Hypoxia-inducible factor 2α regulates macrophage function in mouse models of acute and tumor inflammation

Hypoxia-inducible factor 1α (HIF-1α) and HIF-2α display unique and sometimes opposing activities in regulating cellular energy homeostasis, cell fate decisions, and oncogenesis. Macrophages exposed to hypoxia accumulate both HIF-1α and HIF-2α, and overexpression of HIF-2α in tumor-associated macrophages (TAMs) is specifically correlated with high-grade human tumors and poor prognosis. However, the precise role of HIF-2α during macrophage-mediated inflammatory responses remains unclear. To fully characterize cellular hypoxic adaptations, distinct functions of HIF-1α versus HIF-2α must be elucidated. We demonstrate here that mice lacking HIF-2α in myeloid cells (Hif2a^Δ/Δ mice) are resistant to lipopolysaccharide-induced endotoxemia and display a marked inability to mount inflammatory responses to cutaneous and peritoneal irritants. Furthermore, HIF-2α directly regulated proinflammatory cytokine/chemokine expression in macrophages activated in vitro. Hif2a^Δ/Δ mice displayed reduced TAM infiltration in independent murine hepatocellular and colitis-associated colon carcinoma models, and this was associated with reduced tumor cell proliferation and progression. Notably, HIF-2α modulated macrophage migration by regulating the expression of the cytokine receptor M-CSFR and the chemokine receptor CXCR4, without altering intracellular ATP levels. Collectively, our data identify HIF-2α as an important regulator of innate immunity, suggesting it may be a useful therapeutic target for treating inflammatory disorders and cancer.     

Patient monitoring and the timing of cardiac arrests and medical emergency team calls in a teaching hospital

Objective To describe the timing of cardiac arrest detection in relation to episodes of Medical Emergency Team (MET) review and routine nursing observations.Design and setting Retrospective observational study in a university-affiliated hospital.Patients 279 cardiac arrests involving ward patientsMeasurements and results Cardiac arrests were allocated to one of 24 1-h intervals (24:00–00:59, 01:00–01:59, etc.). The actual hourly rate of cardiac arrests was related to the expected average hourly rate. Peak levels of cardiac arrest detection occurred during times of routine overnight nursing clinical observations between 02:00 and 03:00 (OR 3.06) and 06:00–07:00 (OR 1.95). The lowest level of cardiac arrest detection occurred between 20:00 and 21:00 (OR 0.42). After introduction of the MET there were 162 cardiac arrests, 28% of which occurred shortly after an initial MET call. The odds ratio for risk of cardiac arrest during periods of lowest MET activation (24:00–08:00) when compared with periods of highest MET activation (16:00–24:00) was 2.26.Conclusions Cardiac arrest detection in our hospital is episodic with peak levels corresponding to periods of overnight routine nursing observations following a period when patient review is likely to be low. After the introduction of the MET there was an inverse link between detection of cardiac arrests and levels of MET activation over the 24-h period. Increased overnight utilization and earlier MET activation may further reduce the incidence of cardiac arrests at our hospital.     

Renal and cardiac function during α 1 -β-blockade in congestive heart failure

The kidney and the neurohormonal systems are essential in the pathogenesis of congestive heart failure (CHF) and the physiologic response. Routine treatment of moderate to severe CHF consists of diuretics, angiotensin-converting enzyme (ACE) inhibition and &#103 -blockade. The need for control of renal function during initiation of ACE-inhibition in patients with CHF is well known. The aim of this study was to investigate whether supplementation by a combined &#102 1 - &#103 -blockade to diuretics and ACE-inhibition might improve cardiac function without reducing renal function. Methods : Fourteen patients treated for moderate to severe CHF with diuretics and ACE inhibitors were investigated at baseline, after 4 months of maximum carvedilol treatment and after withdrawal of carvedilol. Results : Carvedilol lowered blood pressure and heart rate but increased left and right ventricular ejection fractions without changing cardiac output or pulmonary blood volume. At the same time, a minor fall was seen in glomerular filtration rate (GFR), but renal blood flow was unchanged and effective renal plasma flow slightly increased. Carvedilol also lowered the plasma levels of angiotensin II and aldosterone. All changes were reversed after withdrawal of carvedilol. Conclusions : Carvedilol augments ACE-inhibitor-induced vasodilation by lowering blood pressure, and angiotensin II beside reducing heart rate. The heart adapts to the haemodynamic alterations without changes in cardiac output and pulmonary blood volume. GFR is slightly lowered despite no changes in renal blood flow and a slight increase in effective renal plasma flow. The study emphasizes the need for control of renal function during treatment with carvedilol in patients with CHF.     

HIF-1α regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment

Myeloid-derived suppressor cells (MDSCs) are a major component of the immune-suppressive network described in cancer and many other pathological conditions. We demonstrate that although MDSCs from peripheral lymphoid organs and the tumor site share similar phenotype and morphology, these cells display profound functional differences. MDSC from peripheral lymphoid organs suppressed antigen-specific CD8⁺ T cells but failed to inhibit nonspecific T cell function. In sharp contrast, tumor MDSC suppressed both antigen-specific and nonspecific T cell activity. The tumor microenvironment caused rapid and dramatic up-regulation of arginase I and inducible nitric oxide synthase in MDSC, which was accompanied by down-regulation of nicotinamide adenine dinucleotide phosphate–oxidase and reactive oxygen species in these cells. In contrast to MDSC from the spleen, MDSC from the tumor site rapidly differentiated into macrophages. Exposure of spleen MDSC to hypoxia resulted in the conversion of these cells to nonspecific suppressors and their preferential differentiation to macrophages. Hypoxia-inducible factor (HIF) 1α was found to be primarily responsible for the observed effects of the tumor microenvironment on MDSC differentiation and function. Thus, hypoxia via HIF-1α dramatically alters the function of MDSC in the tumor microenvironment and redirects their differentiation toward tumor-associated macrophages, hence providing a mechanistic link between different myeloid suppressive cells in the tumor microenvironment.Myeloid-derived suppressor cells (MDSCs) are one of the major components of the immune-suppressive network responsible for T cell defects in cancer. These cells also contribute to tumor progression via regulation of angiogenesis and tumor cell motility. MDSC is a large group of myeloid cells consisting of immature macrophages (MΦ), granulocytes, and DCs, as well as myeloid cells at earlier stages of differentiation (Sica and Bronte, 2007; Talmadge, 2007; Gabrilovich and Nagaraj, 2009; Peranzoni et al., 2010). In mice, MDSCs express both the myeloid lineage differentiation antigen Gr-1 (Ly6G and Ly6C) and α_M integrin CD11b. Two major groups of MDSCs are now identified: cells with granulocytic (CD11b⁺Ly6G⁺Ly6C^low) and monocytic (CD11b⁺Ly6G⁻Ly6C^high) phenotype (Movahedi et al., 2008; Youn et al., 2008). In humans, MDSCs are generally defined as cells that express CD11b, the common myeloid marker CD33, but lack the expression of markers of mature myeloid and lymphoid cells and the MHC class II molecule HLA-DR (Almand et al., 2001; Zea et al., 2005; Diaz-Montero et al., 2009; Nagaraj et al., 2010). In tumor-free mice, MDSCs represent ∼30% of the normal BM cells and <3% of all nucleated splenocytes. In tumor-bearing mice, this population undergoes dramatic expansion. In many tumor models the proportion of MDSC represents >20% of all splenocytes, and MDSCs are easily detectable in tumors and lymph nodes (Kusmartsev and Gabrilovich, 2006; Rabinovich et al., 2007; Sica and Bronte, 2007; Gabrilovich and Nagaraj, 2009). Similar expansion, albeit to a lesser degree, is observed in patients with cancer. In the presence of appropriate cytokines in vitro and after adoptive transfer in vivo, MDSC can differentiate into mature myeloid cells (Kusmartsev and Gabrilovich, 2003). This differentiation is blocked, however, in the presence of tumor cell–conditioned media or in tumor-bearing hosts (Kusmartsev and Gabrilovich, 2003; Talmadge, 2007).Extensive studies in recent years suggested several mechanisms of MDSC-mediated immune suppression that involve arginine (Bronte and Zanovello, 2005; Rodríguez and Ochoa, 2008) and cysteine (Srivastava et al., 2010) metabolism, expression of some surface molecules (Pan et al., 2010), up-regulation of reactive oxygen species (ROS), and production of different cytokines (Talmadge, 2007; Gabrilovich and Nagaraj, 2009). Practically all these studies were performed with MDSC isolated from peripheral lymphoid organs (mostly spleen).Although an important role of MDSC in tumor-associated immune suppression is well established in recent years, its nature remains unclear. One of the major unresolved questions is the role of MDSCs in peripheral lymphoid organs and tumor tissues as well as their relationship with MΦ and DCs. The main paradox is that, despite the presence of a large number of MDSCs in spleens and lymph nodes of tumor-bearing mice and in the peripheral blood of cancer patients with advanced disease, T cells mostly retain the ability to respond to different tumor-nonspecific stimuli including viruses, lectins, costimulatory molecules, IL-2, and stimulation with CD3- and CD28-specific antibodies (Fricke et al., 2007; Monu and Frey, 2007; Nagaraj et al., 2007). In a sharp contrast, T cells directly isolated from tumors display profound defects in their ability to respond to those stimuli (Rabinowich et al., 1996; Lopez et al., 1998; Reichert et al., 2002). Some evidence may suggest that MDSC-mediated immune suppression in peripheral lymphoid organs could be mainly tumor antigen specific. MDSCs mediate antigen-specific T cell tolerance by taking up soluble antigens, including tumor-associated antigens, processing them, and presenting them to CD8⁺ T cells in the context of MHC class I (Kusmartsev et al., 2005; Movahedi et al., 2008).The role of tumor-associated MDSC remains largely obscure. Despite the fact that the cells with the phenotype of MDSC are abundantly present in tumor tissues but are often referred to as monocytes, MΦ, or granulocytes, the relationship (or lack thereof) between MDSC and tumor-associated macrophages (TAMs) is unclear. This confusion is derived from the difficulties in clearly defining the phenotype of myeloid cells in the tumor site. This creates a convoluted, rather incoherent picture of the various functions of different myeloid cell subsets within the tumor. Ambiguity of the biological role of and the relationship between different myeloid cell populations within the tumor site severely limits our understanding of the biology of tumor progression and the development of targeted therapeutics.In this study, we addressed this issue by investigating the function and differentiation of MDSC at the tumor site using an experimental model where tumors were generated as ascites. This allowed for a rapid isolation of cells and enabled direct experiments with adoptive transfer of MDSC directly into the tumor microenvironment. In this paper, we report that MDSCs in tumor-bearing mice display a biological dichotomy regulated by the tumor microenvironment. In peripheral lymphoid organs, MDSC primarily caused antigen-specific T cell nonresponsiveness, which was mediated by ROS. In contrast, within tumor microenvironment, MDSC with the same phenotype and morphology had low ROS levels but dramatically up-regulated NO production and arginase activity, which caused suppression of antigen-nonspecific T cell functions. In contrast to MDSC from the spleen, MDSC in the tumor microenvironment rapidly differentiated primarily into TAM. This process was mediated primarily by hypoxia via hypoxia-inducible factor (HIF) 1α. Our data thus help to explain the differences in the nature of T cell suppression between tumor and peripheral lymphoid organs in tumor-bearing hosts and suggest a regulatory pathway of myeloid cell function in the tumor microenvironment.     

Contribution of the extracellular cAMP-adenosine pathway to dual coupling of β2-adrenoceptors to Gs and Gi proteins in mouse skeletal muscle

β(2)-Adrenoceptor (β(2)-AR) agonists increase skeletal muscle contractile force via activation of G(s) protein/adenylyl cyclases (AC) and increased generation of cAMP. Herein, we evaluated the possible dual coupling of β(2)-AR to G(s) and G(i) proteins and the influence of the β(2)-AR/G(s)-G(i)/cAMP signaling cascade on skeletal muscle contraction. Assuming that the increment of intracellular cAMP is followed by cAMP efflux and extracellular generation of adenosine, the contribution of the extracellular cAMP-adenosine pathway on the β(2)-AR inotropic response was also addressed. The effects of clenbuterol/fenoterol (β(2)-AR agonists), forskolin (AC activator), cAMP/8-bromo-cAMP, and adenosine were evaluated on isometric contractility of mouse diaphragm muscle induced by supramaximal direct electrical stimulation (0.1 Hz, 2 ms duration). Clenbuterol/fenoterol (10-1000 μM), 1 μM forskolin, and 20 μM rolipram induced transient positive inotropic effects that peaked 30 min after stimulation onset, declining to 10 to 20% of peak levels in 30 min. The late descending phase of the β(2)-AR agonist inotropic effect was mimicked by either cAMP or adenosine and abolished by preincubation of diaphragm with pertussis toxin (PTX) (G(i) signaling inhibitor) or the organic anion transporter inhibitor probenecid, indicating a delayed coupling of β(2)-AR to G(i) protein which depends on cAMP efflux. Remarkably, the PTX-sensitive β(2)-AR inotropic effect was inhibited by the A(1) adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine and ecto-5'-phosphodiesterase inhibitor α,β-methyleneadenosine 5'-diphosphate sodium salt, indicating that β(2)-AR coupling to G(i) is indirect and dependent on A(1) receptor activation. The involvement of the extracellular cAMP-adenosine pathway in β(2)-AR signaling would provide a negative feedback loop that may limit stimulatory G protein-coupled receptor positive inotropism and potential deleterious effects of excessive contractile response.     

Signaling via the prostaglandin E₂ receptor EP4 exerts neuronal and vascular protection in a mouse model of cerebral ischemia

Stroke is the third leading cause of death in the United States. Fewer than 5% of patients benefit from the only intervention approved to treat stroke. Thus, there is an enormous need to identify new therapeutic targets. The role of inducible cyclooxygenase (COX-2) activity in stroke and other neurologic diseases is complex, as both activation and sustained inhibition can engender cerebral injury. Whether COX-2 induces cerebroprotective or injurious effects is probably dependent on which downstream prostaglandin receptors are activated. Here, we investigated the function of the PGE2 receptor EP4 in a mouse model of cerebral ischemia. Systemic administration of a selective EP4 agonist after ischemia reduced infarct volume and ameliorated long-term behavioral deficits. Expression of EP4 was robust in neurons and markedly induced in endothelial cells after ischemia-reperfusion, suggesting that neuronal and/or endothelial EP4 signaling imparts cerebroprotection. Conditional genetic inactivation of neuronal EP4 worsened stroke outcome, consistent with an endogenous protective role of neuronal EP4 signaling in vivo. However, endothelial deletion of EP4 also worsened stroke injury and decreased cerebral reperfusion. Systemic administration of an EP4 agonist increased levels of activated eNOS in cerebral microvessels, an effect that was abolished with conditional deletion of endothelial EP4. Thus, our data support the concept of targeting protective prostaglandin receptors therapeutically after stroke.     

Cardiovascular disease is associated with high-fat-diet-induced liver damage and up-regulation of the hepatic expression of hypoxia-inducible factor 1α in a rat model

CVD (cardiovascular disease) is associated with abnormal liver enzymes, and NAFLD (non-alcoholic fatty liver disease) is independently associated with cardiovascular risk. To gain insights into the molecular events underlying the association between liver enzymes and CVD, we developed an HFD (high-fat diet)-induced NAFLD in the SHR (spontaneously hypertensive rat) and its control WKY (Wistar-Kyoto) rat strain. We hypothesized that hepatic induction of Hif1a (hypoxia-inducible factor 1α) might be the link between CVD and liver injury. Male SHRs (n=13) and WKY rats (n=14) at 16?weeks of age were divided into two experimental groups: standard chow diet and HFD (10?weeks). HFD-fed rats, irrespective of the strain, developed NAFLD; however, only HFD-SHRs had focus of lobular inflammation and high levels of hepatic TNFα (tumour necrosis factor α). SHRs had significantly higher liver weight and ALT (alanine aminotransferase) levels, irrespective of NAFLD. Liver abundance of Hif1a mRNA and Hif1α protein were overexpressed in SHRs (P<0.04) and were significantly correlated with ALT levels (R=0.50, P<0.006). This effect was not reverted by a direct acting splanchnic vasodilator (hydralazine). Angiogenesis may be induced by the HFD, but the disease model showed significantly higher hepatic Vegf (vascular endothelial growth factor) levels (P<0.025) even in absence of dietary insult. Hif1a mRNA overexpression was not observed in other tissues. Liver mRNA of Nr1d1 (nuclear receptor subfamily 1, group D, member 1; P<0.04), Ppara [Ppar (peroxisome-proliferatoractivated receptor) α; P<0.05], Pparg (Pparγ; P<0.001) and Sirt1 (Sirtuin 1; P<0.001) were significantly upregulated in SHRs, irrespective of NAFLD. Sirt1 and Hif1a mRNAs were significantly correlated (R=0.71, P<0.00002). In conclusion, CVD is associated with Hif1a-related liver damage, hepatomegaly and reprogramming of liver metabolism, probably to compensate metabolic demands.     

Soluble TNFα receptor type I and hepcidin as determinants of development of anemia in the long-term follow-up of heart failure patients

BackgroundAnemia is common in patients with chronic heart failure (CHF) and is associated with a worse prognosis. This study aims to identify the biological mechanisms which reflect evolutionary changes in the hemoglobin concentrations in heart failure patients who are still not anaemic.MethodsFifty-nine patients (54 ± 14 years, 83% males) with CHF (LVEF 28 ± 10%), who did not have anemia, and had not received any previous transfusions, were included. The parameters studied were: iron metabolism (ferritin, iron, transferrin, soluble transferrin receptor (sTfR), hepcidin); inflammation (C-reactive protein, soluble TNFα receptor I (sTNFRI), interleukin 6); and myocardial stress (NT-proBNP, high sensitivity TnT, growth differentiation factor 15). All parameters were measured on inclusion and 1 year after inclusion.ResultsBaseline hemoglobin (g/dL) was 14.7 ± 1.5 and at 1 year of follow-up it showed a significant decrease of ? 0.4 (RIC: ? 0.7 to ? 0.06) (p = 0.02). At baseline, only the sTNFRI was a predictor of a decrease in hemoglobin 1 year later (p = 0.007). During follow-up, the increase in sTNFRI (p = 0.002, r = ? 0.39) and hepcidin (p = 0.006, r = ? 0.35) were both associated with a decrease in hemoglobin. Similarly, the patients who became anemic (13%) had higher levels of hepcidin (p = 0.001) and sTNFRI (p = 0.008). The remaining parameters did not show any relationship with the evolution in the hemoglobin.ConclusionsIn CHF patients without anemia, the increase in the inflammatory state (sTNFRI) and the following deterioration in the iron metabolism (hepcidin) were the main determinants of a decrease in hemoglobin and the appearance of anemia in the long term follow-up period.     

Neuroprotective effects of 17β-estradiol after hypovolemic cardiac arrest in immature piglets: the role of nitric oxide and peroxidation

We recently reported that cerebral and cardiac injuries are mitigated in immature female piglets after severe hemorrhage with subsequent cardiac arrest. Female sex was also associated with a smaller increase in the cerebral expression of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). In the current study, we tested the hypothesis that exogenously administered 17β-estradiol (E?) can improve neurological outcome by NOS modulation. Thirty-nine sexually immature piglets were bled to a mean arterial pressure of 35 mmHg over 15 min. Fifty micrograms per kilogram of E? was then administered to 10 male and 10 female animals (estradiol group), whereas control animals (n = 10 males and 9 females) received equal volume of normal saline. The animals were then subjected to ventricular fibrillation (4 min) followed by up to 15 min of open-chest cardiopulmonary resuscitation. Vasopressin 0.4 U · kg?1 and amiodarone 0.5 mg · kg?1 were given, and 3 mL · kg?1 of 7.5% saline with 6% dextran was administered over 20 min. All surviving animals were killed after 3 h, and their brains examined for histological injury and NOS expression. No significant differences were observed in survival or hemodynamics between the groups. Compared with the control group, animals in the E? group exhibited a significantly smaller increase in nNOS and iNOS expression, a smaller blood-brain-barrier disruption, and a mitigated neuronal injury. There was a significant correlation between nNOS and iNOS levels and neuronal injury. Interestingly, estradiol attenuated cerebral damage (including lower activation of nNOS and iNOS) both in male and female piglets. In conclusion, in our immature piglet model of hypovolemic cardiac arrest, E? downregulates iNOS and nNOS expression and results in decreased blood-brain-barrier permeability disruption and smaller neuronal injury.     

IFN-λ receptor 1 expression is induced in chronic hepatitis C and correlates with the IFN-λ3 genotype and with nonresponsiveness to IFN-α therapies

The molecular mechanisms that link IFN-λ3 genotypes to differential induction of interferon (IFN)-stimulated genes (ISGs) in the liver of patients with chronic hepatitis C (CHC) are not known. We measured the expression of IFN-λ and of the specific IFN-λ receptor chain (IFN-λR1) in 122 liver biopsies of patients with CHC and 53 control samples. The IFN-λ3 genotype was not associated with differential expression of IFN-λ, but rather IFN-λR1. In a series of 30 primary human hepatocyte (PHH) samples, IFN-λR1 expression was low but could be induced with IFN-α. IFN-α–induced IFN-λR1 expression was significantly stronger in PHHs carrying the minor IFN-λ3 allele. The analysis of liver biopsies of patients with CHC revealed a strong association of high IFN-λR1 expression with elevated ISG expression, with IFN-λ3 minor alleles, and with nonresponse to pegylated IFN-α and ribavirin. The findings provide a missing link between the IFN-λ3 genotype and the associated phenotype of treatment nonresponse.Hepatitis C virus (HCV) infects an estimated 130–170 million persons worldwide. Chronic hepatitis C (CHC) can lead to cirrhosis and hepatocellular carcinoma (Lauer and Walker, 2001). The treatment for CHC consists of pegylated IFN (peg-IFN)-α and ribavirin, complemented with the HCV protease inhibitors telaprevir or boceprevir for HCV genotype 1 (Ghany et al., 2011). A substantial proportion of patients do not respond to this treatment. The virological response to peg-IFN-α depends on the degree of activation of the endogenous IFN system in the liver (Chen et al., 2005; Asselah et al., 2008; Sarasin-Filipowicz et al., 2008; Dill et al., 2011). Paradoxically, patients with a strong host response to HCV infection whose immune system reacts with a sustained induction of hundreds of IFN-stimulated genes (ISGs) in the liver, in the following designated preactivated patients, are poor responders to peg-IFN-α treatments. Nonresponse to peg-IFN-α in preactivated patients is most likely the consequence of the permanent up-regulation of USP18, an inhibitor of IFN-α signaling through the Jak–STAT pathways (Malakhova et al., 2006; Sarasin-Filipowicz et al., 2009; Dill et al., 2012).The molecular mechanisms responsible for sustained ISG induction in the liver are unknown. The presence of a T cell infiltrate in the liver of CHC patients suggests that IFN-γ could stimulate ISG induction. However, several groups have reported that the CD8⁺ T cell response is dysfunctional in regard to IFN-γ secretion (Thimme et al., 2012). Indeed, gene set enrichment analysis revealed a significant enrichment of IFN-α–induced genes in liver biopsies of preactivated patients with CHC, contrary to the late phase of acute hepatitis C that is characterized by a strong enrichment of IFN-γ–induced ISGs (Dill et al., 2012). However, IFN-αs are probably not the drivers of ISG induction because IFN-α signaling is subject to a strong negative feedback inhibition by USP18 that would prevent long-lasting activation of ISGs (Malakhova et al., 2006; Sarasin-Filipowicz et al., 2009). Interestingly, IFN-β signaling is not affected by USP18 to the same extent as IFN-α, despite the fact that both IFN-α and IFN-β belong to the type I IFN family and bind to and signal through the same receptor, IFNAR (Makowska et al., 2011). IFN-β produced by HCV-infected hepatocytes therefore remains a serious contender for being the driver of ISG expression in CHC. However, neither type I nor type II IFNs have been consistently detected in liver biopsies of preactivated patients with CHC.The discovery, in 2009, that allelic variants near the IFN-λ3 (IL28B) gene are strongly associated with spontaneous clearance of HCV and with response to treatment with peg-IFN-α and ribavirin provided compelling evidence that the type III IFN system is a key component of the host reaction to the virus (Ge et al., 2009; Suppiah et al., 2009; Tanaka et al., 2009; Thomas et al., 2009; Rauch et al., 2010). The molecular mechanisms that link the allelic variants of the IFN-λ3 gene locus to nonresponse to peg-IFN-α and ribavirin are not fully understood, but the significant association of the minor alleles of the IFN-λ3 polymorphism with the induction of ISGs in the liver demonstrates that the IFN-λ system must be critically involved in regulating the innate immune response to HCV (Honda et al., 2010; Urban et al., 2010). More recently, a novel TT/-G polymorphism near the IFN-λ3 gene has been described that is even more strongly associated with spontaneous clearance and treatment response to peg-IFN-α and ribavirin in CHC (Bibert et al., 2013; Prokunina-Olsson et al., 2013). The frameshift variant -G creates a novel 179-aa open reading frame that was designated IFN-λ4 on the basis of its protein sequence homology with type III IFNs (Prokunina-Olsson et al., 2013). Recombinant IFN-λ4 signals through the IFN-λ receptor and has antiviral activity against HCV and coronaviruses in cell culture (Hamming et al., 2013). It is presently not known whether IFN-λ4 is actually produced and functionally important in the HCV-infected human liver. Interestingly, the newly discovered TT/-G polymorphism impacts IFN-λ production in PBMCs. Treatment of PBMCs from patients with at least one -G allelic variant with poly(I:C) resulted in significant lower induction of IFN-λ3 compared with PBMCs from patients homozygous for the major TT allele (Bibert et al., 2013). How lower amounts of IFN-λ3 can be linked to increased expression of ISGs in the liver or how IFN-λ4 could cause a long-lasting ISG induction is presently unknown, but these exciting new findings further point to an important role of the IFN-λ gene locus in the control of the host response to HCV. We therefore analyzed the expression of IFN-λs and their receptor IFN-λR1 (IL28Rα) in liver biopsies and primary human hepatocytes (PHHs) from patients with different IFN-λ3 genotypes.We found a very low expression of IFN-λR1 in PHHs and in liver biopsies from patients who were not infected with HCV. In PHHs, IFN-λR1 expression was induced by IFN-α, leading to substantially improved responses of the Jak–STAT signaling pathway to IFN-λ. The magnitude of IFN-α–induced IFN-λR1 expression was significantly associated with the IFN-λ3 genotype. IFN-λR1 expression was significantly higher in liver biopsies from patients with CHC compared with uninfected controls. Within the group of patients with CHC, IFN-λR1 expression significantly correlated with the IFN-λ3 genotype. We conclude that the IFN-λ3 genotype affects hepatic ISG expression not through differential expression of IFN-λ3, but through differential expression of its receptor IFN-λR1. Moreover, ex vivo treatment of liver biopsies from patients with high-level expression of IFN-λR1 showed strong responses to IFN-λ and poor responses to IFN-α, whereas the contrary was found in patients with low-level expression of IFN-λR1. These findings provide a rationale for treating peg-IFN-α nonresponder patients with peg-IFN-λ.     

Tamoxifen regulation of bone growth and endocrine function in the ovariectomized rat: discrimination of responses involving estrogen receptor α/estrogen receptor β, G protein-coupled estrogen receptor, or estrogen-related receptor γ using fulvestrant (ICI 182780)

Tamoxifen is a selective estrogen receptor (ER) modulator, but it is also a deactivating ligand for estrogen-related receptor-γ (ERRγ) and a full agonist for the G protein-coupled estrogen receptor (GPER). Fulvestrant is a selective ER down-regulator that lacks agonist effects on ERα/ERβ, is inactive on ERRγ, but acts as a full agonist on GPER. Fulvestrant effects on tamoxifen actions on uterine and somatic growth, bone, the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis, and pituitary prolactin were analyzed to pharmacologically discriminate tamoxifen effects that may be mediated by ERα/ERβ versus ERRγ versus GPER. Ovariectomized rats received tamoxifen (0.6 mg/kg/daily) plus fulvestrant at 0, 3, 6, or 12 mg/kg/daily for 5 weeks; controls received vehicle or 6 mg/kg fulvestrant daily. Tamoxifen effects to increase uterine weight, decrease serum IGF-I, increase pituitary prolactin, and increase bone mineral density could be fully blocked by fulvestrant, indicating mediation by ERα/ERβ. Tamoxifen effects to decrease pituitary GH, tibia length, and body weight were only partially blocked by fulvestrant, indicating involvement of mechanisms unrelated to ERα/ERβ. Fulvestrant did not inhibit tamoxifen actions to reduce total pituitary protein, again indicating effects not mediated by ERα/ERβ. Tamoxifen actions to reduce serum GH were mimicked rather than inhibited by fulvestrant, pharmacological features consistent with GPER involvement. However, fulvestrant alone increased IGF-I and also blocked tamoxifen-evoked IGF-I decreases; thus fulvestrant effects on serum GH might reflect increased IGF-I feedback inhibition. Fulvestrant alone had no effect on the other parameters. The findings indicate that mechanisms unrelated to ERα/ERβ contribute to tamoxifen effects on body weight, bone growth, and pituitary function.     

Effects of thymosin β4 and its N-terminal fragment Ac-SDKP on TGF-β-treated human lung fibroblasts and in the mouse model of bleomycin-induced lung fibrosis

Thymosin β4 (Tβ4) and its amino-terminal fragment comprising N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) have been reported to act as anti-inflammatory and anti-fibrotic agents in vitro and in vivo. In recent papers, we have shown that Tβ4 exerts a widely protective role in mice treated with bleomycin, and in particular, we have demonstrated its inhibitory effects on both inflammation and early fibrosis.

Objectives: In this study, the putative anti-proliferative and anti-fibrogenic effects of Tβ4 and Ac-SDKP were evaluated in vitro. In addition, the effects of Tβ4 up to 21 days were evaluated in the bleomycin mouse model of lung fibrosis.

Methods: We utilized both control and TGF-β-stimulated primary human lung fibroblasts isolated from both idiopathic pulmonary fibrosis (IPF) and control tissues. The in vivo effects of Tβ4 were assessed in CD1 mice treated with bleomycin.

Results: In the in vitro experiments, we observed significant anti-proliferative effects of Ac-SDKP in IPF fibroblasts. In those cells, Ac-SDKP significantly inhibited TGF-β-induced α-SMA and collagen expression, hallmarks of fibroblast differentiation into myofibroblasts triggered by TGF-β. In vivo, despite its previously described protective role in mice treated with bleomycin at 7 days, Tβ4 failed to prevent fibrosis induced by the drug at 14 and 21 days.

Conclusion: We conclude that, compared to Tβ4, Ac-SDKP may have greater potential as an anti-fibrotic agent in the lung. Further in vivo experiments are warranted.     

Efficacy and safety of the α4β2 neuronal nicotinic receptor agonist ABT-894 in patients with diabetic peripheral neuropathic pain

Preclinical and clinical studies suggest that neuronal nicotinic receptor (NNR) agonists may be a novel and effective therapy for numerous painful conditions. Analgesic efficacy and safety of the highly selective α(4)β(2) NNR agonist ABT-894 was evaluated in 2 separate randomized, double-blind, multicenter, placebo-controlled clinical trials in patients with diabetic peripheral neuropathic pain (DPNP). Study 1 (280 patients randomized) tested 1, 2, and 4 mg ABT-894 twice daily compared with placebo and 60 mg duloxetine once per day over 8 weeks of treatment. Study 2 (124 patients randomized) tested 6 mg ABT-894 twice daily vs placebo for 8 weeks. The primary efficacy outcome measure in both studies was the weekly mean of the 24-hour average pain score recorded in each patient's diary. In both trials, none of the ABT-894 dose groups showed efficacy compared with placebo, whereas duloxetine achieved a statistically significant improvement over placebo in Study 1. All dose levels of ABT-894 were well tolerated, and no significant safety issues were identified. These results are in contrast to the outcome of a previously reported study of DPNP using the less selective α(4)β(2) NNR agonist ABT-594, which demonstrated efficacy compared with placebo, albeit with significant tolerability limitations. The failure of the highly selective α(4)β(2) NNR agonist ABT-894 indicates that it may not be possible to define a therapeutic index for this mechanism or that selectively targeting α(4)β(2) NNRs may not be a viable approach to treating neuropathic pain.     

Differential sensitivity to LPS-induced myocardial dysfunction in the isolated brown Norway and Dahl S rat hearts: roles of mitochondrial function, NF-κB activation, and TNF-α production

Recently, we reported that Brown Norway (BN) rats were more resistant to lipopolysaccharide (LPS)-induced myocardial dysfunction than Dahl S (SS) rats. This differential sensitivity was exemplified by reduced production of proinflammatory cytokines and diminished nuclear factor-κB pathway activation. To further clarify the mechanisms of different susceptibility of these two strains to endotoxin, this study was designed to examine the alterations of cardiac and mitochondrial bioenergetics, proinflammatory cytokines, and signaling pathways after hearts were isolated and exposed to LPS ex vivo. Isolated BN and SS hearts were perfused with LPS (4 μg/mL) for 30 min in the Langendorff preparation. Lipopolysaccharide depressed cardiac function as evident by reduced left ventricular developed pressure and decreased peak rate of contraction and relaxation in SS hearts but not in BN hearts. These findings are consistent with our previous in-vivo data. Under complex I substrates, a higher oxygen consumption and hydrogen peroxide (H2O2) production were observed in mitochondria from SS hearts than those from BN hearts. Lipopolysaccharide significantly increased H2O2 levels in both SS and BN heart mitochondria; however, the increase in oxygen consumption and H2O2 production in BN heart mitochondria was much lower than that in SS heart mitochondria. In addition, LPS significantly decreased complex I activity in SS hearts but not in BN hearts. Furthermore, LPS induced higher levels of tumor necrosis factor-α and increased phosphorylation of IκκB and p65 more in SS hearts than in BN hearts. Our results clearly demonstrate that less mitochondrial dysfunction combined with a reduced production of tumor necrosis factor-α and diminished activation of nuclear factor-κB are involved in the mechanisms by which isolated BN hearts were more resistant to LPS-induced myocardial dysfunction.