A cross-sectional clinical study in women to investigate possible genotoxicity and hematological abnormalities related to the use of black cohosh botanical dietary supplements.

Black cohosh (BC; Actaea racemosa L.), a top-selling botanical dietary supplement, is marketed to women primarily to ameliorate a variety of gynecological symptoms. Due to widespread usage, limited safety information, and sporadic reports of hepatotoxicity, the Division of the National Toxicology Program (DNTP) initially evaluated BC extract in female rats and mice. Following administration of up to 1000 mg/kg/day BC extract by gavage for 90 days, dose-related increases in micronucleated peripheral blood erythrocytes were observed, along with a nonregenerative macrocytic anemia resembling megaloblastic anemia in humans. Because both micronuclei and megaloblastic anemia may signal disruption of folate metabolism, and inadequate folate levels in early pregnancy can adversely affect neurodevelopment, the DNTP conducted a pilot cross-sectional study comparing erythrocyte micronucleus frequencies, folate and B12 levels, and a variety of hematological and clinical chemistry parameters between women who used BC and BC-naïve women. Twenty-three women were enrolled in the BC-exposed group and 28 in the BC-naïve group. Use of any brand of BC-only supplement for at least 3 months was required for inclusion in the BC-exposed group. Supplements were analyzed for chemical composition to allow cross-product comparisons. All participants were healthy, with no known exposures (e.g., x-rays, certain medications) that could influence study endpoints. Findings revealed no increased micronucleus frequencies and no hematological abnormalities in women who used BC supplements. Although reassuring, a larger, prospective study with fewer confounders (e.g., BC product diversity and duration of use) providing greater power to detect subtle effects would increase confidence in these findings.

Expanded assessment of xenobiotic associations with antinuclear antibodies in the United States, 1988-2012.

BACKGROUND: The prevalence of autoimmunity in the U.S. has increased recently for undetermined reasons. Little is known about associations between autoimmunity and environmental causes. OBJECTIVES: In a large representative sample of the U.S. population, we expanded our prior exploratory study of how exposures to selected xenobiotics and dioxin-like (DL) mixtures relate to antinuclear antibodies (ANA), the most common biomarker of autoimmunity. METHODS: We analyzed cross-sectional data on 12,058 participants aged ≥ 12 years from three time periods of the National Health and Nutrition Examination Survey between 1988 and 2012, of whom 14% were ANA-positive. We used lognormal regression models and censored-data methods to estimate ANA associations with xenobiotic concentrations overall and in sex, age, and race/ethnicity subgroups. Our analyses adjusted for potential confounders and appropriately handled concentrations below detection limits. RESULTS: Observed ANA associations were positive for most DL compounds and nonDL polychlorinated biphenyls (PCBs), negative for most phthalates, and mixed for other xenobiotic classes. After correcting for multiple comparisons, some associations remained statistically significant. In subgroup analyses, the most significant finding was a positive ANA association with N-acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine (MHB2) in males, followed by positive associations with 2,2',3,5'-tetrachlorobiphenyl (PCB 44), 2,2',4,5'-tetrachlorobiphenyl (PCB 49), and 2,2',3,4',5',6-hexachlorobiphenyl (PCB 149) in 12-19 year-olds, and with 3,4,4',5-tetrachlorobiphenyl (PCB 81), 2,2',3,3',4,4',5,5',6-nonachlorobiphenyl (PCB 206), and N-acetyl-S-(phenyl)-L-cysteine (PMA) in Mexican Americans. Negative associations were found with mono-benzyl phthalate (MBzP) in 20-49 year-olds and mono-n-butyl phthalate (MnBP) in 12-19 year-olds. In overall analyses, combining stratum-specific results across race/ethnicity strata revealed a positive ANA association with PCB 81 and a negative ANA association with N-acetyl-S-(2-hydroxyethyl)-L-cysteine (HEMA). DISCUSSION: This study identified potential associations between ANA and various xenobiotics. Further investigation to confirm these observations and elucidate effects of certain xenobiotics on immune regulation could have important mechanistic, preventive, and treatment implications for a variety of immune-mediated disorders.

Systemic exposure and urinary excretion of vanadium following perinatal subchronic exposure to vanadyl sulfate and sodium metavanadate via drinking water.

Vanadium is a ubiquitous environmental contaminant although there are limited data to assess potential adverse human health impact following oral exposure. In support of studies investigating the subchronic toxicity of vanadyl sulfate (V4+) and sodium metavanadate (V5+) following perinatal exposure via drinking water in male and female rats, we have determined the internal exposure and urinary excretion of total vanadium at the end of study. Water consumption decreased with increasing exposure concentration following exposure to both compounds. Plasma and urine vanadium concentration normalized to total vanadium consumed per day increased with the exposure concentration of vanadyl sulfate and sodium metavanadate suggesting absorption increased as the exposure concentration increased. Additionally, females had higher concentrations than males (in plasma only for vanadyl sulfate exposure). Animals exposed to sodium metavanadate had up to 3-fold higher vanadium concentration in plasma and urine compared to vanadyl sulfate exposed animals, when normalized to total vanadium consumed per day, demonstrating differential absorption, distribution, metabolism, and excretion properties between V5+ and V4+ compounds. These data will aid in the interpretation of animal toxicity data of V4+ and V5+ compounds and determine the relevance of animal toxicity findings to human exposures.

Fluorinated graphene nanomaterial causes potential mechanical perturbations to a biomembrane.

Fluorinated graphene (F-GRA) has attracted great interest in biomedical applications. In this context, the direct interaction between F-GRA and various biomolecules is a vital process guiding the bio-function of this nanomaterial. Nevertheless, information regarding the interaction of F-GRA with biomolecules is scarce, particularly at the molecular level. In this study, using an in silico approach, we investigate the adsorption of F-GRA to a phospholipid bilayer to evaluate the potential effect of the nanomaterial to a biomembrane and its mechanism. Our results indicate that F-GRA can either slightly insert into the membrane or parallelly adhere on the membrane surface, different from the complete insertion of graphene. Detailed analysis confirms that the electrostatic forces dominantly mediate the adsorption process. F-GRA in its parallel binding pattern causes a partial enlargement in the membrane thickness via the disruption of the lipids' order parameters, indicating a mild mechanical influence to the membrane structure. Although the potential mechanical perturbation of F-GRA to membrane is detected, this impact is much weaker than graphene. These findings suggest the potentially weak physical perturbations of F-GRA to the cellular membrane, which may establish the basis for the future biomedical applications of this material after proper surface coating.

Whole-body inhalation exposure to 2-ethyltoluene for two weeks produced nasal lesions in rats and mice.

OBJECTIVE: Ethyltoluenes are isolated during crude oil refinement for use in gasoline and commercial products and are ubiquitous in the environment. However, minimal toxicity data are available. Previously, we identified 2-ethyltoluene (2-ET) as the most potent isomer via nose-only inhalation exposure in rodents. Here, we expanded the hazard characterization of 2-ET in two rodent models using whole-body inhalation exposure and evaluated the role of prenatal exposure. METHODS: Time-mated Hsd:Sprague Dawley® SD® rats were exposed to 0, 150, 300, 600, 900, or 1200 ppm 2-ET via inhalation starting on gestation day 6 until parturition. Rat offspring (n = 8/exposure/sex) were exposed to the same concentrations as the respective dams for 2 weeks after weaning. Adult male and female B6C3F1/N mice (n = 5/exposure/sex) were exposed to the same concentrations for 2 weeks. RESULTS AND DISCUSSION: Exposure to ≥600 ppm 2-ET produced acute toxicity in rats and mice characterized by large decreases in survival, body weight, adverse clinical observations, and diffuse nasal olfactory epithelium degeneration (rats) or necrosis (mice). Due to the early removal of groups ≥600 ppm, most endpoint evaluations focused on lower exposure groups. In 150 and 300 ppm exposure groups, reproductive performance and littering were not significantly changed and body weights in exposed rats and mice were 9-18% lower than controls. Atrophy of the olfactory epithelium and nerves was observed in all animals exposed to 150 and 300 ppm. These lesions were more severe in mice than in rats. CONCLUSION: Nasal lesions were observed in all animals after whole-body exposure up to 600 ppm 2-ET for 2 weeks. Future studies should focus on 2-ET metabolism and distribution to better understand species differences and refine hazard characterization of this understudied environmental contaminant.

Characterizing relationship between chemicals and in vitro bioactivities of teas made by six typical processing methods using a single Camellia sinensis cultivar, Meizhan.

Processing method is considered as a major factor that affects biotransformation of phytochemicals in tea and leads to diverse flavor and bioactivity of tea. In the present work, six typical tea manufacturing processings were employed to compare the effect on chemical composition of teas through using leaves of the single tea cultivar - - Camellia sinensis var. Meizhan. And in vitro antioxidant activity, inhibition against α-glucosidase and three lipid metabolism enzymes of these teas were also investigated, the relationships among them were analyzed further. As fresh leaves were processed into six categories of teas, the content of total catechins (TCs) has decreased in varying degrees while theaflavins (TFs) has increased. The antioxidant capacity composite index (ACCI) from high to low were green tea, yellow tea, oolong tea, white tea, dark tea, and black tea with the range from 98.44 to 58.38, which dominated by the content of TCs. Furthermore, all categories of teas possessed an inhibition effect on the pancreatic lipase (PL), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-COA reductase), lecithin cholesterol acyltransferase (LCAT), and α-glucosidase. The inhibition rate of PL and α-glucosidase appears to be positively influenced by TFs content (r =0.863, r =0.857, p < 0.05) while that of LCAT showed significant positive correlations with the content of tea polyphonels (TPs) (r = 0.902, p < 0.01). These results provide a better understanding of the relationships between processing method and chemical components of tea. It is suggested that various tea categories possess potential healthy effects which could serve as promising nutritional supplements.[Figure: see text].

Efficiency of placental transfer of vaccine-elicited antibodies relative to prenatal Tdap vaccination status.

Administration of vaccines during pregnancy provides maternal protection against infectious diseases. This protection is extended to their infants during the first months of life, as pathogen-specific antibodies formed in response to maternal vaccination are transferred across the placenta to the fetus. Notably, Tdap (tetanus-diphtheria-acellular pertussis) vaccination booster is routinely administered to pregnant women both to prevent neonatal tetanus and to ensure that infants have protective levels of pertussis antibodies until they are able to establish their own vaccine-induced levels. Whether infant protection through maternal immunization is merely due to an increase in maternal antibody levels or whether maternal immunization enhances the transfer of vaccine-specific antibodies is unclear. Moreover, the potential impact of prenatal vaccinations on the transplacental transfer of other antibodies, such as antibodies raised as a result of infections or other vaccines administered prior to pregnancy, has not been studied. The goal of this study was to define the impact of maternal vaccination on IgG transplacental transfer efficiency. We analyzed antigen-specific antibody populations and IgG subclass distribution in maternal and cord blood samples from 58 mother-infant pairs. All women received the seasonal inactivated influenza vaccine during pregnancy and 25 women received the Tdap vaccine during the second or third trimester of gestation. Prenatal Tdap vaccination did not impact the efficiency of IgG transplacental transfer; however, it was associated with higher maternal and infant vaccine-elicited Tdap-specific antibody levels, and with a higher proportion of infants with protective levels of antibodies, especially against diphtheria. There was also no difference in the IgG transplacental transfer rate of antibodies against non-Tdap vaccines between the two groups of women. Our results confirm previous reports demonstrating the benefits of prenatal Tdap immunization and indicate that this strategy does not impede the transplacental transfer of other antibodies that are also important for infant protection.

Short-term perinatal toxicity study in sprague Dawley rats with the plasticizer and emerging contaminant N-Butylbenzenesulfonamide.

N-Butylbenzenesulfonamide (NBBS) is a plasticizer and emerging contaminant that has been detected in a wide array of environmental samples. There are very little toxicity data available with which to evaluate potential risk from exposure to NBBS or other structurally-related sulfonamide plasticizers. To address this knowledge gap, NBBS was selected by the National Toxicology Program for evaluation. The current short-term pre- and post-natal (perinatal) study aims to provide preliminary toxicity and gestational transfer data for NBBS. NBBS was administered via dosed feed at concentrations of 0, 625, 1250, 2500, 5000, and 10,000 ppm to time-mated Sprague Dawley (Hsd:Sprague Dawley SD®) rats from gestation day (GD) 6 through postnatal day (PND) 28. The high concentration of 10,000 ppm NBBS was overtly toxic to dams, and the group was removed on GD 17-18. Exposure to NBBS resulted in lower maternal weights during the gestational period in the 5000 and 10,000 ppm groups as compared to control weights. Dams also displayed lower weights in the lactational period, which resolved to control levels by PND 28. NBBS exposure did not affect pregnancy or littering parameters in F0 dams. However, pup survival was lower in the 5000 ppm group, and pup weights were dose-responsively lower than control pup weights with the difference expanding over the postnatal period. The lowest observed effect level (LOEL) based on significantly lower body weights was 5000 ppm NBBS for F0 dams and 2500 ppm NBBS for F1 pups. Preliminary data for NBBS levels indicated that the chemical was transferred from dams to offspring during the gestational period.

Postnatal Effects of Gestational and Lactational Gavage Exposure to Boric Acid in the Developing Sprague Dawley Rat.

Human exposure to boron occurs primarily through diet and drinking water sources. Animal studies have found that reduced fetal weight following gestational exposure to boron (as boric acid) is the most sensitive toxicological effect. However, recent studies suggest that newborns in areas with elevated boron in drinking water may receive levels of exposure that exceed the U.S. EPA oral reference dose for B. Currently, there are no data to inform a boron risk assessment accounting for this developmental window. To address this knowledge gap, the National Toxicology Program evaluated developmental toxicity following pre- and postnatal boron exposure. Time-mated female Sprague Dawley (Hsd: Sprague Dawley SD) rats were administered 0-20 mg B/kg/day (as boric acid) via gavage from gestation day 6 to 21; offspring were dosed via gavage at the same respective dose level from postnatal day (PND) 1 to 28. There were no dose-related effects on dam bodyweight, bodyweight gain, or feed consumption. Clinical findings were limited to low incidences of umbilical hernia in the 20 mg B/kg pups which resolved by study completion. Pup plasma boron concentrations increased in dose-proportional manner and were similar between PND 4 and PND 28. Postnatal weight gain was significantly reduced at 20 mg B/kg, with male and female pups weighing 23% less than the controls on PND 28. These findings demonstrate that postnatal growth in the Sprague Dawley rat is sensitive to boron exposure and highlights the importance of evaluating the potential toxicity of agents with known human exposures during early life stages.

Intrahost Dynamics of Human Cytomegalovirus Variants Acquired by Seronegative Glycoprotein B Vaccinees.

Human cytomegalovirus (HCMV) is the most common congenital infection worldwide and a frequent cause of hearing loss and debilitating neurologic disease in newborn infants. Thus, a vaccine to prevent HCMV-associated congenital disease is a public health priority. One potential strategy is vaccination of women of child bearing age to prevent maternal HCMV acquisition during pregnancy. The glycoprotein B (gB) plus MF59 adjuvant subunit vaccine is the most efficacious tested clinically to date, demonstrating 50% protection against primary HCMV infection in a phase 2 clinical trial. Yet, the impact of gB/MF59-elicited immune responses on the population of viruses acquired by trial participants has not been assessed. In this analysis, we employed quantitative PCR as well as multiple sequencing methodologies to interrogate the magnitude and genetic composition of HCMV populations infecting gB/MF59 vaccinees and placebo recipients. We identified several differences between the viral dynamics in acutely infected vaccinees and placebo recipients. First, viral load was reduced in the saliva of gB vaccinees, though not in whole blood, vaginal fluid, or urine. Additionally, we observed possible anatomic compartmentalization of gB variants in the majority of vaccinees compared to only a single placebo recipient. Finally, we observed reduced acquisition of genetically related gB1, gB2, and gB4 genotype "supergroup" HCMV variants among vaccine recipients, suggesting that the gB1 genotype vaccine construct may have elicited partial protection against HCMV viruses with antigenically similar gB sequences. These findings suggest that gB immunization had a measurable impact on viral intrahost population dynamics and support future analysis of a larger cohort.IMPORTANCE Though not a household name like Zika virus, human cytomegalovirus (HCMV) causes permanent neurologic disability in one newborn child every hour in the United States, which is more than that for Down syndrome, fetal alcohol syndrome, and neural tube defects combined. There are currently no established effective measures to prevent viral transmission to the infant following HCMV infection of a pregnant mother. However, the glycoprotein B (gB)/MF59 vaccine, which aims to prevent pregnant women from acquiring HCMV, is the most successful HCMV vaccine tested clinically to date. Here, we used viral DNA isolated from patients enrolled in a gB vaccine trial who acquired HCMV and identified several impacts that this vaccine had on the size, distribution, and composition of the in vivo viral population. These results have increased our understanding of why the gB/MF59 vaccine was partially efficacious, and such investigations will inform future rational design of a vaccine to prevent congenital HCMV.

HCMV glycoprotein B subunit vaccine efficacy mediated by nonneutralizing antibody effector functions.

Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, frequently causing hearing loss and brain damage in afflicted infants. A vaccine to prevent maternal acquisition of HCMV during pregnancy is necessary to reduce the incidence of infant disease. The glycoprotein B (gB) + MF59 adjuvant subunit vaccine platform is the most successful HCMV vaccine tested to date, demonstrating ∼50% efficacy in preventing HCMV acquisition in multiple phase 2 trials. However, the mechanism of vaccine protection remains unknown. Plasma from 33 postpartum women gB/MF59 vaccinees at peak immunogenicity was tested for gB epitope specificity as well as neutralizing and nonneutralizing anti-HCMV effector functions and compared with an HCMV-seropositive cohort. gB/MF59 vaccination elicited IgG responses with gB-binding magnitude and avidity comparable to natural infection. Additionally, IgG subclass distribution was similar with predominant IgG1 and IgG3 responses induced by gB vaccination and HCMV infection. However, vaccine-elicited antibodies exhibited limited neutralization of the autologous virus, negligible neutralization of multiple heterologous strains, and limited binding responses against gB structural motifs targeted by neutralizing antibodies including AD-1, AD-2, and domain I. Vaccinees had high-magnitude IgG responses against AD-3 linear epitopes, demonstrating immunodominance against this nonneutralizing, cytosolic region. Finally, vaccine-elicited IgG robustly bound membrane-associated gB on the surface of transfected or HCMV-infected cells and mediated virion phagocytosis, although were poor mediators of NK cell activation. Altogether, these data suggest that nonneutralizing antibody functions, including virion phagocytosis, likely played a role in the observed 50% vaccine-mediated protection against HCMV acquisition.

Predominant envelope variable loop 2-specific and gp120-specific antibody-dependent cellular cytotoxicity antibody responses in acutely SIV-infected African green monkeys.

BACKGROUND: The initial envelope (Env)-specific antibody response in acutely HIV-1-infected individuals and simian immunodeficiency virus (SIV)-infected rhesus monkeys (RMs) is dominated by non-neutralizing antibodies targeting Env gp41. In contrast, natural primate SIV hosts, such as African green monkeys (AGMs), develop a predominant Env gp120-specific antibody response to SIV infection. However, the fine-epitope specificity and function of SIV Env-specific plasma IgG, and their potential role on autologous virus co-evolution in SIV-infected AGMs and RMs remain unclear. RESULTS: Unlike the dominant linear gp41-specific IgG responses in RMs, SIV-infected AGMs demonstrated a unique linear variable loop 2 (V2)-specific plasma IgG response that arose concurrently with high gp120-directed antibody-dependent cellular cytotoxicity (ADCC) activity, and SIVsab-infected cell binding responses during acute infection. Moreover, SIV variants isolated from SIV-infected AGMs exhibited high amino acid mutation frequencies within the Env V1V2 loop compared to those of RMs. Notably, the linear V2-specific IgG epitope in AGMs overlaps with an analogous region of the HIV V2 loop containing the K169 mutation epitope identified in breakthrough viruses from RV144 vaccinees. CONCLUSION: Vaccine-elicited Env V2-specific IgG responses have been proposed as an immune correlate of reduced risk in HIV-1/SIV acquisition in humans and RMs. Yet the pathways to elicit these potentially-protective V2-specific IgG responses remain unclear. In this study, we demonstrate that SIV-infected AGMs, which are the natural hosts of SIV, exhibited high plasma linear V2-specific IgG binding responses that arose concurrently with SIV Env gp120-directed ADCC-mediating, and SIV-infected cell plasma IgG binding responses during acute SIV infection, which were not present in acutely SIV-infected RMs. The linear V2-specific antibody response in AGMs targets an overlapping epitope of the proposed site of vaccine-induced immune pressure defined in the moderately protective RV144 HIV-1 vaccine trial. Identifying host factors that control the early elicitation of Env V2-specific IgG and ADCC antibody responses in these natural SIV hosts could inform vaccination strategies aimed at rapidly inducing potentially-protective HIV-1 Env-specific responses in humans.

Synthesis of Zr2WP2O12/ZrO2 Composites with Adjustable Thermal Expansion.

Zr2WP2O12/ZrO2 composites were fabricated by solid state reaction with the goal of tailoring the thermal expansion coefficient. XRD, SEM and TMA were used to investigate the composition, microstructure, and thermal expansion behavior of Zr2WP2O12/ZrO2 composites with different mass ratio. Relative densities of all the resulting Zr2WP2O12/ZrO2 samples were also tested by Archimedes' methods. The obtained Zr2WP2O12/ZrO2 composites were comprised of orthorhombic Zr2WP2O12 and monoclinic ZrO2. As the increase of the Zr2WP2O12, the relative densities of Zr2WP2O12/ZrO2 ceramic composites increased gradually. The coefficient of thermal expansion of the Zr2WP2O12/ZrO2 composites can be tailored from 4.1 × 10-6 K-1 to -3.3 × 10-6 K-1 by changing the content of Zr2WP2O12. The 2:1 Zr2WP2O12/ZrO2 specimen shows close to zero thermal expansion from 25 to 700°C with an average linear thermal expansion coefficient of -0.09 × 10-6 K-1. These adjustable and near zero expansion ceramic composites will have great potential application in many fields.

Loss of pericyte smoothened activity in mice with genetic deficiency of leptin.

BACKGROUND: Obesity is associated with multiple diseases, but it is unclear how obesity promotes progressive tissue damage. Recovery from injury requires repair, an energy-expensive process that is coupled to energy availability at the cellular level. The satiety factor, leptin, is a key component of the sensor that matches cellular energy utilization to available energy supplies. Leptin deficiency signals energy depletion, whereas activating the Hedgehog pathway drives energy-consuming activities. Tissue repair is impaired in mice that are obese due to genetic leptin deficiency. Tissue repair is also blocked and obesity enhanced by inhibiting Hedgehog activity. We evaluated the hypothesis that loss of leptin silences Hedgehog signaling in pericytes, multipotent leptin-target cells that regulate a variety of responses that are often defective in obesity, including tissue repair and adipocyte differentiation. RESULTS: We found that pericytes from liver and white adipose tissue require leptin to maintain expression of the Hedgehog co-receptor, Smoothened, which controls the activities of Hedgehog-regulated Gli transcription factors that orchestrate gene expression programs that dictate pericyte fate. Smoothened suppression prevents liver pericytes from being reprogrammed into myofibroblasts, but stimulates adipose-derived pericytes to become white adipocytes. Progressive Hedgehog pathway decay promotes senescence in leptin-deficient liver pericytes, which, in turn, generate paracrine signals that cause neighboring hepatocytes to become fatty and less proliferative, enhancing vulnerability to liver damage. CONCLUSIONS: Leptin-responsive pericytes evaluate energy availability to inform tissue construction by modulating Hedgehog pathway activity and thus, are at the root of progressive obesity-related tissue pathology. Leptin deficiency inhibits Hedgehog signaling in pericytes to trigger a pericytopathy that promotes both adiposity and obesity-related tissue damage.

Hedgehog regulates yes-associated protein 1 in regenerating mouse liver.

UNLABELLED: Adult liver regeneration requires induction and suppression of proliferative activity in multiple types of liver cells. The mechanisms that orchestrate the global changes in gene expression that are required for proliferative activity to change within individual liver cells, and that coordinate proliferative activity among different types of liver cells, are not well understood. Morphogenic signaling pathways that are active during fetal development, including Hedgehog and Hippo/Yes-associated protein 1 (Yap1), regulate liver regeneration in adulthood. Cirrhosis and liver cancer result when these pathways become dysregulated, but relatively little is known about the mechanisms that coordinate and control morphogenic signaling during effective liver regeneration. We evaluated the hypothesis that the Hedgehog pathway controls Yap1 activation during liver regeneration by studying intact mice and cultured liver cells. In cultured hepatic stellate cells (HSCs), disrupting Hedgehog signaling blocked activation of Yap1, and knocking down Yap1 inhibited induction of both Yap1- and Hedgehog-regulated genes that enable HSC to become myofibroblasts (MFs). In mice, disrupting Hedgehog signaling in MFs inhibited liver regeneration after partial hepactectomy (PH). Reduced proliferative activity in the liver epithelial compartment resulted from loss of stroma-derived paracrine signals that activate Yap1 and the Hedgehog pathway in hepatocytes. This prevented hepatocytes from up-regulating Yap1- and Hedgehog-regulated transcription factors that normally promote their proliferation. CONCLUSIONS: Morphogenic signaling in HSCs is necessary to reprogram hepatocytes to regenerate the liver epithelial compartment post-PH. This discovery identifies novel molecules that might be targeted to correct defective repair during cirrhosis and liver cancer. (Hepatology 2016;64:232-244).

Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis (NASH) fibrosis.

INTRODUCTION: Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH. METHODS: Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis. RESULTS: MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFß mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice. CONCLUSION: OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.

Vitamin B5 and N-Acetylcysteine in Nonalcoholic Steatohepatitis: A Preclinical Study in a Dietary Mouse Model.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and second indication for liver transplantation in the Western world. Effective therapy is still not available. Previously we showed a critical role for caspase-2 in the pathogenesis of nonalcoholic steatohepatitis (NASH), the potentially progressive form of NAFLD. An imbalance between free coenzyme A (CoA) and acyl-CoA ratio is known to induce caspase-2 activation. OBJECTIVES: We aimed to evaluate CoA metabolism and the effects of supplementation with CoA precursors, pantothenate and cysteine, in mouse models of NASH. METHODS: CoA metabolism was evaluated in methionine-choline deficient (MCD) and Western diet mouse models of NASH. MCD diet-fed mice were treated with pantothenate and N-acetylcysteine or placebo to determine effects on NASH. RESULTS: Liver free CoA content was reduced, pantothenate kinase (PANK), the rate-limiting enzyme in the CoA biosynthesis pathway, was down-regulated, and CoA degrading enzymes were increased in mice with NASH. Decreased hepatic free CoA content was associated with increased caspase-2 activity and correlated with worse liver cell apoptosis, inflammation, and fibrosis. Treatment with pantothenate and N-acetylcysteine did not inhibit caspase-2 activation, improve NASH, normalize PANK expression, or restore free CoA levels in MCD diet-fed mice. CONCLUSION: In mice with NASH, hepatic CoA metabolism is impaired, leading to decreased free CoA content, activation of caspase-2, and increased liver cell apoptosis. Dietary supplementation with CoA precursors did not restore CoA levels or improve NASH, suggesting that alternative approaches are necessary to normalize free CoA during NASH.

Schistosome-induced cholangiocyte proliferation and osteopontin secretion correlate with fibrosis and portal hypertension in human and murine schistosomiasis mansoni.

Schistosomiasis is a major cause of portal hypertension worldwide. It associates with portal fibrosis that develops during chronic infection. The mechanisms by which the pathogen evokes these host responses remain unclear. We evaluated the hypothesis that schistosome eggs release factors that directly stimulate liver cells to produce osteopontin (OPN), a pro-fibrogenic protein that stimulates hepatic stellate cells to become myofibroblasts. We also investigated the utility of OPN as a biomarker of fibrosis and/or severity of portal hypertension. Cultured cholangiocytes, Kupffer cells and hepatic stellate cells were treated with soluble egg antigen (SEA); OPN production was quantified by quantitative reverse transcriptase polymerase chain reaction (qRTPCR) and ELISA; cell proliferation was assessed by BrdU (5-bromo-2'-deoxyuridine). Mice were infected with Schistosoma mansoni for 6 or 16 weeks to cause early or advanced fibrosis. Liver OPN was evaluated by qRTPCR and immunohistochemistry (IHC) and correlated with liver fibrosis and serum OPN. Livers from patients with schistosomiasis mansoni (early fibrosis n=15; advanced fibrosis n=72) or healthy adults (n=22) were immunostained for OPN and fibrosis markers. Results were correlated with plasma OPN levels and splenic vein pressures. SEA-induced cholangiocyte proliferation and OPN secretion (P<0.001 compared with controls). Cholangiocytes were OPN (+) in Schistosoma-infected mice and humans. Liver and serum OPN levels correlated with fibrosis stage (mice: r=0.861; human r=0.672, P=0.0001) and myofibroblast accumulation (mice: r=0.800; human: r=0.761, P=0.0001). Numbers of OPN (+) bile ductules strongly correlated with splenic vein pressure (r=0.778; P=0.001). S. mansoni egg antigens stimulate cholangiocyte proliferation and OPN secretion. OPN levels in liver and blood correlate with fibrosis stage and portal hypertension severity.