proportional hazard models, serum viral load was a more reliable predictor of clinical outcomes than NP viral load; high serum viral load was associated with higher risk for death and mechanical ventilation/death and lower likelihood of hospital discharge (high vs. negative viral load hazard ratios [95% confidence interval {CI}] were 2.87 [1.57-5.25], 3.86 [2.23-6.68], and 0.23 [0.14-0.36], respectively, in COVACTA and 8.11 [2.95-22.26], 10.29 [4.5-23.55], and 0.21 [0.15-0.29], respectively, in REMDACTA) and high serum viral load correlated with levels of inflammatory cytokines and lung damage biomarkers. High anti-SARS-CoV-2 spike protein antibody (ACOV2S) levels were associated with higher likelihood of hospital discharge (high vs. below the limit of quantification hazard ratios [95% CI] were 2.55 [1.59-4.08] for COVACTA and 1.54 [1.13-2.09] for REMDACTA). These results support the role of baseline SARS-CoV-2 serum viral load and ACOV2S antibody titers in predicting clinical outcomes for patients hospitalized with COVID-19.

Genetic diversity estimates for the Caenorhabditis Intervention Testing Program screening panel.

The Caenorhabditis Intervention Testing Program (CITP) was founded on the principle that compounds with positive effects across a genetically diverse test-set should have an increased probability of engaging conserved biochemical pathways with mammalian translational potential. To fulfill its mandate, the CITP uses a genetic diversity panel of Caenorhabditis strains for assaying longevity effects of candidate compounds. The panel comprises 22 strains from three different species, collected globally, to achieve inter-population genetic diversity. The three represented species, C. elegans, C. briggsae, and C. tropicalis, are all sequential hermaphrodites, which simplifies experimental procedures while maximizing intra-population homogeneity. Here, we present estimates of the genetic diversity encapsulated by the constituent strains in the panel based on their most recently published and publicly available whole-genome sequences, as well as two newly generated genomic data sets. We observed average genome-wide nucleotide diversity (π) within the C. elegans (1.2e-3), C. briggsae (7.5e-3), and C. tropicalis strains (2.6e-3) greater than estimates for human populations, and comparable to that found in mouse populations. Our analysis supports the assumption that the CITP screening panel encompasses broad genetic diversity, suggesting that lifespan-extending chemicals with efficacy across the panel should be enriched for interventions that function on conserved processes that are shared across genetic backgrounds. While the diversity panel was established by the CITP for studying longevity interventions, the panel may prove useful for the broader research community when seeking broadly efficacious interventions for any phenotype with potential genetic background effects.

Octacalcium Phosphate for Bone Tissue Engineering: Synthesis, Modification, and In Vitro Biocompatibility Assessment.

Octacalcium phosphate (OCP, Ca8H2(PO4)6·5H2O) is known to be a possible precursor of biological hydroxyapatite formation of organic bone tissue. OCP has higher biocompatibility and osseointegration rate compared to other calcium phosphates. In this work, the synthesis of low-temperature calcium phosphate compounds and substituted forms of those at physiological temperatures is shown. Strontium is used to improve bioactive properties of the material. Strontium was inserted into the OCP structure by ionic substitution in solutions. The processes of phase formation of low-temperature OCP with theoretical substitution of strontium for calcium up to 50 at.% in conditions close to physiological, i.e., temperature 35-37 °C and normal pressure, were described. The effect of strontium substitution range on changes in the crystal lattice of materials, the microstructural features, surface morphology and biological properties in vitro has been established. The results of the study indicate the effectiveness of using strontium in OCP for improving biocompatibility of OCP based composite materials intended for bone repair.

Fenebrutinib in H1 antihistamine-refractory chronic spontaneous urticaria: a randomized phase 2 trial.

Bruton's tyrosine kinase (BTK) is crucial for FcεRI-mediated mast cell activation and essential for autoantibody production by B cells in chronic spontaneous urticaria (CSU). Fenebrutinib, an orally administered, potent, highly selective, reversible BTK inhibitor, may be effective in CSU. This double-blind, placebo-controlled, phase 2 trial (EudraCT ID 2016-004624-35 ) randomized 93 adults with antihistamine-refractory CSU to 50 mg daily, 150 mg daily and 200 mg twice daily of fenebrutinib or placebo for 8 weeks. The primary end point was change from baseline in urticaria activity score over 7 d (UAS7) at week 8. Secondary end points were the change from baseline in UAS7 at week 4 and the proportion of patients well-controlled (UAS7 ≤ 6) at week 8. Fenebrutinib efficacy in patients with type IIb autoimmunity and effects on IgG-anti-FcεRI were exploratory end points. Safety was also evaluated. The primary end point was met, with dose-dependent improvements in UAS7 at week 8 occurring at 200 mg twice daily and 150 mg daily, but not at 50 mg daily of fenebrutinib versus placebo. Asymptomatic, reversible grade 2 and 3 liver transaminase elevations occurred in the fenebrutinib 150 mg daily and 200 mg twice daily groups (2 patients each). Fenebrutinib diminished disease activity in patients with antihistamine-refractory CSU, including more patients with refractory type IIb autoimmunity. These results support the potential use of BTK inhibition in antihistamine-refractory CSU.

In Vitro Study of Octacalcium Phosphate Behavior in Different Model Solutions.

Octacalcium phosphate (OCP), a new-generation bone substitute material, is a considered precursor of the biological bone apatite. The two-layered structure of OCP contains the apatitic and hydrated layers and is intensively involved in ion-exchange surface reactions, which results in OCP hydrolysis to hydroxyapatite and adsorption of ions or molecular groups presented in the environment. During various in vitro procedures, such as biomaterial solubility, additive release studies, or the functionalization technique, several model solutions are applied. The composition of the environmental solution affects the degree and rate of OCP hydrolysis, its surface reactivity, and further in vitro and in vivo properties. The performed study was aimed to track the structural changes of OCP-based materials while treating in the most popular model solutions of pH values 7.2-7.4: simulated body fluid (SBF), Dulbecco's phosphate-buffered saline (DPBS), supersaturated calcification solution (SCS), normal saline (NS), and Dulbecco's modified Eagle's medium (DMEM). Various degrees of OCP hydrolysis and/or precipitate formation were achieved through soaking initial OCP granules in the model solutions. Detailed data of X-ray diffraction, Fourier-transform infrared spectroscopy, atomic emission spectrometry with inductively coupled plasma, and scanning electron microscopy are presented. Cultivation of osteosarcoma cells was implemented on OCP pre-treated in DMEM for 1-28 days. It was shown that NS mostly degraded the OCP structure. DPBS slightly changed the OCP structure during the first treatment term, and during further terms, the crystals got thinner and OCP hydrolysis took place. Treatment in SBF and SCS caused the precipitate formation along with OCP hydrolysis, with a larger contribution of SCS solution to precipitation. Pre-treating in DMEM enhanced the cytocompatibility of materials. As a result, on performing the in vitro procedures, careful selection of the contact solution should be made to avoid the changes in materials structure and properties and get adequate results.

Diversity and host associations of Myrsidea chewing lice (Phthiraptera: Menoponidae) in the tropical rainforest of Malaysian Borneo.

The tropical rainforests of Sundaland are a global biodiversity hotspot increasingly threatened by human activities. While parasitic insects are an important component of the ecosystem, their diversity and parasite-host relations are poorly understood in the tropics. We investigated parasites of passerine birds, the chewing lice of the speciose genus MyrsideaWaterston, 1915 (Phthiraptera: Menoponidae) in a natural rainforest community of Malaysian Borneo. Based on morphology, we registered 10 species of lice from 14 bird species of six different host families. This indicated a high degree of host specificity and that the complexity of the system could be underestimated with the potential for cryptic lineages/species to be present. We tested the species boundaries by combining morphological, genetic and host speciation diversity. The phylogenetic relationships of lice were investigated by analyzing the partial mitochondrial cytochrome oxidase I (COI) and the nuclear elongation factor alpha (EF-1α) genes sequences of the species. This revealed a monophyletic group of Myrsidea lineages from seven hosts of the avian family Pycnonotidae, one host of Timaliidae and one host of Pellorneidae. However, species delimitation methods supported the species boundaries hypothesized by morphological studies and confirmed that four species of Myrsidea are not single host specific. Cophylogenetic analysis by both distance-based test ParaFit and event-based method Jane confirmed overall congruence between the phylogenies of Myrsidea and their hosts. In total we recorded three cospeciation events for 14 host-parasite associations. However only one host-parasite link (M. carmenae and their hosts Terpsiphone affinis and Hypothymis azurea) was significant after the multiple testing correction in ParaFit. Four new species are described: Myrsidea carmenae sp.n. ex Hypothymis azurea and Terpsiphone affinis, Myrsidea franciscae sp.n. ex Rhipidura javanica, Myrsidea ramoni sp.n. ex Copsychus malabaricus stricklandii, and Myrsidea victoriae sp.n. ex. Turdinus sepiarius.

Degradation of the Repetitive Genomic Landscape in a Close Relative of Caenorhabditis elegans.

The abundance, diversity, and genomic distribution of repetitive elements is highly variable among species. These patterns are thought to be driven in part by reproductive mode and the interaction of selection and recombination, and recombination rates typically vary by chromosomal position. In the nematode Caenorhabditis elegans, repetitive elements are enriched at chromosome arms and depleted on centers, and this mirrors the chromosomal distributions of other genomic features such as recombination rate. How conserved is this genomic landscape of repeats, and what evolutionary forces maintain it? To address this, we compared the genomic organization of repetitive elements across five Caenorhabditis species with chromosome-level assemblies. As previously reported, repeat content is enriched on chromosome arms in most Caenorhabditis species, and no obvious patterns of repeat content associated with reproductive mode were observed. However, the fig-associated C. inopinata has experienced repetitive element expansion and reveals no association of global repeat density with chromosome position. Patterns of repeat superfamily specific distributions reveal this global pattern is driven largely by a few repeat superfamilies that in C. inopinata have expanded in number and have weak associations with chromosome position. Additionally, 15% of predicted protein-coding genes in C. inopinata align to transposon-related proteins. When these are excluded, C. inopinata has no enrichment of genes in chromosome centers, in contrast to its close relatives who all have such clusters. Forward evolutionary simulations reveal that chromosomal heterogeneity in recombination rate alone can generate structured repetitive genomic landscapes when insertions are weakly deleterious, whereas chromosomal heterogeneity in the fitness effects of transposon insertion can promote such landscapes across a variety of evolutionary scenarios. Thus, patterns of gene density along chromosomes likely contribute to global repetitive landscapes in this group, although other historical or genomic factors are needed to explain the idiosyncrasy of genomic organization of various transposable element taxa within C. inopinata. Taken together, these results highlight the power of comparative genomics and evolutionary simulations in testing hypotheses regarding the causes of genome organization.

Chromosome-Level Assembly of the Caenorhabditis remanei Genome Reveals Conserved Patterns of Nematode Genome Organization.

The nematode Caenorhabditis elegans is one of the key model systems in biology, including possessing the first fully assembled animal genome. Whereas C. elegans is a self-reproducing hermaphrodite with fairly limited within-population variation, its relative C. remanei is an outcrossing species with much more extensive genetic variation, making it an ideal parallel model system for evolutionary genetic investigations. Here, we greatly improve on previous assemblies by generating a chromosome-level assembly of the entire C. remanei genome (124.8 Mb of total size) using long-read sequencing and chromatin conformation capture data. Like other fully assembled genomes in the genus, we find that the C. remanei genome displays a high degree of synteny with C. elegans despite multiple within-chromosome rearrangements. Both genomes have high gene density in central regions of chromosomes relative to chromosome ends and the opposite pattern for the accumulation of repetitive elements. C. elegans and C. remanei also show similar patterns of interchromosome interactions, with the central regions of chromosomes appearing to interact with one another more than the distal ends. The new C. remanei genome presented here greatly augments the use of the Caenorhabditis as a platform for comparative genomics and serves as a basis for molecular population genetics within this highly diverse species.

Cancer-related gene expression is associated with disease severity and modifiable lifestyle factors in non-alcoholic fatty liver disease.

OBJECTIVE: The aim of this study was to determine whether hepatic gene expression related to hepatocellular carcinoma (HCC) is associated with disease severity and modifiable lifestyle factors in non-alcoholic fatty liver disease (NAFLD). METHODS: In a cross-sectional study, the associations between hepatic gene expression and liver histology, insulin resistance, anthropometrics, diet, and physical activity were assessed in patients with non-alcoholic steatohepatitis (NASH; n = 19) or simple steatosis (SS; n = 20). In a group of patients with NASH, we then conducted a 1-y, single-arm, pilot study using ω-3 polyunsaturated fatty acid (PUFA) supplementation to determine whether changes in hepatic PUFA content would have a modulating effect on hepatic gene expression and would affect liver histology. RESULTS: In the cross-sectional study, histological features of disease severity correlated with AKR1B10, ANXA2, PEG10, SPP1, STMN2, MT1A, and MT1B in NASH and with EEF1A2, PEG10, and SPP1 in SS. In addition, PEG10, SPP1, ANXA2, and STMN2 expression correlated positively with insulin resistance in NASH. SPP1 and UBD correlated strongly with body mass index in SS. Associations between ENPP2, AKR1B10, SPP1, UBD, and waist circumference depended on sex and diagnosis. Several genes correlated with protein, fat, or carbohydrate intake. PEG10 correlated positively with physical activity in NASH and inversely with plasma vitamin C in both groups. Despite increased erythrocyte and hepatic ω-3 PUFA, supplementation did not alter hepatic gene expression and liver histology. CONCLUSIONS: HCC-related gene expression was associated with liver histology, body mass index, waist circumference, diet, and physical activity but was not affected by ω-3 PUFA supplementation.

Altered hepatic genes related to retinol metabolism and plasma retinol in patients with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD), especially non-alcoholic steatohepatitis (NASH) is a chronic liver disease commonly associated with hepatic fibrosis. NASH patients have an increased risk for hepatocellular carcinoma (HCC). An altered retinol metabolism is one of the pathways involved in the process of hepatic fibrosis, and enzymes involved in retinol metabolism have been associated with HCC. We aimed to determine the association between plasma retinol levels and hepatic expression of genes related to retinol metabolism, as well as to assess the hepatic expression of transcription factors regulated by retinoic acid in patients with NAFLD. Cross-sectional study where hepatic gene expression (Illumina microarray) and plasma retinol levels (HPLC) were measured in 17 patients with simple steatosis (SS), 15 with NASH, and 22 living liver donors (LD) as controls. Plasma retinol levels were higher in SS (1.53 ± 0.44 µmol/L) and NASH (1.51 ± 0.56 µmol/L) compared to LD (1.21 ± 0.38 µmol/L; p<0.05). AKR1B10 was highly overexpressed in NASH compared to SS (+6.2-fold) and LD (+9.9-fold; p = 4.89E-11). Retinaldehyde dehydrogenase 1 family, member A2 (ALDH1A2) and retinaldehyde dehydrogenase 1 family, member A3 (ALDH1A3), key enzymes for retinoic acid synthesis, were underexpressed in SS (-1.48 and -2.3-fold, respectively) and NASH (-1.47 and -2.6-fold, respectively) versus LD. In NASH, hepatic ALDH1A2 and ALDH1A3 were underexpressed and inversely correlated with plasma retinol levels, which may reduce retinoic acid in the liver. This, in addition to changes in expression of other genes involved in retinol metabolism, suggests a role for altered retinol homeostasis in NASH.

Markers of activated inflammatory cells are associated with disease severity and intestinal microbiota in adults with non­alcoholic fatty liver disease.

Several mechanisms contribute to the pathogenesis of non­alcoholic fatty liver disease (NAFLD). The intestinal microbiota (IM) and liver immune cells (LIC) may serve a role, but there has been no previous study assessing potential associations between IM and LIC. The aim of the present study was to investigate whether there are differences in LIC markers between patients with NAFLD and healthy controls (HC), and to determine whether these markers are associated with specific IM. The present prospective, cross­sectional study examined a cohort of adults with liver biopsy­confirmed NAFLD and HC. Clinical and laboratory data were collected. Fecal IM was assessed by quantitative polymerase chain reaction and LIC, by immunohistochemistry. NAFLD activity score (NAS) was used for disease severity. Liver immune cell counts were increased in patients with NAFLD (n=34) vs. HC (n=8) and this was associated with disease severity. Hematopoietic cell marker cluster of differentiation (CD)45+ and Kupffer cell marker CD163+ were higher in NAFLD compared with HC, and those with an NAS ≥5 had higher levels of CD20+ cells, a marker of B cells, vs. a NAS of 0 or 1­4. Additionally, from those patients (5 HC, 34 NAFLD), IM was measured. Specific immune cells in portal or lobular areas correlated with specific fecal IM, suggesting a potential association between IM and liver inflammation in patients with NAFLD. Specifically, Faecalibacterium prausnitzii was negatively correlated with CD45+ (r= ­0.394; P=0.015) and CD163+ (r= ­0.371; P=0.022) cells in the portal tract and Prevotella was negatively correlated with CD20+ (r= ­0.353; P=0.028) cells in the liver lobule. Other taxa exhibited no correlation. In conclusion, the present study demonstrated a potential association between IM and liver inflammation in NAFLD.