Doxycycline Significantly Enhances Induction of Induced Pluripotent Stem Cells to Endoderm by Enhancing Survival Through Protein Kinase B Phosphorylation.

BACKGROUND AND AIMS: Induced pluripotent stem cells (iPSCs) provide an important tool for the generation of patient-derived cells, including hepatocyte-like cells, by developmental cues through an endoderm intermediate. However, most iPSC lines fail to differentiate into endoderm, with induction resulting in apoptosis. APPROACH AND RESULTS: To address this issue, we built upon published methods to develop an improved protocol. We discovered that doxycycline dramatically enhances the efficiency of iPSCs to endoderm differentiation by inhibiting apoptosis and promoting proliferation through the protein kinase B pathway. We tested this protocol in >70 iPSC lines, 90% of which consistently formed complete sheets of endoderm. Endoderm generated by our method achieves similar transcriptomic profiles, expression of endoderm protein markers, and the ability to be further differentiated to downstream lineages. CONCLUSIONS: Furthermore, this method achieves a 4-fold increase in endoderm cell number and will accelerate studies of human diseases in vitro and facilitate the expansion of iPSC-derived cells for transplantation studies.

Polycystic ovary syndrome (PCOS) is associated with NASH severity and advanced fibrosis.

BACKGROUND: Polycystic ovary syndrome (PCOS) affects 10% of reproductive-aged women, and is marked by irregular menses and high androgens. PCOS is a known risk factor for imaging-confirmed steatosis, and we now aim to evaluate whether PCOS influences histologic severity of non-alcoholic fatty liver disease (NAFLD). METHODS: Retrospective study of women ages 18-45 years with biopsy-confirmed NAFLD between 2008 and 2019. Metabolic comorbidities were captured within 6 months of biopsy. Histologic features of non-alcoholic steatohepatitis (NASH) were independently evaluated by two pathologists blinded to PCOS status. RESULTS: Among 102 women meeting study criteria, 36% (n = 37) had PCOS; median age was 35 years; 27% were white, 6% black, 19% Asian and 47% reported Hispanic ethnicity. Women with PCOS had higher LDL (122 vs 102 mg/dL, P = .05) and body mass index(BMI) (38 vs 33 kg/cm2 , P < .01). NASH was present in 76% of women with PCOS vs 66% without PCOS (P = .3), and a higher proportion with PCOS had severe ballooning (32% vs 13%, P = .02), presence of any fibrosis (84% vs 66%, P = .06) and advanced fibrosis (16% vs 6%, P = .10). Adjusted for age and BMI, PCOS remained associated with severe hepatocyte ballooning (OR 3.4, 95% CI 1.1-10.6, P = .03) and advanced fibrosis (OR 7.1, 95% CI 1.3-39, P = .02). Among women with advanced fibrosis, median age was 5 years younger in those with as compared to those without PCOS (40 vs 45 years, P = .02). CONCLUSION: Polycystic ovary syndrome is independently associated with more severe NASH, including advanced fibrosis. Hepatologists should routinely inquire about PCOS in reproductive-aged women with NAFLD, and evaluate for more severe liver disease in this population.

Contrasting responses of Kupffer cells and inflammatory mononuclear phagocytes to biliary obstruction in a mouse model of cholestatic liver injury.

BACKGROUND: Biliary obstruction and cholestasis are serious complications of many liver diseases. Although resident hepatic macrophages (Kupffer cells) are frequently implicated in disease progression, most studies fail to differentiate the contribution of Kupffer cells and inflammatory mononuclear phagocytes (iMNPs) that infiltrate the liver subsequent to obstruction. AIM: This study was undertaken to examine the roles and potential interactions of these two disparate mononuclear phagocyte populations in hepatic injury attending cholestasis. METHODS: Female, C57Bl/6 mice were injected with magnetic beads on day 3 prior to sham operation or bile duct ligation (BDL) to facilitate subsequent Kupffer cell isolation. Three days post-surgery, animals were euthanized, and bead-containing Kupffer cells and iMNPs were separated, purified and analysed. To examine the ability of Kupffer cells to modulate iMNP activity, iMNPs were isolated from the livers of intact and Kupffer cell-depleted mice on day 3 post-surgery and compared. RESULTS: Purified Kupffer cells and iMNP populations obtained from BDL mice exhibited heterogeneous morphologies rendering them visually indistinguishable. iMNPs, however, were characterized by the increased expression of Ly-6C and CD11b and the elevated production of chemokines/cytokines characteristic of inflammatory cells. In the absence of Kupffer cells, iMNPs immigrating to the liver following BDL exhibited significant decreases in CD11b and Ly-6C expression, and in pro-inflammatory chemokine/cytokine production. CONCLUSIONS: Kupffer cells and iMNPs exhibit disparate biological responses to biliary obstruction and cholestasis. Kupffer cells play a key role in regulating iMNP influx and activity.

Neonatal Hepatic Myeloid Progenitors Expand and Propagate Liver Injury in Mice.

BACKGROUND: Biliary atresia (BA) is a progressive pediatric inflammatory disease of the liver that leads to cirrhosis and necessitates liver transplantation. The rapid progression from liver injury to liver failure in children with BA suggests that factors specific to the perinatal hepatic environment are important for disease propagation. Hematopoietic stem and progenitor cells (HSPCs) reside in the fetal liver and are known to serve as central hubs of inflammation. We hypothesized that HSPCs are critical for the propagation of perinatal liver injury (PLI). METHODS: Newborn BALB/c mice were injected with rhesus rotavirus (RRV) to induce PLI or with PBS as control. Livers were compared using histology and flow cytometry. To determine the effects of HSPCs on PLI, RRV-infected neonatal mice were administered anti-CD47 and anti-CD117 to deplete HSPCs. RESULTS: PLI significantly increased the number of common myeloid progenitors and the number of CD34+ hematopoietic progenitors. Elimination of HSPCs through antibody-mediated myeloablation rescued animals from PLI and significantly increased survival (RRV+isotype control 36.4% vs. RRV+myeloablation 77.8%, Chi-test = 0.003). CONCLUSIONS: HSPCs expand as a result of RRV infection and propagate PLI. Targeting of HSPCs may be useful in preventing and treating neonatal inflammatory diseases of the liver such as BA.

The Role of STING in Liver Injury Is Both Stimulus- and Time-Dependent.

STING, Tmem173, is involved in liver injury caused by both infectious and sterile inflammatory models. Its role in toxic liver injury and non-alcoholic fatty liver disease (NAFLD), however, is less clear. While a few groups have investigated its role in NAFLD pathogenesis, results have been conflicting. The objective of this study was to clarify the exact role of STING in toxic liver injury and NAFLD models. Goldenticket mice (Tmem173gt), which lack STING protein, were subjected to either a toxic liver injury with tunicamycin (TM) or one of two dietary models of non-alcoholic fatty liver disease: high fructose feeding or Fructose-Palmitate-Cholesterol (FPC) feeding. Three days after TM injection, Tmem173gt mice demonstrated less liver injury (average ALT of 54 ± 5 IU/L) than control mice (average ALT 108 ± 24 IU/L). In contrast, no significant differences in liver injury were seen between WT and Tmem173gt mice fed either high fructose or FPC. Tmem173gt mice only distinguished themselves from WT mice in their increased insulin resistance. In conclusion, while STING appears to play a role in toxic liver injury mediated by TM, it plays little to no role in two dietary models of NAFLD. The exact role of STING appears to be stimulus-dependent.

Targeting acid ceramidase ameliorates fibrosis in mouse models of non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) is a common cause of liver disease worldwide, and is characterized by the accumulation of fat in the liver. Non-alcoholic steatohepatitis (NASH), an advanced form of NAFLD, is a leading cause of liver transplantation. Fibrosis is the histologic feature most associated with liver-related morbidity and mortality in patients with NASH, and treatment options remain limited. In previous studies, we discovered that acid ceramidase (aCDase) is a potent antifibrotic target using human hepatic stellate cells (HSCs) and models of hepatic fibrogenesis. Using two dietary mouse models, we demonstrate that depletion of aCDase in HSC reduces fibrosis without worsening metabolic features of NASH, including steatosis, inflammation, and insulin resistance. Consistently, pharmacologic inhibition of aCDase ameliorates fibrosis but does not alter metabolic parameters. The findings suggest that targeting aCDase is a viable therapeutic option to reduce fibrosis in patients with NASH.

NK cells suppress experimental cholestatic liver injury by an interleukin-6-mediated, Kupffer cell-dependent mechanism.

BACKGROUND & AIMS: Natural killer (NK) cells are innate immune effector cells first characterized by their ability to lyse susceptible tumor cells. Recent studies demonstrated their role in initiating and modulating adaptive immunity. NK cells represent a larger percentage of the lymphoid population in liver than other organs, suggesting that hepatic NK cells express some unique function. Here, we examined the response of NK cells to liver injury that occurs in a mouse model of biliary obstruction. METHODS: Bile duct ligations (BDL) were performed in mice previously depleted or not depleted of NK cells. NK cell activation, interleukin (IL)-6 mRNA expression and protein production by Kupffer cells, and the ability of exogenous IL-6 to ameliorate liver injury in NK cell-depleted mice, were determined. RESULTS: The number of activated hepatic NK cells increased markedly following BDL. Activation was suppressed in mice rendered Kupffer cell-depleted prior to ligation. Increased liver injury occurred in NK cell-depleted mice correlating with a reduction in IL-6 production. Purified Kupffer cells, obtained from NK cell-depleted or anti-interferon (IFN)-γ monoclonal antibody-pretreated mice following BDL, produced less IL-6 in culture than did Kupffer cells derived from control animals. In culture, hepatic NK cells derived from BDL mice stimulated IFN-γ-dependent IL-6 production by Kupffer cells; splenic NK cells obtained from the same animals had a negligible effect. Treatment with recombinant murine IL-6 reduced liver injury in BDL, NK cell-depleted mice. CONCLUSIONS: Hepatic NK cells suppress cholestatic liver injury by stimulating Kupffer cell-dependent IL-6 production.

ER Disposal Pathways in Chronic Liver Disease: Protective, Pathogenic, and Potential Therapeutic Targets.

The endoplasmic reticulum is a central player in liver pathophysiology. Chronic injury to the ER through increased lipid content, alcohol metabolism, or accumulation of misfolded proteins causes ER stress, dysregulated hepatocyte function, inflammation, and worsened disease pathogenesis. A key adaptation of the ER to resolve stress is the removal of excess or misfolded proteins. Degradation of intra-luminal or ER membrane proteins occurs through distinct mechanisms that include ER-associated Degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD), which includes macro-ER-phagy, micro-ER-phagy, and Atg8/LC-3-dependent vesicular delivery. All three of these processes are critical for removing misfolded or unfolded protein aggregates, and re-establishing ER homeostasis following expansion/stress, which is critical for liver function and adaptation to injury. Despite playing a key role in resolving ER stress, the contribution of these degradative processes to liver physiology and pathophysiology is understudied. Analysis of publicly available datasets from diseased livers revealed that numerous genes involved in ER-related degradative pathways are dysregulated; however, their roles and regulation in disease progression are not well defined. Here we discuss the dynamic regulation of ER-related protein disposal pathways in chronic liver disease and cell-type specific roles, as well as potentially targetable mechanisms for treatment of chronic liver disease.

Hepatocyte-specific deletion of XBP1 sensitizes mice to liver injury through hyperactivation of IRE1α.

X-box binding protein-1 (XBP1) is a transcription factor that plays a central role in controlling cellular responses to endoplasmic reticulum (ER) stress. Under stress conditions, the transcriptionally active form of XBP1 is generated via splicing of Xbp1 mRNA by the ER-resident protein inositol-requiring enzyme-1 (IRE1α). Genetic deletion of XBP1 has multiple consequences: some resulting from the loss of the transcription factor per se, and others related to compensatory activation of IRE1α. The objective of the current study was to investigate the effects of XBP1 deletion in adult mouse liver and determine to what extent they are direct or indirect. XBP1 was deleted from hepatocytes in adult Xbp1fl/fl mice using AAV8-Transthyretin-Cre (Xbp1Δhep). Xbp1Δhep mice exhibited no liver disease at baseline, but developed acute biochemical and histologic liver injury in response to a dietary challenge with fructose for 4 weeks. Fructose-mediated liver injury in Xbp1Δhep mice coincided with heightened IRE1α activity, as demonstrated by Xbp1 mRNA splicing, JNK activation, and regulated IRE1α-dependent RNA decay (RIDD). Activation of eIF2α was also evident, with associated up-regulation of the pro-apoptotic molecules CHOP, BIM, and PUMA. To determine whether the adverse consequences of liver-specific XBP1 deletion were due to XBP1 loss or heightened IRE1α activity, we repeated a fructose challenge in mice with liver-specific deletion of both XBP1 and IRE1α (Xbp1Δhep;Ire1aΔhep). Xbp1Δhep;Ire1aΔhep mice were protected from fructose-mediated liver injury and failed to exhibit any of the signs of ER stress seen in mice lacking XBP1 alone. The protective effect of IRE1α deletion persisted even with long-term exposure to fructose. Xbp1Δhep mice developed liver fibrosis at 16 weeks, but Xbp1Δhep;Ire1aΔhep mice did not. Overall, the results indicate that the deleterious effects of hepatocyte-specific XBP1 deletion are due primarily to hyperactivation of IRE1α. They support further exploration of IRE1α as a contributor to acute and chronic liver diseases.

Targeting acid ceramidase inhibits YAP/TAZ signaling to reduce fibrosis in mice.

Hepatic stellate cells (HSCs) drive hepatic fibrosis. Therapies that inactivate HSCs have clinical potential as antifibrotic agents. We previously identified acid ceramidase (aCDase) as an antifibrotic target. We showed that tricyclic antidepressants (TCAs) reduce hepatic fibrosis by inhibiting aCDase and increasing the bioactive sphingolipid ceramide. We now demonstrate that targeting aCDase inhibits YAP/TAZ activity by potentiating its phosphorylation-mediated proteasomal degradation via the ubiquitin ligase adaptor protein ß-TrCP. In mouse models of fibrosis, pharmacologic inhibition of aCDase or genetic knockout of aCDase in HSCs reduces fibrosis, stromal stiffness, and YAP/TAZ activity. In patients with advanced fibrosis, aCDase expression in HSCs is increased. Consistently, a signature of the genes most down-regulated by ceramide identifies patients with advanced fibrosis who could benefit from aCDase targeting. The findings implicate ceramide as a critical regulator of YAP/TAZ signaling and HSC activation and highlight aCDase as a therapeutic target for the treatment of fibrosis.

Macronutrients and the Adipose-Liver Axis in Obesity and Fatty Liver.

Macronutrient metabolism is a highly orchestrated process, with adipose tissue and liver each playing central roles in nutrient uptake, processing, transport, and storage. These 2 tissues form an important metabolic circuit, particularly as it relates to lipids as the primary storage form of excess energy. The function of the circuit is influenced by many factors, including the quantity and type of nutrients consumed and their impact on the overall health of the tissues. In this review we begin with a brief summary of the homeostatic disposition of lipids between adipose tissue and liver and how these processes can become dysregulated in obesity. We then explore how specific dietary nutrients and nutrient combinations can exert unique influences on the liver-adipose tissue axis.

The Association of Hispanic Ethnicity with Nonalcoholic Fatty Liver Disease in Polycystic Ovary Syndrome.

Polycystic Ovary Syndrome (PCOS) affects 10-15% of reproductive age women and is a recognized risk factor for nonalcoholic fatty liver disease (NAFLD). The more severe form of NAFLD, known as nonalcoholic steatohepatitis (NASH), results in liver inflammation with or without fibrosis, and is now a leading cause of cirrhosis. Ethnic differences are apparent in NAFLD, with higher prevalence in Hispanics, although the role of Hispanic ethnicity on risk for NAFLD/NASH in women with PCOS is not known. OBJECTIVE: The aim of this study was to evaluate ethnic differences in the prevalence and risk of NAFLD/NASH in women with PCOS. STUDY DESIGN: Among PCOS women followed in a large academic medical center the association of Hispanic ethnicity with elevated biomarkers of NASH, including plasma cytokeratin 18 (CK18) M30 fragments and/or ALT levels (n=303), was assessed. Prevalence of hepatic steatosis by Controlled Attenuation Parameter (CAP) imaging was also evaluated in a subset of PCOS women (n=35). RESULTS: The median cohort age (n=303) was 28 years (IQR 8), and 15.5% (n=47) were Hispanic, the majority of whom reported white race (94%). Most Hispanic women had hepatic steatosis on imaging, which was markedly higher than in non-Hispanics (83% vs 24%, p=0.005). Approximately 17% of PCOS women had elevated ALT or elevated CK18, which was more common in Hispanics than non-Hispanics, at 34% vs 14%, respectively, p=0.002. On univariate analysis, Hispanic ethnicity was associated with two-fold higher odds of NASH (OR 2.0, 95% CI 1.0-3.9, p=0.038), and the association persisted after adjustment for HOMA-IR and waist circumference (AOR 3.1, 95% CI 1.1-8.9, p=0.034). CONCLUSION: NAFLD/NASH is an important condition to be considered by PCOS providers and Hispanic women with PCOS are a particularly high-risk group that may warrant routine screening.

CD18 deficiency improves liver injury in the MCD model of steatohepatitis.

Neutrophils and macrophages are important constituents of the hepatic inflammatory infiltrate in non-alcoholic steatohepatitis. These innate immune cells express CD18, an adhesion molecule that facilitates leukocyte activation. In the context of fatty liver, activation of infiltrated leukocytes is believed to enhance hepatocellular injury. The objective of this study was to determine the degree to which activated innate immune cells promote steatohepatitis by comparing hepatic outcomes in wild-type and CD18-mutant mice fed a methionine-choline-deficient (MCD) diet. After 3 weeks of MCD feeding, hepatocyte injury, based on serum ALT elevation, was 40% lower in CD18-mutant than wild-type mice. Leukocyte infiltration into the liver was not impaired in CD18-mutant mice, but leukocyte activation was markedly reduced, as shown by the lack of evidence of oxidant production. Despite having reduced hepatocellular injury, CD18-mutant mice developed significantly more hepatic steatosis than wild-type mice after MCD feeding. This coincided with greater hepatic induction of pro-inflammatory and lipogenic genes as well as a modest reduction in hepatic expression of adipose triglyceride lipase. Overall, the data indicate that CD18 deficiency curbs MCD-mediated liver injury by limiting the activation of innate immune cells in the liver without compromising intrahepatic cytokine activation. Reduced liver injury occurs at the expense of increased hepatic steatosis, which suggests that in addition to damaging hepatocytes, infiltrating leukocytes may influence lipid homeostasis in the liver.

Specific Macronutrients Exert Unique Influences on the Adipose-Liver Axis to Promote Hepatic Steatosis in Mice.

BACKGROUND & AIMS: The factors that distinguish metabolically healthy obesity from metabolically unhealthy obesity are not well understood. Diet has been implicated as a determinant of the unhealthy obesity phenotype, but which aspects of the diet induce dysmetabolism are unknown. The goal of this study was to investigate whether specific macronutrients or macronutrient combinations provoke dysmetabolism in the context of isocaloric, high-energy diets. METHODS: Mice were fed 4 high-energy diets identical in calorie and nutrient content but different in nutrient composition for 3 weeks to 6 months. The test diets contained 42% carbohydrate (sucrose or starch) and 42% fat (oleate or palmitate). Weight and glucose tolerance were monitored; blood and tissues were collected for histology, gene expression, and immunophenotyping. RESULTS: Mice gained weight on all 4 test diets but differed significantly in other metabolic outcomes. Animals fed the starch-oleate diet developed more severe hepatic steatosis than those on other formulas. Stable isotope incorporation showed that the excess hepatic steatosis in starch-oleate-fed mice derived from exaggerated adipose tissue lipolysis. In these mice, adipose tissue lipolysis coincided with adipocyte necrosis and inflammation. Notably, the liver and adipose tissue abnormalities provoked by starch-oleate feeding were reproduced when mice were fed a mixed-nutrient Western diet with 42% carbohydrate and 42% fat. CONCLUSIONS: The macronutrient composition of the diet exerts a significant influence on metabolic outcome, independent of calories and nutrient proportions. Starch-oleate appears to cause hepatic steatosis by inducing progressive adipose tissue injury. Starch-oleate phenocopies the effect of a Western diet; consequently, it may provide clues to the mechanism whereby specific nutrients cause metabolically unhealthy obesity.

Isocaloric manipulation of macronutrients within a high-carbohydrate/moderate-fat diet induces unique effects on hepatic lipogenesis, steatosis and liver injury.

Diets containing excess carbohydrate and fat promote hepatic steatosis and steatohepatitis in mice. Little is known, however, about the impact of specific carbohydrate/fat combinations on liver outcome. This study was designed to determine whether high-energy diets with identical caloric density but different carbohydrate and fat composition have unique effects on the liver. Four experimental diets were formulated with 60%kcal carbohydrate and 20%kcal fat, each in nearly pure form from a single source: starch-oleate, starch-palmitate, sucrose-oleate and sucrose-palmitate. The diets were fed to mice for 3 or 12 weeks for analysis of lipid metabolism and liver injury. All mice developed hepatic steatosis over 12 weeks, but mice fed the sucrose-palmitate diet accumulated more hepatic lipid than those in the other three experimental groups. The exaggerated lipid accumulation in sucrose-palmitate-fed mice was attributable to a disproportionate rise in hepatic de novo lipogenesis. These mice accrued more hepatic palmitate and exhibited more evidence of liver injury than any of the other experimental groups. Interestingly, lipogenic gene expression in mice fed the custom diets did not correlate with actual de novo lipogenesis. In addition, de novo lipogenesis rose in all mice between 3 and 12 weeks, without feedback inhibition from hepatic steatosis. The pairing of simple sugar (sucrose) and saturated fat (palmitate) in a high-carbohydrate/moderate-fat diet induces more de novo lipogenesis and liver injury than other carbohydrate/fat combinations. Diet-induced liver injury correlates positively with hepatic de novo lipogenesis and is not predictable by isolated analysis of lipogenic gene expression.

Mechanisms of Liver Injury in Non-Alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a disorder marked by alterations in hepatic lipid homeostasis as well as liver injury in the form of cell death, inflammation and fibrosis. Research into the pathophysiology of NASH is dynamic. New concepts from the fields of cell biology, microbiology, immunology and genetics are being tested for their applicability to NASH; discoveries in each of these areas are enriching our understanding of this complex disease. This review summarizes how recent developments from the bench and bedside are merging with more traditional concepts to reshape our view of NASH pathogenesis. Highlights include human studies that emphasize the role of de novo lipogenesis in NASH and experimental work uncovering a role for the inflammasome in NASH. Genetic predispositions to NASH are being clarified, and intestinal microbiome is emerging as a determinant of fatty liver. These unique ideas are now taking their place within an integrated picture of NASH pathogenesis.

Cross-activating invariant NKT cells and kupffer cells suppress cholestatic liver injury in a mouse model of biliary obstruction.

Both Kupffer cells and invariant natural killer T (iNKT) cells suppress neutrophil-dependent liver injury in a mouse model of biliary obstruction. We hypothesize that these roles are interdependent and require iNKT cell-Kupffer cell cross-activation. Female, wild-type and iNKT cell-deficient C57Bl/6 mice were injected with magnetic beads 3 days prior to bile duct ligation (BDL) in order to facilitate subsequent Kupffer cell isolation. On day three post-BDL, the animals were euthanized and the livers dissected. Necrosis was scored; Kupffer cells were isolated and cell surface marker expression (flow cytometry), mRNA expression (qtPCR), nitric oxide (NO (.) ) production (Griess reaction), and protein secretion (cytometric bead-array or ELISAs) were determined. To address the potential role of NO (.) in suppressing neutrophil accumulation, a group of WT mice received 1400W, a specific inducible nitric oxide synthase (iNOS) inhibitor, prior to BDL. To clarify the mechanisms underlying Kupffer cell-iNKT cell cross-activation, WT animals were administered anti-IFN-γ or anti-lymphocyte function-associated antigen (LFA)-1 antibody prior to BDL. Compared to their WT counterparts, Kupffer cells obtained from BDL iNKT cell-deficient mice expressed lower iNOS mRNA levels, produced less NO (.) , and secreted more neutrophil chemoattractants. Both iNOS inhibition and IFN-γ neutralization increased neutrophil accumulation in the livers of BDL WT mice. Anti-LFA-1 pre-treatment reduced iNKT cell accumulation in these same animals. These data indicate that the LFA-1-dependent cross-activation of iNKT cells and Kupffer cells inhibits neutrophil accumulation and cholestatic liver injury.

Targeting the diverse immunological functions expressed by hepatic NKT cells.

INTRODUCTION: NKT cells comprise approximately 30% of the hepatic lymphoid population in mice (∼ 50% in humans). Most mouse hepatic NKT cells [invariant (i)NKT cells] express T cell receptors, composed of invariant Vα14Jα18 chains. Unlike conventional T cells, iNKT cells recognize glycolipids presented in association with MHC class Ib (CD1d) molecules. Purportedly, iNKT cells serve key functions in several immunological events; the nature of these is often unclear. The consequences of hepatic iNKT cell activation can be beneficial or detrimental. α-Galactosylceramide stimulates the production of IFN-γ and IL-4. The reciprocal suppression exhibited by these cytokines limits the potential therapeutic value of α-galactosylceramide. Efforts are ongoing to develop α-galactosylceramide analogs that modulate iNKT cell activity and selectively promote IFN-γ or IL-4. AREAS COVERED: An overview of hepatic iNKT cells and their purported role in liver disease. Efforts to develop therapeutic agents that promote their beneficial contributions. EXPERT OPINION: While a growing body of literature documents the differential effects of α-GalCer analogs on IFN-γ and IL-4 production, the effects of these analogs on other iNKT cell activities remain to be determined. An exhaustive examination of the effects of these analogs on inflammation and liver injury in animal models remains prior to considering their utility in clinical trials.

Effects of recovery from immobilization stress on striatal preprodynorphin- and kappa opioid receptor-mRNA levels of the male rat.

Previously, we have reported that brain regions that are thought to be involved in motivated behavior are altered in animals undergoing repeated exposures to immobilization stress. The goal of the present study was to determine the effects of recovery from this type of stress on these same mesolimbic brain regions. For this purpose, adult male Sprague-Dawley rats were initially exposed to immobilization stress either once (2 h) or repeatedly (2 h×10 days). Rats were then either allowed to recover from the stressor for a shorter (2 days) or longer period of time (9 days) in their home cages. At the end of this recovery period, rats were euthanized and trunk blood and brains were processed for serum corticosterone (CORT) and neurochemistry, respectively. Brain mRNA levels were determined via in situ hybridization for the opioid preprodynorphin (DYN) and its cognate receptor (kappa, KOR), in striatal and accumbal subregions. A pattern of selective transcriptional activation emerged in the four resultant treatment conditions where a short recovery from either a single or repeated exposure to immobilization produced increases in KOR-mRNA levels in striatal and nucleus accumbens (Acb) subregions. Relative to controls, these differences were diminished after a longer recovery period. Interestingly, DYN-mRNA levels were unchanged after the shorter recovery period and after single or repeated immobilizations but appeared to be induced after a longer recovery period after repeated immobilizations. A relative amount of weight loss occurred after immobilization following repeated but not single exposure to stress. In addition, only those rats recovering from repeated stress exposures had higher CORT levels compared with non-immobilized controls. These results suggest that recovery from immobilization stress may alter the motivational system after as little as a single immobilization and that a possible dysphoric effect on appetitive behavior may be reflected by an altered striatal dynorphin system.