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+Current address: Endocrinology and Diabetes, Institute of Human Development, AV Hill Building University of Manchester, Manchester, M13 9PT, UK.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Pre-eclampsia is a pregnancy-specific disorder characterised by hypertension and proteinuria, which in severe cases results in multi-system disturbances. The maternal syndrome is associated with a pro-inflammatory state, consisting of leukocyte activation, which is thought to contribute to the widespread endothelial dysfunction. We previously showed increased activation of NADPH oxidase in pre-eclampsia, in both neutrophils and B-lymphoblast cell lines (B-LCLs). In this study, the mechanism by which NADPH oxidase activity is increased in pre-eclampsia was further investigated. NADPH oxidase activity was found to be increased in phorbol-12-myristate-13-acetate (PMA) stimulated B-LCLs isolated from women with pre-eclampsia. This correlated with an increase in protein kinase C (PKC) substrate phosphorylation, p47-phox phosphorylation (a regulatory component of NADPH oxidase) and p47-phox directed-kinase activity. Using ion exchange and hydroxyapatite chromatography we identified a major peak of PMA regulated p47-phox kinase activity. Chromatography fractions were probed for PKC isoforms. We found the major peak of p47-phox kinase activity could not be separated from the elution profile of PKC epsilon. Using a peptide inhibitor of PKC epsilon, PMA-induced reactive oxygen species (ROS) production could be reduced to that of a normal B-LCL. These data suggest a pro-inflammatory role for PKC epsilon in the pathogenesis of pre-eclampsia.Pre-eclampsia is a pregnancy-specific disorder and a leading cause of maternal morbidity and perinatal death [1]. It is associated with hypertension and proteinuria; however, its origins are early in gestation, where poor implantation and remodelling of the spiral arteries leads to placental underperfusion and hypoxia. This results in the release of placental factors, of which there are many, e.g., soluble vascular endothelial growth factor (VEGF) receptor (sflt) [2]. The maternal syndrome results from a widespread endothelial dysfunction, possibly as a direct consequence of an excessive maternal inflammatory response [3]. The activation of leukocytes, such as neutrophils, is thought to contribute to this inflammatory process through the release of toxic substances, such as reactive oxygen species (ROS) and other microvascular disturbances [4,5].To provide an appropriate response to a specific stimulus, leukocytes may undergo a priming process that enhances ROS production to agonists, such as tumour necrosis factor (TNF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) [6]. This process is largely controlled by kinases, including phosphatidylinositol-3-kinase (PI3K) and p38MAPK. These kinases have been shown to induce the phosphorylation of the regulatory components of NADPH oxidase, leading to an enhanced oxidative burst [7].NADPH oxidase consists of a cytosolic trimer of p40-phox, p47-phox and p67-phox, becoming phosphorylated upon activation and then translocating to the cell membrane. A fourth cytosolic subunit, the small guanine binding protein, cytosolic Rac, is found and translocates independently. The membrane component consists of p22-phox and gp91-phox, which bind the cytosolic components upon activation. However, the p47-phox subunit is the most widely accepted regulatory point. This protein is heavily phosphorylated in its C-terminal quarter by numerous kinases, such as PKC [8] and Akt [9].We previously showed in a pre-eclampsia lymphoblast model ROS production was increased in response to phorbol-12-myristate-13-acetate (PMA) [10]. In the current study we show that P47-phox phosphorylation was also increased on PKC phosphorylation sites, correlating with increased ROS production in the pre-eclamptic B-lymphoblast cell lines (B-LCLs). P47-kinase activity was also increased, therefore we determined the kinase responsible for this process. The novel PKC isoform, PKCε, was found to be responsible for this increase and hence highlights an important role for this kinase in the pathogenesis of pre-eclampsia.Acrylamide (Flowgen Bioscience Ltd., Nottingham, UK), protein G sepharose, β-glycerophosphate, and RO318330 (Merck Biosciences; Nottingham, UK), anti-phospho PKC substrate antibody (Cell Signalling Technologies; Inc., MA, USA), mouse anti-p47-phox antibody and anti-PKC antibodies (BD Biosciences; Belgium), Resource S, Q and heparin columns, horse radish peroxidase (HRP)-coupled rabbit secondary antibody, Hybond-C nitrocellulose membrane and ECL kit (Amersham Biosciences; Little Chalfont, UK), methylacridinium ester (MAE; 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methylacridinium 9-carboxylate fluorosulphonate) (Molecular Light Technology; Cardiff, UK). Microlite 2 microtitre plates were supplied by Dynex Technologies Inc. and hydroxyapatite columns were from Bio-Rad Laboratories; Hemel Hempstead, UK. All other materials were obtained from Sigma (Poole, UK).B-lymphoblast cell lines (B-LCLs) (Epstein-Barr virus transformed), isolated and cultured from third-trimester and postpartum pre-eclamptic and normal women, were characterised in a previous study [10]. This study group consisted of 20 cell lines from either normal (NT) or pre-eclamptic (PET) (10 third trimester and postpartum) women.Luminol chemiluminescence (CL) was performed as previously described [11]. ROS production was measured from 2 × 105 cells, stimulated by the addition of 0.001−1 μmol/L PMA. The total oxidative capacity was determined from the area under the curve (AUC) and expressed as relative light units (RLU min−1). NADPH oxidase activity in cell lysates was measured as previously described [12]. Protein concentration was determined by the fluorescamine assay [13]. ROS production was measured from 50 μg of total lysate in a reaction buffer containing 10 μmol/L lucigenin. ROS production was stimulated with NADPH, GTPγS and SDS. CL was measured continuously for 40 min. NADPH oxidase activity was determined from the AUC and expressed as RLU min−1 50 µg protein content−1 [12].B lymphoblasts (3 × 106/mL) were stimulated with PMA (final concentration 1 μM) for the indicated time and lysed on ice by the addition of a triton lysis buffer [11]. Cells were then fractionated by previously described methods [14]. 35 μg of protein was analysed using 10% SDS-polyacrylamide gels and immunoblotting [11] with an anti-phospho PKC substrate, anti-PKCα, anti-PKCβ, anti-PKCδ, anti-PKCε and pan-PKC antibodies. Blots were visualised using a HRP-coupled secondary antibody and an ECL detection system. Hyperfilms were quantified by densitometry using a BioRad densitometer. To test for equal loading, all blots were stripped and probed with anti-tubulin or rabbit anti-p47-phox antibodies. For immunoprecipitation, 500 μg of total cell protein was incubated overnight with 0.5 μg of a mouse anti-p47-phox or PKCε antibody as described in [11].P47-phox and p67-phox were expressed in sf9 cells and purified as previously described using a baculovirus expression system (constructs were a kind gift from JD Lambeth, Emory University, Atlanta, Georgia, GA, USA) [15]. Microlite 2 microtitre plates were coated with p47-phox (0.5 μg per well) in phosphate buffered saline (PBS) overnight. After washing in PBS, the plates were blocked with PBS supplemented with 1% (w/v) bovine serum albumin (BSA) for 1 h. Before use, the plates were washed with PBS. Whole cell lysates were prepared from B lymphoblasts as described above. From each cell lysate a 5 μg/mL solution (diluted in a HEPES-Tween solution (25 mmol/L HEPES, pH 7.3, with 0.01% (v/v) Tween-20) was prepared. 6 μL of this sample was added to each well and the volume made up to 80 μL with HEPES-Tween solution. The plates were then warmed to 37 °C for 10 min and in some experiments either Ro31-8220 (1 μmol/L) or mPKC peptide inhibitor (50 μmol/L) was also included. PMA (1 μmol/L), diacylglycerol analogue (DAG analogue) (OC-DAG (1,2-Dioctanoyl-sn-glycerol)) (200 μmol/L), phosphatidylserine (PS) (0.1 mg/mL) and calcium (1 mmol/L) were also included (modified from [16]). Reactions were initiated by the addition of 20 μL of a pre-warmed kinase buffer (final concentration: 20 mmol/L Tris, pH 7.5, 25 mmol/L MgCl2, 10 mmol/L β-glycerophosphate, 0.5 mmol/L sodium orthovanadate, 0.5 mmol/L dithiothreitol (DTT) and 1 mmol/L adenosine triphosphate (ATP) and incubated for 30 min at 37 °C. The reaction was terminated by washing the plate in a modified PBS-tween [17]. Phosphorylated p47-phox was detected using an anti-phospho PKC substrate antibody. The anti-phospho PKC substrate antibody is thought to preferentially bind phosphorylated serine residues with an arginine or lysine residue in the −2 and +2 positions and with a hydrophobic residue at the +1 position [18]. Bound antibody was detected using MAE-labelled streptavidin. The CL-signal (expressed as RLU) was detected using a Dynex MLX luminometer [17] Radioactive kinase assays were carried in similar buffer conditions as described above using a [32P] ATP (25 μM ATP final concentration).Cytosol was prepared as previously described [14] and applied to a Resource Q cation-exchange column and washed with 5 column volumes of 5 mmol/L Tris (pH 7.4). The column was eluted with a linear gradient of 0–1 mol/L NaCl. The majority of p47-phox protein kinase activity eluted at 0.2–0.5 mol/L NaCl. Fractions containing PMA-regulated p47-phox kinase activity were subjected to hydroxyapatite chromatography.1 × 107 cells were equilibrated at a reduced temperature by two sequential 2-minute incubations, each with 1 mL PBS. The first PBS incubation is carried out at room temperature; the second, with chilled PBS (4 °C). 1.3 mL of a permeabilization buffer (20 mmol/L HEPES, pH 7.4, 10 mmol/L EGTA, 140 mmol/L KCl) was then added. 200 μL aliquots were incubated with the PKCε Translocation Inhibitor Peptide (EAVSLKPT) or a peptide negative control (LSETKPAV) (1 mmol/L). 200 µL of Saponin-permeabilization buffer (20 mmol/L HEPES, pH 7.4, 10 mmol/L EGTA, 140 mmol/L KCl, 12 mmol/L ATP and 100 µg/mL saponin were then added. Cells were further incubated for 10-min on ice and washed four times with 1 mL of chilled PBS. Cells were incubated on ice for 20 min (recovery period), the chilled PBS was removed, 1 mL of room temperature PBS was added and the cells were placed at room temperature for 2 minutes, after which complete cell media was added back to the cells at 37 °C. The cells were further incubated for 30 min at 37 °C before use. The cells were stimulated with 10 nmol/L PMA and ROS production measured as described above.All data are presented as mean ± SEM and values were compared using Minitab software (Pennsylvania, PA, USA) with either a Mann-Whitney test or one-way ANOVA, followed by Tukey’s post hoc test. Statistical significance was declared at the p < 0.05 level. Areas under the curves (AUC) were calculated using the trapezoidal rule. For Western blotting analysis, all data were normalized to the control group.We previously showed that PMA-stimulated ROS production was increased in B-lymphoblast cell lines (B-LCLs) isolated from third trimester and postpartum pre-eclamptic women [10]. The 10 normal and pre-eclamptic B-LCLs (5 third trimester and 5 postpartum) used in this study were all stable cultures displaying increased ROS production in response to PMA, as measured by luminol-CL (Figure 1A,B). ROS production could not be detected in unstimulated cells (not shown). There were no significant differences between pregnancy status and ROS production in either group (p > 0.05).NADPH oxidase activity could also be detected in cell homogenates from B-LCLs using lucigenin-CL (which measures superoxide [O2−] production). O2− production could be measured in cell lysates stimulated with NADPH (with and without SDS and GTPγS). There were no significant differences between normal and pre-eclamptic B-LCLs when NADPH oxidase activity was stimulated in this fashion (Figure 1C).Pre-eclamptic B-lymphoblast cell lines (B-LCLs) display enhanced reactive oxygen species (ROS) production in response to phorbol-12-myristate-13-acetate (PMA). (A) Whole cell ROS production was measured from B-LCLs stimulated with 0.01 μmol/L or 1 μmol/L PMA added at time = 0 min and measured by luminol-CL (shown as a representative chemiluminescent (CL) recording). (B) The area under the curve (AUC) was calculated for each CL-recording. Each column represents the mean ± SEM for combined results from 10 normal (NT) and 10 pre-eclamptic (PET) B-LCLs stimulated with either PMA dose. (C) Cell-free activation in normal and pre-eclamptic B-LCL, cell lysates were stimulated with a combination of NADPH or NADPH with SDS and GTPγS, n = 6 for each cell type. Statistical significance determined using a Mann-Whitney test.PMA stimulation in normal and pre-eclamptic B-LCLs resulted in the rapid phosphorylation of cellular PKC substrates, as detected using an anti-phospho PKC substrate antibody. Both pre-eclamptic and normal cell lines showed increased phosphorylation on numerous proteins in response to PMA. However, the 60 and 50 kDa regions were the most consistently phosphorylated and easy to identify (Figure 2A). Analysis of the optical density in the 50 kDa region was found to be increased in pre-eclamptic B-LCLs (mean ± SEM; normal 1.7 ± 1.0 vs. pre-eclamptic 6.3 ± 4.3 AU, p = 0.0028) (Figure 2B). The phosphorylation of the p50 region in response to PMA could be inhibited by pre-treating the cells with 1 μmol/L Ro31-8220 (an inhibitor of protein kinase C [PKC] (Figure 2C and Figure S1). The protein in the 60 kDa region was less affected by Ro31-8220. The sensitivity towards Ro31-8220 did not differ between the two disease states, inhibited by 93.5 ± 6.3% and 93.2 ± 3.5% for normal and pre-eclamptic B-LCLs (mean ± SEM, n = 3) (Figure 2D).P47-phox immunoprecipitates were prepared from PMA stimulated normal and pre-eclamptic B-LCLs. Immunoblotting with an anti-phospho PKC substrate antibody revealed that p47-phox phosphorylation was increased in pre-eclamptic B-LCLs (mean ± SEM; normal 0.8 ± 0.6 vs. pre-eclamptic 2.0 ± 0.9, p = 0.0058, n = 10) (Figure 3A,B and Figure S3). The basal level of p47-phox phosphorylation was found to be very low and did not differ between the two cell types (p > 0.05).PMA has been shown to stimulate ROS production in B-LCLs and is inhibited by the PKC inhibitor Ro31-8220 [10]. The expression of PKC isoforms were determined by western blotting. The expression of PKCα, PKCβ, PKCδ and PKCε could be measured in these B lymphoblasts, whereas PKCθ and PKCλ were found to be much less. The expression of PKC isoforms α, β, δ and ε (normal vs. pre-eclamptic; mean ± SEM) was found to be similar (PKCα, 3.6 ± 2.6 vs. 5.8 ± 3.8; PKCβ, 2.3 ± 0.7 vs. 2.1 ± 1.4; PKCδ, 3.6 ± 1.8 vs. 2.8 ± 1.1; and PKCε, 2.5 ± 1.3 vs. 2.7 ± 1.8 normalized densitometric arbitrary units). The protein expression of PKCλ and PKCθ isoforms was at a much lower level than the other PKC isoforms, with no statistical differences between the normal and pre-eclamptic groups (See Figure S2). PKC substrate phosphorylation is increased in pre-eclamptic B lymphoblasts. (A) Normal and pre-eclamptic B-LCLs were stimulated with 1 μmol/L PMA for 7 min. PKC substrate phosphorylation was determined by western blotting. Pre-eclamptic B-LCLs showed an increase in phosphorylation in a 50 kDa region (p = 0.0028) (lower tubulin immunoblots show equal loading). (B) Data obtained from immunoblot experiments were analysed using densitometry; each column represents the mean ± SEM from 10 normal and pre-eclamptic B-LCLs. Statistical significance was determined using a Mann-Whitney test (n = 10 for each state). (C) Normal and pre-eclamptic B-LCLs were pre-treated with 1 μmol/L Ro31-8220 for 15 min before stimulation with 1 μmol/L PMA. PKC substrate phosphorylation was determined by western blotting. Data obtained from immunoblots experiments were analysed by densitometry and the effect of Ro31-8220 on PKC phosphorylation of a 50 kDa protein was determined (shown as mean ± SEM, n = 3 for each state). Statistical significance from PMA stimulated control cells was determined using a one-way ANOVA (*, p > 0.05).To determine if p47-phox kinase activity was increased in pre-eclamptic B-LCLs a non-radioactive kinase assay was developed to measure p47-phox kinase activity in whole cell lysates. In resting cells a basal p47-phox kinase activity could be measured, which could be rapidly enhanced by the addition of PMA directly into the kinase assay. The basal p47-phox kinase activity was not found to be significantly different between the two cell types (normal vs. pre-eclamptic, mean ± SEM; 10.1 ± 4.9 vs. 14.1 ± 3.5 × 103 RLU, n = 10). Pre-eclamptic B-LCLs were found to have increased kinase activity in response to PMA, when PMA was added directly into the kinase assay (normal vs. pre-eclamptic, mean ± SEM; 24.8 ± 11 vs. 39.2 ± 12.8 × 103 RLU, p = 0.0128, n = 10) (Figure 4A). A DAG analogue was also able to induce p47-phox phosphorylation, albeit to a lesser extent to that found with PMA (Figure 4A). p67-phox kinase activity could not be detected in cell lysates using the phospho-PKC substrate antibody (not shown). The PMA-sensitive kinase was found in the cytosol of resting cells, whereas membrane fractions contained the most activity if cells were stimulated with PMA prior to cell fractionation (Figure 4B). A radioactive kinase assay was also used to confirm the presence of a PMA-sensitive kinase found in B-LCL cytosol (Figure 4C).p47-phox phosphorylation is increased in pre-eclamptic B-LCLs. (A) Normal and pre-eclamptic B-LCLs were stimulated for 7 minutes with 1 μmol/L PMA. p47-phox was immunoprecipitated from normal and pre-eclamptic B-LCLs (using a mouse anti-p47-phox antibody). Immunoblots were then probed for PKC substrate phosphorylation. The identity of p47-phox and equal loading was determined using a rabbit anti-p47-phox antibody. (B) Data obtained from immunoblot experiments were analysed by densitometry, each column represents the mean ± SEM for 10 normal and pre-eclamptic B-LCLs. Pre-eclamptic B-LCLs showed an increase in p47-phox phosphorylation (p = 0.0058). Statistical significance was determined using a Mann-Whitney test. Dashed line indicates separate blots (see Figure S3 for full image).p47-phox kinase activity is increased in pre-eclamptic B-LCLs. (A) Cell lysates were prepared from normal and pre-eclamptic B-LCLs. p47-phox kinase activity was determined using a non-radioactive kinase assay. Phosphorylated p47-phox was determined using a PKC substrate antibody. Kinase activities from normal and pre-eclamptic B-LCLs are expressed as RLU, with each column representing the mean kinase activity (±SEM). Pre-eclamptic B-LCLs display an increase in PMA-induced p47-phox kinase activity (p = 0.0128). Statistical significance was determined using a Mann-Whitney test (n = 10 for each state). The effect of a DAG analogue on p47-phox kinase activity was also tested (shown as mean RLU ± SEM, n = 3). (B) p47-phox kinase activity was determined in membrane and cytosol fractions from pre-eclamptic B lymphoblasts (stimulated with PMA before (cells pre-treated with PMA) and after fractionation (PMA added into the kinase assay)), each column represents the mean (±SEM) kinase activity for 3 pre-eclamptic B-LCLs. (C) PMA-induced kinase activity was determined in cytosol prepared from normal and pre-eclamptic B lymphoblasts and measured using a radioactive kinase assay (n = 3), kinase activity was determined using recombinant p47-phox and [32P] ATP. An autoradiogram for phosphorylated p47-phox is shown. (D) The effect of a mPKC peptide inhibitor (50 μmol/L) and phosphatidylserine (PS) 0.1 mg/ml on p47-phox activity was measured in whole cell lysates; each column represents the mean kinase activity (RLU ± SEM) for 3 pre-eclamptic B-LCLs. (E) The effect of Ro31-8220 (1 μmol/L) on PMA-induced p47-phox kinase activity measured in whole cell lysates; each column represents the mean kinase activity (RLU ± SEM) for 3 pre-eclamptic B-LCLs. Statistical significance from PMA stimulated control lysates was determined using a one-way ANOVA (*, p < 0.05).A peptide inhibitor of PKCα and β (mPKC inhibitor) did not inhibit PMA-induced p47-kinase activity (Figure 4D). If the PKC cofactor PS was added into the assay, kinase activity was not increased. Kinase activity was also found to be calcium independent (not shown) and all subsequent reactions were carried out in the presence of EGTA. PMA-induced p47-phox kinase activity was inhibited by the non-specific PKC inhibitor, Ro31-8220 (using 1 mmol/L ATP) (Figure 4E).The apparent increase in kinase activity in the pre-eclamptic cell lines was further investigated. Cytosol was applied to a Q-column and fractionated. Kinase activity and PKC levels in each fraction were then tested. The major peak of activity was strongly associated with the presence of PKCε. Using a pan-PKC antibody (for PKCα, βI, βII and γ) the remaining peak of activity could be determined (Figure 5A–C). The major peak of activity was further purified on a hydroxyapatite column, where the peak of activity was found to correlate with the presence of PKCε (Figure 5D–F).Identification of a major p47-phox kinase in pre-eclamptic B-LCLs. Cytosolic preparations were fractionated by ion exchange chromatography (using a Resource Q column as described in the material and methods), kinase activity (non-radioactive kinase assay) (A) and the expression profile of PKC isoforms (B and C) was determined for each fraction. Fractions 4 and 5 from the ion exchange column were subjected to fractionation using a hydroxyapatite column, with kinase activity (non-radioactive D and E using [32P] ATP) and the presence of PKCε (F) determined for each fraction.B-LCLs proved to be very difficult to transfect, making the use of gene knockdown methods such as siRNA a challenge. The most appropriate method to reduce the activation of PKCε in the cells was to use an inhibitor of PKCε translocation, previously shown to be effective and delivered in to the cell by a transient permeablization method (saponin). Using this peptide (and a scrambled control peptide), ROS production was found to be reduced (by greater than 50%) (Figure 6A,B). We were unable to measure NADPH oxidase activity in normal (NT) cells by this method, possibly due to the low levels of enzyme activity in this group. PKCε translocation was also found to be unchanged between the two cell types (Figure 6C). The basal level of PKCε association was also found not to be significantly different in the two cell types. The translocation inhibitor was found to effectively inhibit membrane translocation of PKCε (Figure 6D). ROS production in these cells was found to be inhibited by apocynin (a general inhibitor of NADPH oxidase) and as expected the PKC inhibitors Ro318220 and Go6796. Interestingly, there was a difference between the two PKC inhibitors (Figure 6E). Go6796 has been previously shown to be ineffective against PKCε [19].Pre-eclampsia is associated with leukocyte activation [3] which is thought to contribute towards the disease process through numerous mechanisms, including the activation of NADPH oxidase and subsequent ROS production. Oxidative stress and a pro-inflammatory state are features of the maternal syndrome. However, leukocyte activation and entrapment in the microcirculation may affect whole organ perfusion pressure, leading to organ damage [20,21]. Neutrophils isolated from women with pre-eclampsia have been found to be hyper-responsive to agonists, such as formyl-methionyl-leucyl-phenylalanine (fMLP) [4,22,23] and PMA [4]. B-LCLs isolated from women with pre-eclampsia also show this difference in response to PMA. Interestingly, this phenomenon was still evident 6 months after parturition. Our previous work has suggested that these differences were not due to any changes in the expression levels of the subunits of NADPH oxidase [10] and in this study we show data that implicates the activation of PKCε.The role of PKC in vascular disease has been well established. PKCε has been shown to play an important role in the pathogenesis of cardiovascular disease (reviewed in [24]). In particular, the progression of cardiac hypertrophy, failure [25], and fibrosis [26]. Interestingly, pre-eclampsia has been shown to be associated with multiple postpartum cardiovascular impairments, such as left ventricular dysfunction and hypertension [27]. However, to date the only association of PKCε in pre-eclampsia has been demonstrated with endothelial cells that treated with serum from pre-eclamptic women, showing an increase in membrane association of PKCε. Therefore, increased PKCε activation has the potential to contribute to the long term cardiovascular disturbances seen in pre-eclampsia.The effect of PKCε inhibition on ROS production in pre-eclamptic B-LCLs. (A) Pre-eclamptic B-LCLs were permeabilized with saponin and incubated with either a PKCε translocation inhibitor peptide (EAVSLKPT) or a scrambled negative control peptide (LSETKPAV). Whole cell ROS production was measured from B-LCLs stimulated with 0.01 μmol/L PMA added at time = 0 minutes and measured by luminol-CL (shown as a representative chemiluminescent (CL) recording). (B) The AUC was calculated for each CL-recording and represented as %PMA response (mean ± SEM) for combined results from 6 pre-eclamptic B-LCLs. Statistical significance was determined by a One-way ANOVA. (C) Membrane translocation of PKCε in normal and pre-eclamptic B-LCLs. (D) The effect of the PKCε translocation inhibitor peptide on PMA-induced translocation of PKCε. (E) Pre-eclamptic B-LCLs were treated with either apocynin, Ro318220 or Go6796 before being stimulated with 1 μmol/L PMA, here represented as %PMA response.Firstly, the increased responsiveness to PMA was not due to a detectable change in PKC isoform expression. However, neutrophils isolated from diabetic patients showed an increase in ROS production in response to PMA and which correlated with the increased expression of PKC [28]. Neutrophils isolated from patients with rheumatoid arthritis have been shown to be hyper-responsive to PMA [29] and phosphorylation on serine 345 mediates the priming effect [30]. Secondly, we found that phosphorylation of PKC substrates revealed that a protein in a region of 50 kDa was significantly increased. Moreover, p47-phox phosphorylation and kinase activity also was increased in PMA-stimulated B-LCLs isolated from pre-eclamptic subjects. These results suggested that the pre-eclamptic phenotype was associated with an increase in PKC activity or an associated kinase.Finally, we showed that kinase activity was increased in B-LCLs isolated from pre-eclamptic subjects. Cell fractionation experiments revealed a kinase located in the cytosol of B lymphoblasts, which could be directly stimulated by adding PMA into the kinase assay. Translocation of this kinase to membrane fractions was demonstrated by PMA stimulation of cells. The activity of this kinase was also found to be calcium-independent. Cytosolic fractions from pre-eclamptic B-LCLs were subjected to ion exchange and hydroxyapatite chromatography. This suggested that the major peak of kinase activity in these cells was associated with the presence of PKCε.The importance of p47-phox phosphorylation has been previously demonstrated in B-LCLs [31,32,33]. The major sites of phosphorylation in the NADPH oxidase complex are serine residues in the C-terminal quarter of p47-phox (9 serines between residues 303 and 379 [31]). We used an anti-phospho-(ser) PKC substrate antibody (detecting phosphorylated serines surround by a basic residue at 2−/2+ and a hydrophobic residue at +1) to detect enhanced PKC activity and p47-phox phosphorylation in cells from pre-eclamptic subjects. However, the basal level of p47-phox phosphorylation in B-LCLs isolated from pre-eclamptic or normal subjects was very low or on different phosphorylation sites to those detected by the antibody. This antibody theoretically detects phosphorylation on a subset of functionally important serine residues, serine 303, 304 and 328 of p47-phox [18], but may also detect other phosphorylated serine residues with a similar motif. Serines 303 and 304 are heavily phosphorylated after cell activation and are both required for oxidase activity, while serine 328 has been shown to be a major phosphorylation site for PKC isoforms [34]. Phosphorylation of p47-phox initiates the activation of NADPH oxidase, allowing the translocation of the cytosolic subunits to the cell membrane. Upon cell stimulation, cytosolic p47-phox is phosphorylated on serine 359 and/or 370. Following membrane translocation of partially phosphorylated p47-phox, serines 303 and/or 304 are then phosphorylated [33].Novel PKC isoforms have been previously shown to be critical in the activation of NADPH oxidase in monocytes [35]. However, the direct activation of NADPH oxidase by PKCε has not been extensively studied. PKCε has a similar substrate specificity to other PKC isoforms; however, it shows less specificity with only a strong requirement for a basic/charged residue at −3 and −2 positions from the target serine [36]. However, kinase substrate specificity can be influenced by many additional interactions in vitro.We used a peptide inhibitor of PKCε, which inhibits the membrane translocation of the enzyme rather than activity per se [37], its specificity has been widely demonstrated [38]. This inhibitor was able to reduce ROS production in the pre-eclamptic cells by 50%, which may account for the priming mechanism in the pre-eclamptic cells. ROS production was completely inhibited by Ro318220 and to a much lesser extent Go6796. Go6796 is not an effective inhibitor of PKCε [19]; however, caution should be taken when analysing data regarding the specificity of these compounds. PKCε activation has been implemented in multiple cardiovascular disorders [39,40], where interestingly its activation has also been associated with a protective role. The mechanism by which PKCε activity is increased in pre-eclamptic B-LCLs is unclear. The levels of PKCε and its membrane translocation did not reach significance in pre-eclamptic B-LCLs; however, this could contribute to the priming mechanism for increase ROS production. Similar mechanisms have been demonstrated in neutrophils [30]. Further investigation into the regulation of PKCε in B-LCLs is warranted. Human umbilical endothelial cells that are treated with serum from pre-eclamptic women showed that there was an increase in membrane association of PKCε [41], which further indicates a possible role for this kinase. PKCε is essential for VEGF-stimulated phosphorylation of Akt, eNOS and the catalytic activity of NO synthase in endothelial cells [42], factors that are also important in pre-eclampsia [5].These data indicate that PKCε is responsible for the enhanced NADPH oxidase activity associated with leukocytes in pre-eclampsia. Leukocyte activation and the subsequent ROS production (from NADPH oxidase) are thought to be a contributory factor in the underlying endothelial dysfunction associated with pre-eclampsia. Further investigations to elucidate the nature of PKCε may reveal future therapeutic targets that may limit leukocyte activation and the production of free radicals from leukocytes in pre-eclampsia.Supplementary materials can be accessed at: http://www.mdpi.com/2079-9721/1/1/1/s1.This work was supported by the British Heart Foundation. LN was supported by the Leicester NIHR Cardiovascular Biomedical Research Unit.The authors declare no conflict of interest. 

Med-MDPI/diseases/diseases-01-01-00018.txt

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The introduction of antiretroviral therapy (ART) in the management of HIV infection has resulted in a significant reduction in the morbidity and mortality associated with the disease. The fact that a nearly perfect adherence is required in ART has remained a major challenge to people infected with HIV. This review underscores the impact of adherence to antiretroviral therapy and highlights recent advances in adherence monitoring and enhancement among people infected with HIV who are on lifelong antiretroviral therapy.Medical knowledge around HIV/AIDS has increased significantly over the years and good progress has been made in the treatment of HIV as a manageable life-threatening chronic condition using antiretroviral therapy (ART). The treatment of the disease extends beyond knowledge development among people infected with HIV; a partnership between them and healthcare providers is required, with the HIV-infected person assuming the major responsibility of self-care that will result in adherence and a good clinical outcome [1]. This is the reason why medication adherence is described as the extent to which the individual’s behaviour corresponds to the prescribed medical advice of the health care provider [2]. This review provides an overview on adherence to ART among people infected with HIV by describing the pattern of adherence to treatment. It also includes the consequences of non-adherence to treatment, and the barriers and facilitators of ART adherence. Furthermore, the various modalities of adherence monitoring and assessment are described in detail, together with recent advances in strategies and tools used in enhancing adherence to antiretroviral therapy.Medication adherence is a major challenge in chronic medical condition. Adherence levels change over time. Clinical experience and research indicate that adherence is a “moving target”; the longer a patient stays on treatment the poorer the adherence is likely to become [3,4]. Generally, adherence rates are higher among patients who are taking medications for acute medical conditions compared to those with chronic medical conditions [5]. In addition to this, adherence levels among patients with chronic diseases, no matter how impressive adherence is initially, have been reported to drop dramatically after six months [6]. HIV-infected persons have been shown to adhere better than the general population; the average adherence to chronic medications among the general population is 50% which is far below the self-report adherence rate of between 55–77% among patients on ART [7]. Despite this, non-adherence is common among patients on ART, which is estimated to be between 30–50%, and more than 10% of patients usually miss one or more of their daily doses of antiretroviral drugs [8]. Adherence level among adolescents is usually lower than adults and elderly patients on ART [9,10]. In a study done in the USA among adolescents on antiretroviral drugs (ARVs), only 28% took all their prescribed ARVs in the previous month [10]. Another cohort study in nine countries within the Southern Africa region also reported poor adherence among adolescents [11]. A survey done among HIV patients in India also revealed poor adherence among people who were aged below 40 years [3]. The explanatory factors responsible for this difference may be due to the fact that older individuals are more likely to have prior experience taking medication for age-related diseases and may have already become accustomed to such. Another reason may be related to the fact that lifestyle adjustments necessary for successful adherence are often less burdensome for adults compared to young patients [3]. Numerous interventional studies have been done in the past to address the problem of non-adherence among patients [6,12,13]. The unfortunate thing is that most of these interventions seem to have only modest impact [12,13,14]. The lack of appropriate theories to explain and predict non-adherence among patients on chronic medications may have a role to play in the slow progress made in developing interventions aimed at enhancing ART adherence [6,15].The study of Paterson et al. [16] has been used widely in establishing the level required for optimal adherence necessary to maintain viral suppression (>95%). In the study, the authors reported virologic failure (HIV RNA>400 copies/mL) in 22% of patients with adherence level of 95% and above, 61% in patients with adherence level of 80–94.9% and 80% virologic failure in patients whose adherence level fell below 80% [16]. Any adherence below 95% in ART has been linked to treatment failure. The risk of developing resistance to ARV is also shown to be highest between 80–90% level of adherence [17]. This is because the virus is not likely to develop resistance at low concentrations of the drugs but at a high concentration which is suboptimal for suppression of viral replication and the chances of viral resistance are very high [17,18]. The earlier estimates of adherence levels necessary for viral suppression were done among patients receiving the unboosted protease inhibitors (PI) regimens which have been described as less potent than the non-nucleoside reverse transcriptase inhibitors (NNRTI) based-regimens [19,20]. Recent studies on adherence levels required to maintain good viral suppression with NNRTI-based regimens revealed that viral suppression was still achievable in patients with an adherence level below 80% [21,22]. A South African study showed that there was improvement in patients’ virologic outcomes as adherence to NNRTI-based regimens went beyond 50% [21]. In addition, 73% of the patients in the study achieved maximal viral suppression with adherence levels of 90–100% [21]. This study shows that the NNRTI-based regimens may be an appropriate alternative to the PI-based regimens where adherence between 70–94% is anticipated, although the authors warned that adherence should be enhanced in any patients on ART irrespective of the patterns of adherence among populations groups [21]. Although the methods used in assessing adherence in some of these studies may not allow for the results to be generalized to other settings, what remains unclear is whether these results would be sustainable in the longer term as a very high adherence level is required indefinitely to maintain a continuous good clinical outcome in ART [16,22]. Several studies in sub-Saharan Africa have reported low adherence among patients on ART. In a sample of 109 patients on ART in Botswana, only 54% of the patients were adherent by self-report, although this increased marginally to 56% when provider assessment was used [23]. In another study in Ethiopia conducted among 400 patients on ART [24], about 24% were non-adherent when combined indicators of dose, time and dietary instructions were used. The percentage of non-adherent patients increased to 27% after reassessment three months later [24]. A Nigerian study that assessed ART adherence among patients on subsidized ART programme reported an alarming 75% of the study participants as non-adherent to their medications, patients in this study cited various reasons for their actions, ranging from ARVs side-effects and non-availability of ARVs to forgetfulness [25]. A more recent KwaZulu-Natal study among 735 HIV-infected persons found 30% of the participants to be non-adherent to dose, schedule and dietary instructions [26]. Another South African study revealed that only 50% of patients on ART reported taking at least 95% of their prescribed ARVs [27]. A major review on studies conducted on ART adherence that involved 72 developed countries and 12 developing countries, five of which are African, estimated adherence level among people living with HIV/AIDS in Sub-Saharan Africa at 77%, surprisingly higher than 55% in North America. The authors stated further that non-adherence to ART in adult populations ranged between 33 and 88%, depending on the measure of adherence employed [7]. These percentages are low considering that 95% adherence is required to minimize development of viral resistance and to achieve optimal benefit of ART [16]. Adherence to ART is a good predictor of clinical outcome among patients on ART [16,17,18]. Adherence to combination ARV therapy has been shown to inhibit HIV replication which has resulted in the steady decline in HIV/AIDS related morbidity and mortality [28,29,30]. Lower levels of adherence have been demonstrated to achieve treatment goals in chronic medical conditions like hypertension and diabetes where moderate adherence has been considered adequate for treatment outcomes [14]. This is quite different in HIV/AIDS management where an adherence level greater than 95% has been described as the requirement for maximal virological suppression especially ART regimens that contain the protease inhibitors [16,31]. Although recent studies indicate moderate adherence at 80–90% could suppress viral replication with the non-nucleotide reverse transcriptase inhibitors [21,22]; This finding has to be interpreted with caution since the risk of viral resistance and mortality is high at such adherence levels [32]. Unlike other chronic diseases where non-adherence is somehow tolerated, the high rate of viral replication and mutation in HIV means that high levels of adherence have to be maintained all times [18]. Non-adherence to ART results in inadequate suppression of viral replication in the body which allows the virus to continuously replicate and deplete the T-Helper cells; this destroys the immune system and allows the disease to progress at a faster rate [33]. Non-adherence is associated with repeated hospital admissions, development of opportunistic infections, poor quality of life, loss of productivity and premature mortality [32,34,35,36]. Development of resistance to ART is another consequence of non-adherence because the virus is prone to developing resistance to ARV when exposed to suboptimal concentrations of the drug [33,37]. These resistant strains could be transmitted to other persons thereby decreasing treatment options and worsening the HIV epidemic. Furthermore, various comparative studies among patients with poor adherence to ART showed that patients whose adherence levels were below 75% were three times more likely to die compared to those whose adherence levels were above 75% [21,38,39]. Although, one has to bear in mind that many of the patients in these studies were initiated on ART with advanced stage of HIV/AIDS and with very low CD4 counts. The most important lesson from these studies is that poor adherence in advanced stages of the disease carries a high risk of mortality.The management of HIV/AIDS with ART has resulted in significant clinical outcomes; but the complicated dosing requirements, ART adverse drug reactions and socio-economic factors often constitute a challenge to patients [24,40,41]. Although various studies in sub-Saharan Africa have shown that high levels of adherence, viral suppression and good clinical outcome are achievable in these resource-limited settings [7,11,32,42,43,44,45], it is very challenging to adhere to ART, and strict adherence is not common. [46] This does not mean adherence should be a barrier to ART initiation in developing countries with limited resources if there is adequate access to ART, proper patient education, social support, and issues around social barriers are well addressed [47]. The results obtained from some of the adherence studies in resource-limited settings should be interpreted with caution, as there is a possibility of the results skewing towards better adherence. The reason for this is because a large majority of participants in these studies received free ARVs, frees information and support from dedicated clinical staff, a beneficial experience that does not reflect the true situation of things in most resource-constraint settings [11,24,26,30,32,42,44]. Many studies have been done on factors associated with adherence in patients taking antiretroviral medication, while some have focused on socio-demographic characteristics like race, age, income and level of education; some focused have on health beliefs of patients; and others have concentrated on the interaction between patients and health care providers [24,27,30,40,41]. Although some of the factors identified in these studies are somehow predictive of adherence, none of them has been shown to be associated with adherence across studies.Socio-demographic factors such as age, gender, socio-economic status, level of education, income and ethnicity have been used in studies to understand their influence on adherence [48,49]. It has not been possible to absolutely predict adherence based on all these demographic characteristics as no consistent correlation has been found between demographic characteristics and patient adherence levels [3,48]. Psychosocial factors like drugs and alcohol use, social stability, depression and psychiatric illness have also been used in other studies to find out if there were correlation between any of them and adherence to ART [10,49,50,51]. What studies have found are barriers to adherence such as substance abuse, unstable housing, depression, mental illness, fear of disclosure of HIV status, decreased quality of life, work and family responsibility and past history of non-adherence [7,9,52]. Although socio-demographic factors have not been consistently correlated with adherence to ART in several studies, they can be used to identify particular populations that may benefit more extensively from targeted interventions that address specific barriers to adherence [3,49]. The importance of these findings in ART initiation is that denying an individual the benefit of ART based on the assumption that the person would not adhere due to his or her demographic characteristics is a futile exercise as no data are available to substantiate such action [4].Other socioeconomic factors implicated as part of barriers to adherence to ART in a number of studies in Africa are the cost of ART, availability and accessibility to medications [23,43,53,54,55,56]. There are also some indirect costs associated with ART which influence adherence; these are the time taken off work, the time spent in hospital and inability to fend for one’s family during bouts of opportunistic infections [43,57]. The access to drugs while away from home is another factor that has been identified which affects adherence among patients on ART [58,59]. Certain structural factors tend to influence adherence to ART, which are issues that are beyond the control of the patients [60]. Structural approach to adherence does not see adherence as a simple individual behaviour but as one that occurs within social and environmental contexts. In other words, it is difficult for some people to adhere to ART because the adherence barriers are not generated at an individual level [60]. Some of these are out-of-pocket cost of health services and medication, transportation, homelessness, lack of access to food and water and poor accessibility to health facilities. All these are structural barriers to adherence that have to be addressed at the societal level in order to improve adherence [23,43,60]. Reduction in these structural barriers to healthcare has been shown to improve patient’s adherence levels in ART in some interventional studies to enhance adherence to ART [60]. ART is a complex treatment that is characterized by pill burden, dietary and fluid restrictions and timing of medication intake. The complexity of drug regimens is one of the causes of non-adherence among patients on chronic medication [3,52]. This has also been reported among HIV patients taking ARV; daily dosing regimens of three times or more are associated with non-adherence [60,61,62]. The pill burden is a major challenge since combination ART is used and it often contains 2–20 pills that have to be taken in a day together with the other factors to be considered like timing of dosages and food requirements [9]. The numerous and potentially debilitating side-effects contribute to irregular drug use and deliberate discontinuation of medication intake by some patients [23,63]. Regimens with significant side-effect profiles are usually associated with poor adherence [3,52]. Health care providers should consider the circumstances of patients while prescribing ART [64]; a potent combination therapy may not fit into a patient’s daily schedule and may therefore affect the adherence to such medication. The rate of adherence to once-daily antiretroviral regimens has been found to be better than the rate of adherence to twice-daily regimens [65]. Regimen-based strategies that could improve long term adherence among patients include simplification of ART regimens which will assist in the long term adherence to treatment and maintaining efficacy of treatment [3,9,51,61,64]. The clinical outcomes achieved with the use of ART are linked to adherence levels [3]. Although the Swiss HIV Cohort Study revealed that long standing HIV disease was among the predictors of worsening adherence [52], prior opportunistic infections in an HIV patient before initiating ART have the potential of influencing adherence as the patient may perceive the disease to be severe enough to require good adherence to treatment in order to achieve the desired treatment outcome [3]. Providers’ characteristics and clinical settings affect patients’ adherence; overall patients’ satisfaction with the level of care has been found to correlate with increased adherence [66]. The aspects of clinical setting that could positively influence adherence are a friendly and supportive environment, non-judgmental health care providers, convenient appointment schedule and confidentiality in service provision [67,68]. Long waiting times, poor staff attitudes, intermittent drug availability and other procedural barriers decrease patients’ adherence to ART and also results in poor clinic attendance [53,54]. Continuous access to health care services and medications by patients also influences treatment adherence [53]. The patient-provider relationship is another factor that has been well researched in terms of adherence to ART; a good patient-provider relationship results in patient’s trust and confidence in the provider which in turn influences good adherence [66,67]. The burden posed by adherence is not limited to the patient alone but extends to the health care provider; therefore, the relationship between the patient and the health provider should be that of a therapeutic alliance where both parties work toward a common goal of improving the health of the patient. This is why frequent change in health care providers is associated with poor adherence [52].Discussing ART initiation with an informed patient is an effective strategy that enhances patient-provider interaction. It enables an alliance formation which allows the patient and the provider to define therapy goals, side-effects, medication management and adherence monitoring [64]. Social support and ability to disclose one’s status are important issues to be considered when assessing treatment adherence irrespective of the context [32]. A study on adherence of patients to chronic medications recommends that therapeutic alliance between patients and providers should be strengthened, time should be devoted to address adherence among patients, and patients should be well assessed to identify barriers to treatment adherence [69]. Although adherence is one of the modifiable factors in ART, monitoring it is difficult among patients, since no standard method exists to monitor adherence in ART, and therefore, multiple approaches are often used [7,11,68]. Generally, ART adherence is calculated as the percentage of doses taken over those prescribed within a given period [2]. Specifically, medication adherence is defined as the percentage of prescribed medications taken in any form for a specified period; instructions adherence is defined as the percentage of medications for which the correct special instructions were followed at each prescribed dose [2,15]. Adequate monitoring of adherence is vital in preventing treatment failure and development of resistance to antiretroviral drugs [70]. Some of the commonest methods are self-report, electronic device monitoring, pills count, pharmacy refill tracking, biological markers, provider estimation and therapeutic drug monitoring. Self-report remains the main measure of adherence globally although other techniques have been used widely; it involves asking patients to report their adherence periodically [71]. Studies have found correlation with actual medication intake in research done on self-report as a method of adherence monitoring [44,71]. In the Ugandan study that used three-day self-report adherence, median adherence correlated with other measures of adherence [44]. The major drawback of this method is overestimation of adherence by patients due to desirability bias [72]. Adherence guidelines stipulate that accuracy of adherence self-report could be achieved if patients are approached in a non-judgmental way during assessment [70]. Pill count as a measure of adherence involves calculating the percentage of the number of pills prescribed and dispensed for the period between hospital appointments with the number of pills returned at the following appointment [42]. It is done during patients’ visits to health facilities or unannounced at patients’ home. This technique is cheap and correlates well with adherence measured using viral load [42], but its major shortcoming is manipulation of pills by the patient. Pill dumping prior to hospital visits has been documented which will result in overestimation of adherence for the patient. Unannounced pill count is more reliable but it has the tendency to affect the trust between the patient and health care provider which may eventually hinder adherence [22]. Though they may overestimate adherence; pill counts and self-report are still the mainstay of measuring adherence in resource-limited settings [73].This involves an electronic chip that is implanted on the bottle lid that records the opening and closing of the bottle which contained the prescribed medicines, a computer program is later used to extract information from the lid and the data is then analyzed. This technique assumes that opening of the bottle by the patient coincides with actual intake of the drug. Although this method has shown some correlation with actual drug intake in some studies [16,51], others have described it as an expensive and non-reliable device [73]. A study in Malawi that compared methods of adherence monitoring found no correlation between electronic device monitoring, self-reporting and pill counts [73]. Electronic devices may underestimate or overestimate adherence since it is difficult to know if a patient opening a drug bottle is actually taking the tablets or just opening the bottle, therefore, actual medication intake by patients is not measured by EMD [74]. A study of electronic monitoring devices used among HIV patients on ART revealed that patients who rely on pill boxes are not likely to use EMD because pill boxes are the preferred tools for patients taking numerous medications everyday [74]. Pharmacy record is a simple and effective tool for monitoring adherence in ART and has been proved to be useful in adherence monitoring in a resource-limited setting [32,48]. This method employs pharmacy records to monitor adherence among patients when collecting their medication at the pharmacy. Patients collecting their medication regularly are said to be adherent by the pharmacy. Pharmacy records in resource limited settings are useful tools for measuring adherence; adherence measured by pharmacy records have been shown to correlate with CD4 count and weight gain in patients [72]. Adequate record keeping is necessary for pharmacy refill tracking information to be useful; the major setback is that the method only assumes that patients are adherent based on their empty pill boxes and the regular collection of their medication, it does not measure actual medication intake [32,48,72]. This method also requires patients to be collecting their medication at the same pharmacy for all refills which is not always possible [32,48,72].Viral load level and CD4 counts are used as indicators for treatment outcomes and success. Viral load is regarded as the main indicator of the risk of therapeutic failure and can also be used in measuring adherence [42,75]. Low viral loads and increases in CD4 count are suggestive of good adherence although some patients may have a high viral load despite taking the ART regularly. CD4 is a good measure of adherence and correlated with weight gain, self-report and pharmacy records [72]. Viral load was also found to correlate with pill counts and good clinical outcomes in patients [42]. Pill counts may be a reliable and economical tool for monitoring adherence in resource-limited settings although viral load monitoring has been described as the preferred method [42]. The major drawback is cost and availability in resource limited settings [70]. In the provider estimation method of measuring adherence, the health care providers estimate a patient’s adherence based on factors such as socio-demographic and economic factors. In this method, there is no correlation with actual medication intake; studies have shown that health care providers overestimate adherence to HIV treatment when factors such as demographic characteristics are used to predict adherence because it is very difficult to predict adherence based on demography [16]. Furthermore, consistent clinic attendance does not usually correlate with other measures of adherence among patients on ART [72].Therapeutic drug monitoring (TDM) involves measuring the levels of the drug in the blood stream of the HIV patient. This method is not used routinely as most ARVs have short circulating times in the body, coupled with the fact that it is very expensive [71]. Currently, therapeutic drug monitoring is restricted for research purposes. A study that combined TDM and viral resistance monitoring to find causes of virologic failure in patients on ART revealed that TDM is necessary in patients who developed adverse reactions while on a ritonavir boosting regimen because of the risk of drug toxicity in these patients [29]. TDM assists clinicians in choosing the best regimens for their patients and could be used routinely in clinical practice [29]. A prospective cohort study in Cameroon to compare adherence of fixed-dose combination of nevirapine, stavudine and lamivudine using nevirapine plasma level monitoring and self-report by patients found that self-reported adherence was significantly higher than adherence measures by nevirapine level monitoring [71]. The authors concluded that nevirapine plasma concentration monitoring provides an accurate measurement of adherence compared to self-report, but cautioned that it is not feasible in most clinical settings especially in resource-limited areas due to cost [71].Adherence is very complex and unpredictable among patients on chronic medications; several methods of enhancing adherence exist in ART and are usually used in combination for better results. The interventions used in enhancing adherence among patients are aimed at addressing potential barriers to adherence.This entails simplifying regimen characteristics; simplification of dosing schedules, reduction of pill burden, and adjusting dietary restrictions to match patients' daily activities. In addition, identifying previous ART use and pre-existing medical conditions that could affect ART use and ensuring continuous provision of ART have also been reported to enhance adherence to ART [76]. Studies have demonstrated improved adherence with once or twice daily dosing of ART [77]. Many fixed-dose combinations (FDC) of ARVs are now available and have been shown to improve adherence in patients [65,77,78]. The buddy system has been widely used in resource-limited settings where relatives or friends agree to assist the patient in adhering to the medication [76]. Buddies remind patients to take their medication, encourage them and assist in keeping hospital appointments. This approach has led to improved ART adherence among some HIV-infected persons [76]. Social support not only assists the patient in adhering to ART but can also provide psychological support to the patient which helps them to cope with the disease [79].This is usually the mainstay of ART programmes in any setting; Education and counsellingempowers the patient to be part of the treatment process. Knowledge about the disease, its symptoms, treatment and side-effects of the medications are crucial information that has to be passed on to the patient [80]. During counselling, potential barriers to adherence are identified and addressed. Counselling assists the patient in developing positive beliefs and perception towards the disease [81]. It also helps in setting goals and increases the self-efficacy of the patient [82]. A randomized controlled trial in the USA to compare the effect of person-to-person contact and support adherence with medication alarm techniques revealed improved responses to therapy and good adherence among the group that received interpersonal adherence support [83]. This finding is consistent with the literature where repeated supportive adherence has been described as the most effective intervention because it provides human contact and support [68].These tools are often combined with other behavioural interventions [84]. Pill boxes are containers used for storing the ARV for regular use as prescribed. They enable the patient to take the medication correctly. Electronic versions of pill boxes with reminders to the patient are also available. The major setback of this tool is the lack of confidentiality and privacy associated with it, since some patients do not want people around them to see their medications. Another setback is the task of filling out the boxes; uneducated patients might not be able to do this correctly. Pill charts involve visual display of the pills in terms of their colour, shape, name and dosage of the medication during counselling. This is very useful especially among the uneducated patients [84]. Electronic devices such as beepers, alarms and watches that remind patients to take their medication according to the prescribed schedule are also used to enhance adherence [70]. In addition, telephone calls and mobile-phone text messages have been shown to improve medication adherence in HIV infected individuals [85,86,87,88]. Electronic pagers linked to the internet may also be used to send reminders to patients to take their drugs. Although reminder tools have been reported by patients as one of the facilitators of adherence [85,86,87,88], the major disadvantage of this strategy is the lack of privacy associated with it. Another shortcoming is the cost required to set up this kind of service and the challenges associated with the patient having their mobile phones with them all the time [88]. Medication diaries are very useful in understanding the patterns of drug use by the patient and the reasons for not taking the medication regularly. Diaries are used by the patients to document the time and date of taking the medications and missed doses and the reasons for it. This tool may also be used to identify side-effects or other problems that the patient may encounter in the course of taking ART. Research has shown that some tools appear to be of more benefit to patients when they are combined with patient education or counselling. A large multicenter randomized trial in the USA revealed that reminder devices alone do not enhance adherence and suggests that they should be combined with counselling as part of comprehensive support for patients on ART [83].This technique has been used successfully in TB by asking patients to take medication under the supervision of adherence counsellors, trusted family members or community lay workers [89,90,91]. Directly administered ART can be clinic or home-based, once-daily, twice-weekly, or once-weekly [92]. The challenge in ART is different from TB as timing of medication intake varies across ARVs and the fact that the treatment is for life [93]. Although this approach has shown some benefit in improving adherence, research does not support routine use of directly administered ART [92,93,94]; Modified directly observed therapy (mDOT) has been suggested for use in ART and a home-based mDOT strategy has been used to improve adherence rates in resource-limited settings [92]. However, adherence to ART was only seen in the early weeks of a (mDOT) study in Kenya [94]. Despite its limited use, the benefits of mDOT in ART seen in early stages of treatment helped patients to develop good understanding of the treatment and develop good treatment taking behavior. It also assisted the patients in developing trusting relationships with the health care provider which subsequently improved their adherence to ART [92]. These include interventions that target barriers to ART adherence at the level of health care delivery of HIV services such as food supplements, transportation to health facilities, staffing and integration of services. Research has demonstrated that ART adherence and retention in care are associated with interventions that offer food supplements, address transportation issues and integrated services. Family nutritional support for HIV positive patients on ART improves adherence leading to good clinical outcomes, and this could be integrated into ART adherence interventions as an effective and comprehensive community-based primary care [95]. In a South African study, nurse-initiated ART services have been shown to produce the same outcomes as ART services run by doctors, which indicates that where human resources are limited, nurses can provide ART care [96]. In addition, addressing the issue of transportation to the health facilities among HIV patients and integration of home-based care into ART services led to improved attendance in clinic appointments [97]. ART regimens need to be chosen taking into consideration the patient’s working and family life, especially the cost implication of the drugs to ensure continuous supply so that adherence can be optimized [98].Once initiated, antiretroviral therapy is a lifelong treatment; this has made adherence to ART one of the major challenges facing HIV/AIDS services following the rapid scale-up of ART to provide life-saving treatment to people infected with HIV. Non-adherence in ART is not limited to missing medication intake; it also includes other acts like not following instructions regarding dietary or fluid restrictions and not taking medication at the prescribed time. This underscores the complex nature of adherence in the management of HIV/AIDS as a chronic medical condition. Medication adherence is affected by patients’ beliefs about disease origin and transmission, which often form the basis for stigmatization in HIV/AIDS. Adequate knowledge of the medication and understanding the need for strict adherence, self-efficacy, sense of self-worth, acceptance of HIV status, making use of reminder tools and social support are factors that influence adherence to ART in different settings [59]. Therefore, ART adherence needs to be tackled using a multi-disciplinary approach due to the difficultly in predicting which patients will adhere to treatment and those who will not; as a person’s past adherence is the only predictor of future adherence [4].The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Oxidative stress is a key mechanism causing protein aggregation, cell death and neurodegeneration in the nervous system. The neuronal cytoskeleton, that is, microtubules, actin filaments and neurofilaments, plays a key role in defending the nervous system against oxidative stress-induced damage and is also a target for this damage itself. Microtubules appear particularly susceptible to damage, with oxidative stress downregulating key microtubule-associated proteins [MAPs] and affecting tubulin through aberrant post-translational modifications. Actin filaments utilise oxidative stress for their reorganisation and thus may be less susceptible to deleterious effects. However, because cytoskeletal components are interconnected through crosslinking proteins, damage to one component affects the entire cytoskeletal network. Neurofilaments are phosphorylated under oxidative stress, leading to the formation of protein aggregates reminiscent of those seen in neurodegenerative diseases. Drugs that target the cytoskeleton may thus be of great use in treating various neurodegenerative diseases caused by oxidative stress.Oxidative stress represents an imbalance between the production of reactive oxygen species and the ability of the biological system to detoxify the reactive intermediates or to repair the resultant damage. Oxidative stress leads to the formation of peroxides and free radicals, which damage proteins, lipids and DNA. Oxidative stress is implicated in various neurodegenerative disorders, largely due to the abnormal accumulation of oxidatively damaged macromolecules, including β-amyloid and Tau [1].The neuronal cytoskeleton plays a key role in both protecting cells against oxidative stress and is itself the target of oxidative stress-induced damage. The neuronal cytoskeleton consists of microtubules, actin filaments and neurofilaments (intermediate filament proteins). All three are linked through crosslinking proteins to form a unified whole. These cytoskeletal components are regulated through changes in expression levels, post-translational modifications and the effects of the binding of partner proteins. Oxidative stress affects the regulation of the neuronal cytoskeleton through these regulatory mechanisms, with some changes being protective and others pathogenic. Thus, the cytoskeleton plays a key role in cellular responses to oxidative stress and the onset of oxidative stress-induced neurodegeneration.The stabilisation of microtubules with taxol or destabilisation with vinblastine causes concentration-dependent cell death with apoptotic features in neurons. Treatment with taxol increases the amount of reactive oxygen species, and inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or suppression of gp91 (phox) attenuates taxol-induced neuronal death, although the use of NADPH oxidase inhibitors does not decrease vinblastine-induced cell death [2]. The microtubule depolymerising agent, colchicine, upregulates genes related to oxidative stress, glutathione peroxidise 1 and catalase, in rat cerebellar granule neurons [3]. Colchicine causes loss of cholinergic neurons and cognitive dysfunction that is associated with excessive production of free radicals, including malondialdehyde and nitrite, and a decrease in reduced glutathione [4]. Treatment of primary mesencephalic neurons with microtubule-depolymerising agent stimulates dopamine transporter (DAT) activity, leading to an increased rate of dopamine uptake and increased levels of dopamine-induced oxidative stress along with accelerated cell death. DAT co-immunoprecipitates with α- and β-tubulin, suggesting a functional link between the microtubule cytoskeleton and DAT [5].Oxidative stress causes the aggregation of a number of proteins implicated in neurodegenerative disorders [1]. Mutations that affect the dynein motor machinery are sufficient to cause motor neuron disease. Indeed, decreased dynein function impairs autophagic clearance of aggregate-prone proteins [6], losing its function in aggresome formation and failing to return autophagosomes and lysosomes to the centre of the cell for degradation [7]. An intact microtubule cytoskeleton is essential for aggresome formation, and cytoplasmic dynein/dynactin is responsible for the directed transport of misfolded protein into aggresomes [8]. Thus, breakdown of the microtubule cytoskeleton under oxidative stress will, in turn, increase the load of aggregated protein in a cell, potentially leading to overload and cell death.HDAC6 is responsible for the deacetylation of α-tubulin in the brain. An inhibitor of HDAC6, tubastatin A, induces elevated levels of α-tubulin, but not histone, in primary cortical neuron cultures. Tubastatin A also confers dose-dependent protection of neurons against glutathione depletion-induced oxidative stress [9]. Microtubule acetylation is a post-translational modification that promotes the binding and transport of kinesin-1 in neurons [10]. Multiple organelles are transported along microtubules by kinesins, and the breakdown of this process can lead to increased oxidative stress in neurons.Oxidative stress caused by hydrogen peroxide leads to axonal swelling and beading, consistent with disruption of microtubules by oxidative stress and subsequent hold-up of axonal transport [11]. HDAC6 plays an important role in the modulation of mitochondrial transport. When inhibited with the specific HDAC6 inhibitor, tubacin, more kinesin-1 associates with mitochondria. HDAC6 activity is regulated by GSK3β via phosphorylation [12]. Mitochondria in Alzheimer’s disease brains are perinuclear, with few organelles in distal processes, where they are normally located in healthy cells and are needed for exocytosis, ion channel pumps, synaptic function and other activities. Alzheimer’s disease neurons thus have an increased amount of reactive oxidative species and decreased metabolic capability [13]. Axonal transport of mitochondria and mitochondrial dynamics may also be disrupted in amyotrophic lateral sclerosis [14] and in retinal ganglion cell neurodegeneration [15], where oxidative stress is again a factor. Indeed, mitochondrial dysfunction is thought to play a key role in disease pathogenesis, because neurons are among the most energy-consuming cell types and have a highly developed cell shape [16].Tau protein inhibits kinesin-dependent transport of peroxisomes into neurites, and loss of peroxisomes makes cells vulnerable to oxidative stress [17]. Here, again, tubulin acetylation may play a key role in promoting peroxisomal transport to distal processes, and the oxidative stress-induced breakdown of microtubules may lead to increased oxidative stress in these processes. Finally, Parkinson’s disease toxins, such as rotenone, disrupt vesicle transport along microtubules, leading to increased accumulation of vesicles in the cell body and increased cytosolic concentration of dopamine, due to leakage from the vesicles. Elevated oxidative stress induced by dopamine oxidation may trigger apoptosis of dopaminergic neurons [18].Loss of α-tubulin acetylation and impaired motor-protein transport are implicated in a number of neurological disorders, including Huntington’s disease [19], familial dysautonomia [20] and amyotrophic lateral sclerosis, all of which occur through disruption of the α-tubulin acetylase activity of the Elongator complex [21]. These diseases are also characterised by oxidative stress [22]. It seems likely that the breakdown in transport of cargoes along microtubules due to defects in tubulin acetylation may play a key role in promoting oxidative stress and subsequent neurodegeneration, whereas increased α-tubulin acetylation and microtubule-based transport may act to alleviate oxidative stress. Transcription factor trafficking is also affected in oxidatively-stressed neurons [23].The conjugated aromatic ring of tyrosine is a target for free-radical attack, leading to the formation of 3-nitrotyrosine under conditions of oxidative stress [24]. α-Tubulin is tyrosinated at its C-terminus by the action of a tubulin-tyrosine ligase, and the C-terminal tyrosine can then be removed by a tubulin carboxypeptidase. This post-translational modification prevents the binding of kinesin-1 to microtubules in dendrites, thus navigating it to axonal microtubules [25]. High glucose and hyperglycemia can result in the production of high concentrations of reactive oxygen species with subsequent damage to the central nervous system. Indeed, in neuroblastoma and glioma cells exposed to D-glucose, there is a downregulation of tubulin isoforms and an increase in 3-nitrotyrosine modification of tubulin [26]. There is some controversy over the effect of 3-nitrotyrosine incorporation into tubulin on microtubule function. One study found that 3-nitrotyrosine incorporation into mammalian cells by tubulin-tyrosine ligase was irreversible and caused changes in microtubule organisation, cell morphology and the redistribution of the motor protein, dynein [27]. However, another study found that the incorporation of 3-nitrotyrosine into microtubules was reversible and not detrimental to dividing cells [28]. Therefore, the effect of 3-nitrotyrosine incorporation into microtubules is still an open question, but it may interfere with the correct localisation of neuronal motor proteins and MAPs.Friedreich’s ataxia is caused by mutations in the gene for frataxin, a mitochondrial protein implicated in iron metabolism. Loss of frataxin causes iron overload in tissues and an increase in free-radical production and oxidation. There is a significant rise in the dynamic pool of tubulin, possibly caused by increased protein glutathionylation [29]. Loss of glutathionylation of cytoskeletal proteins may give rise to oxidative stress, as this is a way that cells regulate microtubule cytoskeletal stability [29]. It has been recently suggested that microtubule glutathionylation, an increased intracellular level of oxidized glutathione, may cause an alteration of the cytoskeleton organisation and function, leading to axon degeneration [30].Lipid peroxidation is regarded as a major cause of brain damage by oxygen radicals. The antioxidant, melatonin, prevents lipid peroxidation caused by okadaic acid in neuroblastoma cells and prevents the disruption of the microtubule cytoskeleton [31]. Phosphatidylcholine hydroperoxides (PCOOHs) disrupt both the formation of neurites and neuronal microtubules. Differentiated cells are less susceptible to this form of attack than undifferentiated cells [32]. Another product of lipid peroxidation, acrolein, disrupts microtubules in PC12 cells, as well as sympathetic ganglion cells in vitro [33], and this may be due in part to increased phosphorylation of Tau protein [34]. In a model system of microtubule degradation by lipid-derived free radicals, it was shown that the kinetics of this process is determined by the level of lipid saturation and the presence of free radical scavengers [35]. Oxidised very-low-density lipoprotein and hydrogen peroxide reduced phosphorylation of the microtubule-destabilising protein, stathmin, whereas the lipid peroxidation product, 4-hydroxy-2-nonenal (HNE; an aldehyde), increased it, suggesting that free radicals, but not aldehydes, may play a key role in neuronal cell death caused by lipoprotein oxidation [36]. However, HNE disrupts microtubules and modifies cellular tubulin via Michael addition [37], as well as inhibiting dephosphorylation of Tau protein [38].Oxidative stress leads to changes in the suite of MAPs found in neurons through changes in expression levels, post-translational modification, oxidative damage and breakdown of proteins by proteolysis.Expression levels of various MAPs change under conditions of oxidative stress in neurons. Some of these MAPs—MAP2, Tau and doublecortin, are microtubule-stabilising proteins, and the reduction in their expression levels may serve to destabilise microtubules. A proteomic analysis of proteins differentially regulated under acute oxidative stress showed that a number of MAPs changed their levels of expression. Doublecortin was downregulated. Collapsin response-mediator proteins (CRMPs) were upregulated. α-tubulin 6 was downregulated, and Tau protein and MAP2 were downregulated [39]. Other studies support these findings. Chronic ozone exposure causes an increase of reactive oxygen species and oxidative stress. Ozone causes cell swelling in neurons and a decrease in the MAP doublecortin and, also, chronic loss of brain repair in the hippocampus in adult rats [40].Lanthionine ketimine (LK) is a member of a class of thioethers that are present in the mammalian central nervous system. LK interacts with CRMP2 and decreases CRMP2 coprecipitation with β-tubulin. A cell-permeable LK-ester, LKE, protects against oxidative challenge with hydrogen peroxide [41]. CRMP2 is a GTPase-activating protein, and this activity is important for microtubule assembly and neurite formation [42]. However, the fact that CRMPs are upregulated under oxidative stress and tubulin-associated CRMP decreases following treatment of neurons with neuroprotective LK indicates that CRMP has a role in promoting oxidative stress alterations of the microtubule cytoskeleton.Soluble oligomeric forms of amyloid beta-peptide induce oxidative stress in neurons, and this oxidative stress leads to the proteolysis of MAP1A, MAP1B and MAP2 by caspase-3 and calpain [43]. Quinones modify tubulin, leading to its aggregation into dimers and other oligomers [44], which disrupt microtubule formation. The I93M mutation in ubiquitin C-terminal hydrolase L1 (UCH-L1) is associated with familial Parkinson’s disease. UCH-L1 is a major target of oxidative/carbonyl damage associated with sporadic Parkinson’s disease. Familial Parkinson’s disease-associated and carbonyl-modified UCH-L1 in sporadic Parkinson’s disease share aberrant properties, with both modified forms modulating tubulin polymerisation, and this gain of function may be relevant to both familial and sporadic Parkinson’s disease [45]. M sulfoxides map to neuron-enriched β-tubulin in Alzheimer’s disease brains, suggesting oxidative modification of β-III tubulin as a contributor to the neuronal cytoskeletal disruption characteristic of Alzheimer’s disease [46].Tau protein is implicated in neurodegenerative diseases, including Alzheimer’s disease. Peroxynitrite induces the oxidative crosslinking and site-selective nitration of Tau monomers. This causes the formation of aggregates that are crosslinked by inter-filament bridges, inhibiting the ability of monomeric Tau to promote tubulin assembly [47]. Arg, a member of the Abl family of tyrosine kinases, phosphorylates Tau protein at Y394. Arg plays a key role in both oxidative stress response and neural development. Paired helical filaments that comprise neurofibrillary tangles in Alzheimer’s disease contain an abnormally hyperphosphorylated form of Tau [48]. During mitochondrial inhibition and oxidative stress, actin-depolymerising factor (ADF)/cofilin assemble into rods along the processes of cultured neurons and recruit phosphorylated Tau protein. Microtubules are disrupted in processes that possess rods [49], suggesting that neurofibrillary tangles may be initiated by F-actin bundling caused by oxidative stress [50]. Oxidised products of dopamine (dopamine quinone) also promote Tau polymerisation, again suggesting a link between oxidative damage and the onset of tauopathies [51]. The peptidyl-prolyl cis/trans isomerase Pin1 regulates the function and processing of both Tau proteins and amyloid precursor protein (APP), both of which are implicated in Alzheimer’s disease. In humans, the Alzheimer’s disease brain Pin1 is downregulated by oxidative modification and/or genetic changes [52].Some MAPs appear to function to decrease oxidative stress in neurons. NAPVSIPQ (NAP), an 8-amino acid peptide derived from activity-dependent neuroprotective protein (ADNP), interacts with microtubules and protects microtubules from degradation under oxidative stress, probably inhibiting an early event of apoptosis [53]. NAP specifically binds βIII-tubulin, and taxol reduces NAP-tubulin binding [54]. Parkin is a MAP that is mutated in familial Parkinson’s disease. Elevating Parkin in cells reduces markers of oxidative stress, whereas blocking its expression increases oxidative stress. Parkin also acts as a ubiquitin ligase in the ubiquitin-proteasome system, and mitochondrial function is deficient in mouse and fly Parkin knockdown models. It is unclear which of Parkin’s functions is neuroprotective [55]. Hydrogen peroxide treatment of neuronal and non-neuronal mixed rat retinal cell cultures causes the loss of MAP2 in the neuronal soma, and downregulation of MAP2 increases the vulnerability of retinal neurons to oxidative stress [56].Tensional integrity (tensegrity) architecture has been proposed to be a means by which cells stabilise their shape and sense mechanical signals from the nanoscale to the macroscale [57]. Importantly, these networks are in a state of isometric tension (i.e., they experience a tensile prestress), ensuring that molecular-scale mechanochemical transduction mechanisms proceed simultaneously [58]. In cells, it is suggested that microtubules provide compression-resistant components and actin-filaments, tensional elements, in a unified tensegrity architecture whole. There is evidence to support this theory, with living cells behaving like discrete structures composed of an interconnected network of actin filaments and microtubules when mechanical stresses are applied to cell surface integrin receptors [59].Short Stop (Shot) encodes a neuronally expressed family of proteins required for sensory and motor axon extension in Drosophila melanogaster. Shot isoforms contain an N-terminal F-actin and C-terminal microtubule-binding domains that crosslink F-actin and microtubules in cultured cells. Both the F-actin and microtubule-binding domains are required in the same molecule for axon extension [60]. Shots also play a key role in dendrite formation and axonal terminal arborisation at the neuromuscular junction, forming a complex with the microtubule plus-end-binding (+TIP) protein, EB1 [61]. Thus, there is evidence for the necessity of a linkage between microtubules and actin filaments in neuronal development, possibly due to the reliance of neurons upon cytoskeletal tensegrity architecture to maintain prestress. Actin and neurofilaments are also crosslinked by dystonin, a member of the bullous pemphigoid antigen (BPAG) family of plakins [62]. Disruption of dystonin causes sensory neuropathy in mice and disorganisation of neurofilaments and microtubules [63]. Neurofilaments undergo strain stiffening suggesting that, as well as microtubules, they act as compression-resistant components of neuronal cytoskeletal tensegrity architecture [64]. Any perturbation of actin filaments, microtubules or neurofilaments, for example, by oxidative stress, will lead to a loss of prestress and the ability of the cytoskeleton to function as an integrated whole.Interestingly, although actin and actin-binding proteins respond to oxidative stress, they appear less susceptible than microtubules. This may be in part due to the fact that the actin cytoskeleton actually utilises oxidative stress as a developmental mechanism.Oxidative stress increases cellular actin aggregation and the binding of PINK1 to Parkin, both of which proteins are implicated in Parkinson’s disease. Parkin also associates with actin, suggesting that oxidative stress may disrupt actin organisation through changing the binding substrates of Parkin [65]. Cu2+-induced stress causes glutathione (GSH) adducts of cysteines of synaptosomal actin followed by its deglutathionylation and dimerisation. Torpedo cyclophilin B is able to sustain peroxiredoxin-1 activity and, thus, might be involved in presynaptic defence against oxidative stress affecting G-actin [66]. Oxidative stress results in a rapid dephosphorylation of cellular cofilin (an actin-binding protein) prior to its assembly into rod-shaped inclusions. The ATP-sensitive interaction of cofilin phosphatase chronophin with chaperone hsp90 mediates the formation of cofilin/actin rods [67].TNF alpha and II-1beta stimulate a redox-dependent reorganisation of the actin cytoskeleton into lamellipodia under the regulation of Rac1 and a neuronal NADPH oxidase as the source of reactive oxygen species. However, the persistent presence of reactive oxygen species provoked carbonylation of actin, which coincided with the loss of lamellipodia and the arrest of cellular plasticity [68]. Carbonylation of β-actin was also detected following the production of reactive oxygen species following ischemia-reperfusion in the hippocampus of the macaque monkey [69].Astrocytes and glia modify their actin cytoskeletons to protect neurons against oxidative stress. Homocysteine is an excitatory amino acid that enhances the vulnerability of neuronal cells to oxidative injury. In astrocytes exposed to homocysteine, there is a dramatic change in the actin cytoskeleton and a change in cell morphology to fusiform and/or flattened cells with retracted cytoplasm. Neurons do not appear to change their actin cytoskeletal organisation in response to homocysteine. Thus, astrocytes reorganise their actin cytoskeleton in response to oxidative stress in order to protect neurons from its effects [70]. The oxidant, paraquat, induces rapid differentiation and proliferation of glial cells, including the expression of smooth muscle actin. In pure neuronal cultures, paraquat caused apoptosis of photoreceptors and amacrine neurons, and coculturing neurons with glial cells completely prevented this apoptosis. Thus, neuronal oxidative stress is ameliorated by the reorganisation of the actin cytoskeleton in glial cells [71].Aggregation of neurofilament (NF) protein because of phosphorylation is a key process in neurodegenerative diseases, including Charcot-Marie-Tooth disease [72], Alzheimer’s disease and amyotrophic lateral sclerosis [73]. Injection of quinolinic acid increases phosphorylation of the low molecular weight NF subunit in neurons, whereas N-methyl-d-aspartic acid (NMDA) antagonist MK-801 and the antioxidant, N omega-nitro-l-arginine methyl ester (l-NAME), prevent this hyperphosphorylation. The hyperphosphorylation is targeted at the Ser55 phosphorylating site on the NFL head domain, a regulatory site for NF assembly in vivo [74]. In the spinal cord in Friedreich’s ataxia, there is an abnormal distribution of phosphorylated forms of NFs [75]. Pin1 modulates oxidative stress-induced phosphorylation of high molecular weight NF protein. Cyclin-dependent kinase 5 (cdk5) phosphorylates NF protein, and overexpression of cdk5 inhibits NF axonal transport and induces accumulation of disordered phosphor-NF cables. Oxidative stress recapitulates this phenotype in a cdk5-dependent manner [76].The lipid peroxidation product, acrolein, leads to the generation of protein carbonyl compounds and the formation of NF aggregates by dityrosine crosslinking. These aggregates display thioflavin T reactivity, reminiscent of amyloid [77]. In Alzheimer’s disease brains, there is an increase in N-malondialdehyde-lysine formation targeting NFL proteins, suggesting an important role for lipid-peroxidation-derived protein modification in Alzheimer’s disease pathogenesis [78]. Neuroaxonal spheroid aggregates in leukoencephalopathy contain amyloid and NF proteins, and there is an increase in markers for oxidative stress [79]. The lipid peroxidation product, HNE, causes the formation of adducts in NF proteins, but here, it is suggested that HNE may play a protective role against toxic aldehydes resulting from oxidative damage, due to its consistency from birth to senility and its dependence on phosphorylation [80]. Modification of NFs by oxidative advanced glycation end-products in diabetic neuropathy leads to axonal degeneration [81]. Oxidative stress also affects the neuronal cytoskeleton through proteolysis and/or abnormal structural changes in NFs [82], consistent with antioxidant treatment of dogs, which leads to a decrease in protein carbonyl levels of NF triplet L protein [83].We have demonstrated that the nervous system cytoskeleton plays a key role in oxidative stress responses and the onset of oxidative stress-induced neurodegeneration. Drugs that target the cytoskeleton, for example, the HDAC6 inhibitor, tubastatin, may thus be of great use in treating various neurodegenerative diseases caused by oxidative stress. Diet, too, may be important, with zinc availability, for example, causing alterations in tubulin thiol redox status [84]. The development of a polypeptide inhibitor of cdk5, potentially useful in reducing protein aggregation caused by the phosphorylation of NF and Tau proteins, is undergoing tests in mice [85]. The development of an inhibitor against the Elongator complex, α-tubulin acetylase, would provide key insights into the role of tubulin acetylation in oxidative stress responses, while a proteomic approach to the identification of cytoskeleton-associated proteins under oxidative stress conditions would be of great interest.Abelson murine leukaemia viral oncogene homolog 1actin depolymerising factoractivity-dependent neuroprotective proteinamyloid precursor proteinAbl-related genebullous pemphigoid antigencell division kinase 5collapsin response-mediator proteinsdopamine transporterend binding 1glutathioneglycogen synthase kinasehistone deacetylase 64-hydoxy-2-nonenallanthionine ketimineN omega-nitro-L-arginine methyl estermicrotubule-associated proteinnicotinamide adenine dinucleotide phosphateNAPVSIPQneurofilamentN-methyl-D-aspartic acidphosphatidylcholine hydroperoxidesshort stopubiquitin C-terminal hydrolase L1The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The clustering of cardio-metabolic risk factors, either when called metabolic syndrome (MetS) or not, substantially increases the risk of cardiovascular disease (CVD) and causes mortality. One of the possible mechanisms for this clustering's adverse effect is an increase in arterial stiffness (AS), and in high central aortic blood pressure (CABP), which are significant and independent CVD risk factors. Arterial hypertension was connected to AS long ago; however, other MetS components (obesity, dyslipidaemia, dysglycaemia) or MetS associated abnormalities not included in MetS diagnostic criteria (renal dysfunction, hyperuricaemia, hypercoaglutability, menopause, non alcoholic fatty liver disease, and obstructive sleep apnea) have been implicated too. We discuss the evidence connecting these cardio-metabolic risk factors, which negatively affect AS and finally increase CVD risk. Furthermore, we discuss the impact of possible lifestyle and pharmacological interventions on all these cardio-metabolic risk factors, in an effort to reduce CVD risk and identify features that should be taken into consideration when treating MetS patients with or without arterial hypertension.Arterial compliance plays a key role in the function of the cardiovascular system, given the intermittent contraction of left ventricle (LV) and the endless need of organs and tissues for oxygen and nutrients [1]. The contraction of the LV increases the pressure in large vessels (i.e., the aorta), which, because of their elasticity, store a significant part of the blood ejected from LV contraction. After the closure of the aortic valve, the recoil of large vessels during LV diastole pushes the blood towards the periphery, keeping a steady flow to organs and tissues [1]. Arterial stiffness (AS) and its hemodynamic consequences have been established as predictors of adverse cardiovascular disease (CVD) outcomes [1,2]. AS is closely related to systolic hypertension and has a causal effect on coronary artery disease, stroke, and heart failure, which are the leading causes of morbidity and mortality in western countries [2,3,4]. Although blood pressure (BP) is a major determinant of AS, the latter has an additional predicative potential over and above that of brachial BP, and may be affected by CVD risk factors other than arterial hypertension [5].The research question was: Are all disease conditions related to what is usually described as metabolic syndrome (MetS) considered to be components of the syndrome or are they just related with it in most cases, and have an adverse effect on AS despite the presence of arterial hypertension, which along with age are the key factors for stiff arteries? Many researchers use several definitions of MetS, they use several methods to measure AS, have different endpoints, and there are no studies at all that investigate the relation of all or even the majority of disease states not included in the MetS criteria with AS. As such, it was not possible to write a systemic review on this issue, and we chose to write a narrative review on the subject at hand. This was done because the issue of AS is an important independent CVD risk factor and the attending physician should deal with it if the higher possible reduction of CVD risk is the aim of any intervention in MetS. Narrative reviews are most useful for obtaining a broad perspective on a topic, including sections on the physiology and/or epidemiology of a topic. Authors search for pivotal papers known to the subject expert and respond with their results to review the hypothesis or question. Systemic review and meta-analysis and are not in direct antagonism with narrative reviews. Where systemic review meta-analysis is applicable (many studies available that test the same hypothesis), it is generally the preferred method. However, in this case there are not enough studies with the same hypothesis or methods or endpoints. The literature search strategy was: Studies evaluating relationships of “MetS” or “arterial hypertension” or “glucose intolerance” or “menopause” or “chronic kidney disease” or “hyperuricaemia” or “hypercoagulability” or “non-alcoholic fatty liver disease” or “dyslipidaemia” or “small dense LDL particles” or “obstructive sleep apnea” or “treatment” with “stiffness,” or “arterial stiffness” or “arterial elasticity,” or “pulse wave velocity” or “prediction,” or “cardiovascular risk” or “death” or “mortality” or “outcome” or “events” "diabetes mellitus" were drawn from PubMed and Cochrane databases until May 2013. Data sources were also identified through manually searching the references of articles using PubMed.The effective means to treat the cluster of all 12 cardio-metabolic risk factors analyzed below and their combinations, with or without the presence of arterial hypertension show promise for the control of AS, leading to CVD risk reduction.The elastic properties of arteries differ along the arterial tree, with more elastic proximal arteries and stiffer distal arteries [6]. AS is largely determined by the structure of the tunica media, which contains the elastic components of the aortic wall, such as elastin fibers and collagen [7]. Several factors influence arterial wall structure. Ageing is closely associated with AS through the fragmentation of elastin fibers, the decrease in elastin/collagen ratio, and the calcification of the tunica media [7]. This process is called arteriosclerosis and should be distinguished from atherosclerosis, which is a disease of the intima. Arterial hypertension is a major determinant of arteriosclerosis and arterial stiffening, through changes in the tunica media that take place earlier than that induced by ageing; this is considered to be the “early vascular ageing”. However, AS contributes to the worsening of arterial hypertension within a vicious cycle [7]. AS is caused by other CVD risk factors besides arterial hypertension, such as smoking, diabetes mellitus, mainly type 2 (T2DM), hypercholesterolaemia, and chronic kidney disease (CKD) [5,8,9]. Arterial stiffening is also increased in patients with established atherosclerotic CVD [7]. AS is characterized by increased pulse wave velocity (PWV) and early return in the ascending aorta of reflected waves, during the systolic phase of the pressure waveform. Thus, systolic central aortic blood pressure (CABP), and consequently LV afterload, is increased. These induce LV hypertrophy (and stiffness) and predispose to LV systolic and diastolic dysfunction [7]. Furthermore, in patients with AS central CABP during the diastole is decreased, thus substantially reducing diastolic coronary artery perfusion. The combination of LV hypertrophy and reduced coronary perfusion leads to myocardial oxygen supply/demand mismatch, and further deteriorates LV systolic and diastolic function [10]. This combination of high systolic and low diastolic CABP leads to an increased value of pulse pressure (PP), which expresses AS and appears to be the most powerful measure to identify those hypertensive patients at greatest CVD risk [10].Among the different non-invasive methods used to assess arterial stiffness is the carotid-femoral PWV has emerged as a gold standard due to its accuracy, reproducibility, relative easy measurement, and low costs. Furthermore, PWV has yielded prognostic value beyond and above traditional risk factors. PWV is assessed non-invasive using the Complior, SphygmoCor, Arteriograph or the Vicorder systems with respective software. Electrocardiogram-gated carotid and femoral waveforms were recorded using applanation tonometry. Carotid-femoral path length was measured as the difference between the surface distances joining the suprasternal notch, the umbilicus and the femoral pulse and the suprasternal notch and the carotid pulse [11]. Carotid-femoral transit time was estimated in 8–10 sequential femoral and carotid waveforms as the average time difference between the onset of the femoral and carotid waveforms. The foot of the pulse wave was identified using the intersecting tangent method. PWV was calculated as the carotid-femoral path length divided by the carotid-femoral transit time. This is an established, non-invasive and reproducible method to determine arterial stiff-ness and no adjustments are required for transit time and path length. Most studies use data on PWV deriving from 16,867 subjects and patients from 13 different centers across eight European countries [11]. Of these, 11,092 individuals were free from overt CV disease, did not have diabetes and were untreated by either anti-hypertensive or lipid-lowering drugs and constituted the reference value population. Prior to data pooling, PWV values were converted to a common standard using established conversion formulae. Subjects were categorized by age and further subdivided according to BP categories. PWV increased with age and BP category; the increase with age being more pronounced for higher BP categories and the increase with BP being more important for older subjects. The distribution of PWV with age and BP category is described and reference values for PWV are established. Normal values are proposed based on the PWV values observed in the non-hypertensive subpopulation with no additional CVD risk factors. Normal median values according to age were 6.0 (range 5.2–7.0) m/s in <30 years old and 10.1 (7.6–13.8) m/s for subjects >75 years old. If participants had AH, median values ranged from 7.2 m/s to 13.5 m/s respectively [11]. Among an adult population that varies widely in age, brachial ankle (baPWV) is significantly and independently associated with aortic PWV as well as leg PWV. Second, decreases in aortic PWV with exercise intervention are related to the corresponding changes in baPWV [12]. These results suggest that baPWV may provide qualitatively similar information to those derived from aortic PWV, a well-established index of central arterial stiffness. In male subjects, the upper limits of baPWV values were 1497/1425, 1518/1513, 1716/1726, 1925/1971, and 2310/2115 cm/s, obtained using two different statistical methods for the age groups of 30-39, 40-49, 50-59, 60-69, and 70 and older, respectively [12]. For females, the upper limits of baPWV values were 1427/1411, 1559/1499, 1733/1739, 1958/1974, and 2721/2577 cm/s for the age groups of 30-39, 40-49, 50-59, 60-69, and 70 and older, respectively [12].The MetS is a cluster of CVD and T2DM risk factors [11]. Patients with MetS are at higher risk for vascular events [12,13] and all-cause mortality [14], even in the absence of clinically evident CVD and/or T2DM [15,16]. However, there are concerns suggesting that when confounding factors such as obesity are accounted for, diagnosis of the MetS has a negligible association with the risk of CVD [17]. This approach encouraged the American Diabetes Association and the European Association for the Study of Diabetes to issue a joint statement identifying concerns on the clinical utility of MetS as a syndrome [18].At the end of the day, all parts recognise that the clustering of several CVD risk factors, regardless if they are considered as a syndrome or not, confer an increased CVD risk, even if that is the just the sum of the risks of MetS components. In 2006 the International Diabetes Federation [19] released a worldwide definition of MetS with the following criteria: central obesity [defined as waist circumference (WC) with ethnicity-specific values] plus any two of the following: raised triglycerides: >150 mg/dL, or specific treatment for this lipid abnormality, reduced high density lipoprotein cholesterol (HDL-C): <40 mg/dL in males, <50 mg/dL in females, or specific treatment for this lipid abnormality, raised BP: systolic BP>130 or diastolic BP>85 mm Hg, or treatment of previously diagnosed arterial hypertension, raised fasting plasma glucose (FPG):>100 mg/dL, or previously diagnosed T2DM [19].. In this consensus, obesity is characterized as the main clinical manifestation of MetS and insulin resistance (IR) as the central pathophysiological aberration. There are another 5 MetS definitions. However it is not the aim of this review to investigate and compare the differences of various MetS definitions, because the review is not about MetS and AS, but goes beyond that analyzing in total the effect of 12 cardio-metabolic risk factors and their combinations. It has been proposed that IR induces AS, which in tern leads to increased CVD risk [20]. Prospective studies have shown that patients with the IR syndrome have a greater increase in AS than age-matched controls without the syndrome [21]. Depletion of nitric oxide (NO) or ineffectiveness of NO mediated vasodilatation associated with the progression of IR to T2DM may induce increased AS [22]. Also increased release of endothelin-1 leads to impaired structural properties of the vessel wall [22]. As a result, the renin-angiotensin-aldosterone system (RAAS) is activated in the diseased vascular beds, with up regulation of the angiotensin II type 1 receptor (AT1R). This results in arterial hypertension, which is a component of MetS, while the increased AT1R mediated activity in the vasculature is central to the development of increased AS, which is further deteriorates IR within a vicious cycle [22]. Intervention that aims at AT1R blockade [such as angiotensin receptor inhibitors (ARBs)] may, therefore, be a valuable treatment for early IR, as antagonism of AT1 receptors is beneficial by itself and also allows angiotensin II to act unopposed on AT2R, activation of which is beneficial in treating arterial hypertension and preventing early AS [22]. Moreover, there is data suggesting that in hypertensive, non-diabetic, older adults, IR is associated with AS, independent of glucose tolerance status [23]. In addition, IR is associated with AS, independent of age, baseline systolic BP (SBP), gender and metabolic status [24]. All the above suggest that MetS through IR, its main pathophysiological aberration, induces AS, regardless of age, gender, arterial hypertension or glycaemic control. Dysglycaemia is one of MetS components in all definitions. A meta regression analysis of data from 20 studies with 95,783 individuals followed for 12.4 years showed that there is a continuous positive relationship between FPG and 2 h plasma glucose, following a 75 g oral glucose tolerance test (2-HPG), with incident CVD events [25]. The 2-HPG is a better predictor of death from all causes and CVD than FPG [26]. There also data on an independent association between the impaired fasting glycaemia (IFG), impaired glucose tolerance (IGT), and T2DM with early AS as well as with total and CVD morbidity and mortality [27,28]. It seems that structural mechanisms connect hyperglycaemia with vascular complications even within prediabetes thresholds [27,28]. These relationships are reported as a cumulative effect of MetS on AS as an entity [29,30,31], while there are relative independent strengths of FPG and 2-HPG associations with AS [32,33]. Thus, it seems that there is a clear relationship of premature arterial stiffening with glucose metabolism disorders, within or outside the T2DM range [33], in MetS patients with or without arterial hypertension.Postmenopausal women tend to have MetS because of hormonal and metabolic changes [34]. CVD incidence increases after menopause and this may be related, at least in part, to IR and AS [34]. From the 9,555 postmenopausal (women) participants of a study, 455 had MetS and normal glucose and BP levels [34]. In addition to a full biochemical screening, adiponectin and PWV (to assess AS) were evaluated. IR was estimated by the homeostatic model assessment (HOMA)-IR. The women were stratified into 3 groups according to their HOMA-IR values. There were significant differences in metabolic parameters between the 3 groups. The mean age, body mass index (BMI), WC, FPG, TG, SBP, diastolic BP (DBP), aortic PWV, and peripheral PWV were all increased according to the degree of IR [34]. Moreover, HDL-C and adiponectin levels were decreased according to the degree of IR. Age, BMI, FPG, TG, insulin, SBP, HOMA-IR, aortic PWV, and peripheral PWV were significantly higher in women with central obesity, while HDL-C and adiponectin were significantly lower in these women [34]. PWV (aortic and peripheral) were significantly correlated with age, WC, total cholesterol, SBP, DBP, insulin, and HOMA-IR. The results of multiple regression analysis indicated that SBP, DBP, and HOMA-IR were independently correlated with PWV and therefore to AS [34]. In the normoglycaemic and normotensive postmenopausal group IR was independently and significantly associated with AS as demonstrated by increased aortic and peripheral PWV [34]. These associations between MetS and AS were also shown in premenopausal women with a cluster of cardio-metabolic risk factors [35].People with the MetS have an increased risk, up to 5 times, of developing CKD in comparison with those without the syndrome [36,37], even in children [38]. A meta-analysis of eleven studies (n = 30,146) showed that MetS and its components are associated with the development of estimated glomerular filtration rate (e-GFR) < 60 mL/min per 1.73 m2 (CKD stage 3 or higher) and microalbuminuria or overt proteinuria [36]. Patients with CKD are at an increased risk not only for ESRD but also for CVD [38]. CKD is a CVD predictor and is practically considered as an ischaemic heart disease equivalent, similar to DM [39]. This is due to the independent harmful effect of CKD itself as well as to CKD related CVD risk factors, such as arterial hypertension and dyslipidaemia [40,41,42,43,44]. A major CKD risk factor is increased AS of large arteries, which is independent of other major confounders [39]. Furthermore, increased AS in central arteries may cause further reduction in GFR in subjects with CKD, within a vicious cycle [45]. This suggests that CKD may interact not only with small but also with large arteries, independently of age, BP level, and classical CVD risk factors [45,46]. On the other hand, arterial stiffening is an independent predictor of survival in ESRD in the general population [47]. Given that statins play a role in arterial “destiffening” [40,41,42,43,44,48,49], it has been shown that the dose and the compound play a major role in achieving improvement of GFR, while reducing AS and CVD risk [50]. Elevated serum uric acid (SUA) levels are related to the presence of MetS, while its levels increase with the increasing number of MetS components (arterial hypertension, dyslipidaemia, DM and visceral obesity) [51], to the degree that many researchers suggest that SUA should be considered a MetS component in a future definition [52]. SUA is also closely related to CKD and is an independent CVD risk factor of total and CVD morbidity and mortality, at least in high risk patients [51,52,53]. SUA seems to be significantly correlated with central systolic BP and AS in both genders [54]. Therefore, SUA may contribute to the pathogenesis of arterial hypertension, mainly through increased wave reflections and contribute thus to the increase of CVD risk [54]. This was confirmed by a study suggesting that in both genders, SUA levels are associated with AS, and this relationship is independent of other conventional risk factors for MetS and atherosclerotic CVD [55]. Similar were the results of a large study with postmenopausal women [56]. On the contrary, a study that included relatively young men and women suggested that SUA levels are associated with an increase in AS, which differs between men and women. Women might be more susceptible to vascular damage associated with high SUA levels [57]. Subjects with hyperuricaemia had significantly higher carotid-ankle PWV in both genders; however the carotid-femoral PWV was higher only in women [57]. Nevertheless, in a recent study it was reported that among 779 subjects (393 men and 234 women), witch had normal SUA levels, those with high-normal SUA levels had an increased AS (higher brachial-ankle PWV), after adjusting for confounding factors [58]. Thus, SUA was identified as an AS risk factor even with high-normal levels, inside the normal range [58].It has been shown that the MetS presents relations and inter-relations between metabolic aberrations (in glucose, salt, insulin, and lipid metabolism as well as coagulation factors) and haemodynamic abnormalities (due to RAAS stimulation, sympathetic overactivity, and decreased NO bioavailability), which are followed by anaemia, hypercoagulability, organ ischaemia, AS, arterial hypertension, as well as renal and LV dysfunction [59]. These might be considered to form the “circulatory syndrome” [59,60]. Within or outside this circulatory syndrome frame MetS is related to increased levels of clotting factors (tissue factor, factor VII and fibrinogen) as well as inhibition of the endogenous fibrinolysis (increased plasminogen activator inhibitor-1 and decreased tissue plasminogen activator activity) [60,61]. Fibrinogen levels correlate with lipid [62] and inflammatory parameters [60,62], suggesting an involvement of adipose tissue-generated inflammatory cytokines [60]. Elevated von Willebrand's factor and factor VIII levels combine with endothelial injury, whereas vitamin K-dependent coagulation correlate with triglyceride levels [60]. All the above seem to inter-relate with low grade inflammation and with AS [59,60]. NAFLD is currently the most common form of chronic liver disease. It affects 20%–35% of general population in western countries, while its prevalence increases up to 70%–90% in people with obesity, MetS or T2DM [63]. NAFLD is considered the hepatic manifestation of MetS; it is closely related to other clinical features of MetS, and thus to CVD risk [64]. Several data have documented that NAFLD associates with other CVD risk factors [64,65]. AS was independently associated with the risk for NAFLD, regardless of classical CVD risk factors [64,65]. Moreover, it was shown in a large study (n = 4,467) that not only the presence but also the degree of NAFLD are associated with AS in non-hypertensive, non-diabetic individuals with or without MetS [66].Dyslipidaemia (low HDL-C and/or high triglycerides) is a MetS component. Patients with hypercholesterolaemia have a higher central pulse pressure and stiffer blood vessels than matched controls, despite similar peripheral BP [67,68]. Low-density lipoprotein cholesterol (LDL-C), not a MetS component, seems to be independently associated with AS, while HDL-C is independently (inversely) associated with AS [69]. Statin therapy leads to a reduction in AS, re-enforcing the concept that stiffness is indeed a modifiable CVD risk factor [67]. It has also been shown that PWV, and therefore AS, is increased in carriers of the lecithin cholesterol acetylo-transferase mutation related to low HDL-C [70]. The triglycerides/HDL ratio, a rough measure of the predominance of small-dense LDL particles over large-buoyant ones [71], is an independent determinant of AS in older adults, younger adults, even in adolescents, particularly in these with central obesity, regardless of the presence of arterial hypertension [72,73].OSA is a highly prevalent condition. About 24% of men aged 30–60 years and 9% of adult women are estimated to have OSA; most of them have MetS [74]. The close association between OSA, overweight/obesity and CVD morbidity and mortality is a major health problem [75]. A plethora of studies have indicated a causal relationship between OSA and arterial hypertension, T2DM, and CVD, while the relative risk for all-cause mortality in patients with OSA is 1.5 in comparison to the individuals that do not have OSA [75]. One of the possible mechanisms for this adverse effect of OSA is increased AS [74,76]. Interventional studies showed a substantial improvement in symptoms associated with OSA (daytime sleepiness, snoring, morning headaches, and reduction in BP), in patients receiving continuous positive airway pressure therapy (CPAP) [74,75]. Moreover, the benefits of effective CPAP on reduction of CVD risk and fatal and nonfatal CVD events have been established in multiple large trials [77]. It has also been clearly shown that after CPAP treatment in patients with OSA there was a significant improvement of all indices of AS [74,78,79,80].After all the distressing data presented above, one question pops up: do we have at our disposal effective means to intervene and tackle the clinical consequences of MetS, and particularly those related to AS? There seem to be several effective tools, from lifestyle advice to pharmacological interventions in AS risk factors, which may substantially contribute to the amelioration of CVD risk, mainly the part related to AS. Aerobic exercise training is associated with lower AS, but little information is available on the required intensity or duration of physical activity. There are data suggesting that both moderate and vigorous physical activity have favorable effects on AS in postmenopausal women, even after adjustment for expected confounders [81]. In the Amsterdam Growth and Health Longitudinal Study it was shown that lifetime vigorous, but not light-to-moderate, habitual physical activity is favorably associated with increased arterial compliance, even after adjustments for sex-specific coefficients [82]. Moreover, adoption of Mediterranean diet beneficially affects AS, even after adjustments for potential confounders [83]. This effect of Mediterranean diet is probably related to its beneficial impact on CVD risk factors, such as BP, central obesity, and dyslipidaemia [83]. Also, in the Amsterdam Growth and Health Longitudinal Study, mentioned above, it was shown that lower intake of fiber throughout the course of young life (from adolescence to adulthood) is associated with AS in adulthood, after adjustment for sex, height, total energy intake, and other lifestyle variables [84].Thus, intense physical activity and adoption of a healthy diet reach in fiber (preferably the Mediterranean diet) are powerful tools to prevent or treat MetS and AS, thus reducing CVD risk [85,86]. However, non-adherence to long term lifestyle changes is very usual and raises important issues for their effectiveness, particularly in cases that they have to be adopted for a life-time period, given that in all interventions the key determinant of success is the adherence level [87].The majority of older individuals develop higher CABP mediated by increasing AS. An increased CABP may lead to target organ damage through a variety of mechanisms [88]. Data indicate that lowering brachial arterial BP does not necessarily correlate with equal lowering of CABP or AS. Pulse pressure varies throughout the arterial tree, resulting in a gradient between central and peripheral pressure. CVD risk factors affect the pulse pressure ratio, and that central pressure cannot be reliably inferred from peripheral pressure. However, assessment of CAPB may improve the identification and management of patients with elevated CVD risk. However, assessment of CAPB may improve the identification and management of patients with elevated CVD risk. BP lowering drugs can have substantially different effects on central aortic pressures and hemodynamics despite a similar impact on brachial BP [89]. Moreover, CAPB may be a determinant of clinical outcomes, and differences in CAPB may be a potential mechanism to explain the different clinical outcomes between the two BP treatment arms in ASCOT [89]. Radial artery applanation tonometry and pulse wave analysis were used to derive CAPB and hemodynamic indexes on repeated visits [89]. The integral system software was used to calculate an averaged radial artery waveform and to derive a corresponding central aortic pressure waveform using a previously validated generalized transfer function. Aortic pressure waveforms were subjected to further analysis by the SphygmoCor software to identify the time to the peak/shoulder of the first and second pressure wave components during systole. The pressure at the peak/shoulder of the first component was identified as P1 height (outgoing pressure wave), and the pressure difference between this point and the maximal pressure during systole (ΔP or augmentation) was identified as the reflected pressure wave occurring during systole. Augmentation index (AIx), defined as the ratio of augmentation to central pulse pressure, is expressed as a percentage: 
+		
+       Aix = (ΔP/PP) × 100
+        
+		where P is pressure and PP is pulse pressure. Pulse pressure amplification (PPA) was expressed as the ratio of CAPB to peripheral (brachial) pulse pressure (PPP): PPA = PPP/CPP [89]. Increased AS, independent of brachial arterial BP, appears to be a novel and independent risk factor for CVD and total mortality [90] in healthy older adults and hypertensive patients [88,91]. These emphasize the need for ambulatory (non invasive) measurement of CABP and AS with equipment recording the 24-h brachial BP, heart rate, and PP too [92]. In a study with 211 patients with uncomplicated arterial hypertension, patients were treated for 1 year [92]. Ambulatory AS index, pulse pressure (PP), and aortic PWV were used to assess the effect of drugs on AS [92]. The results showed that administration of antihypertensive drugs with different hemodynamic profiles, ambulatory AS index and aortic PWV behaved similarly and AS was reduced to the same extent [92]. However, studies in animal models suggest that early brief angiotensin enzyme inhibition (ACE-I) decreased arterial pressure and isobaric wall stiffness in spontaneously hypertensive rats; this was associated with marked alteration of wall structure, while the effects persisted after cessation of early treatment [93]. A meta-analysis of short-term trials in humans showed that ACE-Is were more effective than calcium antagonists and placebo on improving AS [94]. In long-term trials, ACE-I, calcium antagonists, beta-blocker, and diuretic reduced significantly PWV compared to placebo [94]. A review of the effects of antihypertensive drugs suggests that RAAS blockers maintain compelling indications in particular CVD conditions, such as microalbuminuria, LV hypertrophy, and AS. The above provide further support to a drug-based antihypertensive strategy [94]. Moreover, the optimal safety profile of several antihypertensive drugs, in particular of ACE-Is, ARBs and renin inhibitors, allows their use in the very early stages of arterial hypertension, as suggested by recent clinical trials [95]. In a meta-analysis with meta-regression of randomized controlled trials, it was demonstrated that ACE-Is reduce AS and wave reflections in patients with different pathological conditions [96]. However, due to the lack of high quality and properly powered randomized controlled trials, it is not clear whether ACE-Is are superior to other antihypertensive agents in their effect on AS [96]. The ability of ACE-Is to reduce AS appears to be independent of their ability to reduce BP [96]. In regard to atenolol, its use as a first choice antihypertensive agent was limited by the results of the Central Artery Function Evaluation (CAFE) study [89]. CAFE was a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT), which examined the impact of two different BP lowering-regimens (the amlodipine+/−perindopril-based vs. the atenolol+/−thiazide-based therapy) on CABP and hemodynamics [89]. CAFE showed that despite similar brachial SBP between treatment groups, there were substantial reductions in CABP with the amlodipine regimen [96]. Cox regression analysis showed that CABP and central PP was significantly associated with the composite outcome of total CVD events and development of renal impairment (CKD) in the CAFE study population [96]. These differences in CABP may be a potential mechanism to explain the different clinical outcomes between the 2 BP treatment arms in ASCOT [89]. A post hoc analysis of ASCOT showed that the combination of atorvastatin (10 mg/d) with the amlodipine+/−perindopril-based therapy produced a huge reduction in CVD events (by 52%) in comparison to placebo [97], while the combination of atorvastatin (10 mg/d) with the atenolol+/−thiazide-based therapy produced a much smaller reduction in CVD events (by only 15%) in comparison to placebo [97]. Given that atorvastatin did not significantly affect the CABP in this large cohort of the CAFE study [97], these results designated the beginning of the end of the use of atenolol as a first choice antihypertensive agent. Statins seem to reduce AS in patients with dyslipidaemia [48,49,68,69,98], with or without CVD [99,100], with menopause [101], CKD [102], in overweight and obese [103], with T2DM [104], with T1DM [105], in healthy men [106], in patients with rheumatoid arthritis [107], and in elderly patients with arterial hypertension [108]. This effect of statins on AS is probably not a drug class effect, as suggested by the fact that the compound and the dose play a significant role in the degree of AS reduction [99,100,101,102,103,104,105,106,107,108]. It seems that potent statins, such as atorvastatin and rosuvastatin are more effective in reducing AS, reducing thus excessive CVD risk [109].Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting between 6% and 8% of women in reproductive age [110]. It is associated not only with reproductive and cosmetic problems, but also with significantly increased risk of metabolic dysfunction including IR with consequent compensatory hyperinsulinaemia, dyslipidaemia, systemic inflammation, increased oxidative stress, and endothelial dysfunction [110]. In the long-term, women with PCOS may develop T2DM, hypertension and atherosclerosis; ultimately, they are more likely to suffer from CVD and cerebrovascular diseases [110]. In patients with IR due to PCOS metformin improved aortic PWV and brachial PWV, CABP, endothelium-dependent and endothelium-independent vascular responses [111,112]. Metformin also reduced weight, waist circumference, and triglycerides and increased adiponectin [111]. Thus it seems that even short-term metformin therapy improves AS and endothelial function in young women with PCOS [111,112]. In addition, metformin has a considerable beneficial effect on endothelial function and AS in patients with MetS [113]. Furthermore, it has been demonstrated that increase in adiponectin level after treatment with the combination of two insulin sensitizers (pioglitazone plus metformin) may improve AS in patients with T2DM [114,115]. On the contrary the addition of liraglutide, a glucagon-like peptide-1 agonist (GLP-1 agonist), in patients with T2DM on metformin monotherapy, improved several CVD risk markers, beyond glycaemic control, but failed to improve AS [116]. Likewise, in a recent comparative study sitagliptin, but not glibenclamide, demonstrated a significant beneficial effect on BMI and triglycerides [117]. However, AS, BP, oxidative stress, and inflammatory status were not significantly changed by adding sitagliptin or glibenclamide to metformin-treated patients with T2DM [116]. From another ankle, metformin treatment was associated with significant decrease in AS after 1 year of treatment in patients with MetS and NAFLD, regardless of glycaemic control [118]. These beneficial effects suggest that metformin exhibit its vascular effects through glycaemic control-independent mechanisms [118]. In patients with T2DM three long-term different insulin regimens achieved overall good metabolic control with significant differences, however, in postprandial glucose increments [119]. The regimens achieving better vascular function (less AS) were those that induced a better postprandial glucose control [119]. Even after switching from premixed human insulin 30/70 containing rapid-acting insulin to biphasic insulin aspart 30/70 preparation [119], the later improved AS more than the former [120]. This improvement of AS may be associated with improvement of postprandial glycaemia [120].This review has the limitation of being narrative instead of systemic. However, conditions mentioned in introduction, mainly the lack of many original papers available that test the same hypothesis, did not allow us to write a systemic review and perform a meta-analysis. AS and CABP are important independent CVD risk factors. A cluster of disease states related to MetS, they are either its components (arterial hypertension obesity, dyslipidaemia, dysglycaemia) or not (renal dysfunction, hyperuricaemia, hypercoaglutability, menopause, non alcoholic fatty liver disease, and obstructive sleep apnea) contribute, in an additive manner, to the increase of AS and thus to excessive CVD risk, often within a vicious cycle. Lifestyle measures, antihypertensive, hypolipidaemic, and hypoglycaemic drugs as well as CPAP in patients with OSA substantially contribute to the reduction of AS and finally to the decline in CVD risk.There is a great unmet need for well planed, prospective, randomized, controlled, and long term interventional trials to assess the clinical benefit of regimens that improve AS. The examination of multifactorial interventions that showed reductions in CVD risk [121,122] might be even more useful and should also be tested. AS and wave reflections have been widely investigated in old and/or hypertensive subjects for several reasons. In subjects >50 years of age, ventricular ejection fraction tends to be reduced, so that AS and amplitude and timing of wave reflections become the main determinants of increased systolic BP and PP. From the haemodynamic factors that influence PP, two have been shown to independently predict CVD risk: aortic stiffness, and early return of reflected waves to the heart, evaluated from pulse wave analysis. Finally, increased PP is a strong predictor of CVD risk in subjects with MetS, and/or all other cardio-metabolic risk factors analyzed above. It seems that in most cases of cardio-metabolic risk factors effective primary treatment of this factor reduces AS and PP. This is not always easy. Thus, symptomatic treatment of common determinants of AS, regardless of the initial cause, such as dyslipidaemia, AH, diabetes, and smoking is the intervention of choice. Large long-term interventional studies are needed to investigate drug classes and particular drugs for their effect on AS and finally CVD and T2DM incidence in patients with a cluster of cardio-metabolic risk factors. Above all, we should abandon the dispute on the existence of MetS. The cluster of all these 12 cardio-metabolic risk factors analyzed above and their combinations indeed increases AS and CVD risk. This can not be disputed by anyone regardless of how we call it. A more rigorous review is needed to confirm the findings of this narrative review.2 h plasma glucose, following a 75 g oral glucose tolerance testangiotensin enzyme inhibitionangiotensin II receptor blockersarterial stiffnessAnglo-Scandinavian Cardiac Outcomes Trialangiotensin II type 1 receptorbody mass indexblood pressurecentral aortic blood pressureCentral Artery Function Evaluation studychronic kidney diseaseglucagon-like peptide-1 agonistcontinuous positive airway pressure therapycardiovascular diseasediastolic blood pressureestimated glomerular filtration rateend stage renal diseasefasting plasma glucosehigh density lipoprotein cholesterolhomeostatic model assessmentimpaired fasting glycaemiainsulin resistancelow-density lipoprotein cholesterolleft ventriclemetabolic syndromenon-alcoholic fatty liver diseasenitric oxideobstructive sleep apneapolycystic ovary syndromepulse pressurepulse wave velocityrenin-angiotensin-aldosterone systemsystolic blood pressureserum uric acidtype 2 diabetes mellituswaist circumferenceThis review was written independently; no company or institution supported the authors financially or by providing a professional writer. Some of the authors have given talks, attended conferences and participated in trials and advisory boards sponsored by various pharmaceutical companies in the past, but they have no conflict of interest.

Med-MDPI/diseases/diseases-02-01-00001.txt

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).First, I would like to warmly welcome the readers, contributors, and editorial board members of Diseases journal as a new entrant to the international periodicals industry.The current offerings of scientific journals are very rich. One may wonder why there should be another journal, as well as what such a journal (Diseases) will be like.Scientific topics (including medical ones) are multiplying quickly. When a multidisciplinary approach is required, existing journals often do not meet the resulting publishing needs. All medical topics’ publishing needs are quite sufficiently satisfied by (extremely) specialized journals. However, day by day, we are losing the holistic perspective that is often required to understand complex mechanisms. Also, the importance of reading and discussing different pathologies in the same journal/issue is, undoubtedly, not only a pleasure (or a brain work-out), but actually an enrichment for the readers (i.e., the investigators). If an open access journal devotes its efforts to these aims, the results can actually be intriguing; such an open access journal can reach a large community and involve said community in both the journal’s launch and in its development.MDPI, which is now operating one of the most well-established, open-access publishing platforms, has never engaged before in such a challenge. Although MDPI is aware of the intrinsic difficulties of such a project, we now have the right colleagues to face the prospective challenges.What will Diseases journal be like? This depends, to a large extent, on the inputs the Editors and Editorial Board will receive from both the contributors and the readers: on the one hand, the Editors will give as much space as possible to minor, but important, rare syndromes and conditions; on the other hand, there will be maximal alertness with respect to identifying conditions of increasing scientific interest on the basis of external warnings. Either molecular, biochemical or clinical approaches are welcome and papers applying an interdisciplinary approach will be the most welcome.This periodicals project is arduously challenging and ambitious, but I am confident that the high level expertise of the Editorial Board Members and of all our reviewers, readers, and colleagues at MDPI will enable us to achieve our expected results.I invite all of you to collaborate to help us make Diseases journal an important journal: your journal.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).In the class Kinetoplastida, we find an order of parasitic protozoans classified as Trypanosomatids. Three major pathogens form part of this order, Trypanosoma cruzi, Trypanosoma brucei, and Leishmania, which are responsible for disease and fatalities in millions of humans worldwide, especially in non-industrialized countries in tropical and sub-tropical regions. In order to develop new drugs and treatments, the physiology of these pathogenic protozoans has been studied in detail, specifically the significance of membrane transporters in host parasites interactions. Aquaporins and Aquaglyceroporins (AQPs) are a part of the major intrinsic proteins (MIPs) super-family. AQPs are characterized for their ability to facilitate the diffusion of water (aquaporin), glycerol (aquaglyceroporin), and other small-uncharged solutes. Furthermore, AQPs have been shown to allow the ubiquitous passage of some metalloids, such as trivalent arsenic and antimony. These trivalent metalloids are the active ingredient of a number of chemotherapeutic agents used against certain cancers and protozoan parasitic infections. Recently, the importance of the AQPs not only in osmotic adaptations but also as a factor in drug resistance of the trypanosomatid parasites has been reported. In this review, we will describe the physiological functions of aquaporins and their effect in drug response across the different trypanosomatids.Trypanosomatid parasites are single celled pathogenic organisms considered a major cause of morbidity and mortality in tropical and subtropical regions worldwide. Pathogens of this order are the Leishmania species (causative agents of leishmaniasis), Trypanosoma brucei (African sleeping sickness), and Trypanosoma cruzi (Chagas disease). A total of 88 countries are reported to be endemic for leishmaniasis, with new case counts approximately 1.5 to 2 million per year [1]. Rapid increase of drug-resistant cases has further aggravated the situation [2]. Sleeping sickness threatens about 60 million people in 36 sub-Saharan Africa countries [3,4]. Fortunately, there is a recent trend toward a lesser number of reported cases [3]. However, patients with African trypanosomiasis are still a major challenge to clinicians, as diagnosis for central nervous system (CNS) involvement is problematic and treatment at CNS-stage can be highly toxic and fatal if left untreated. Chagas disease is a chronic parasitic infection that affects about 8 million people in the Latin American countries [5].The trypanosomatid parasites undergo diverse and remarkably complex life cycles with interspecies transmission, frequently between an insect and a mammal, invasion of various cell types and/or extra- cellular growth in the blood stream, development of dormant stages in the mammalian host and differentiation to transmittable and infectious forms within the insect gut [6]. As they frequently travel between insect and mammalian hosts, they face severe osmotic challenges [7]. In the insect vector, the parasites differentiate into infective forms and migrate to the destined organ for successful perpetuation of the infection whereas within the mammalian host they need to find appropriate intra and/or extracellular milieu to evade the host defense system followed by disease progression. Several studies in other systems indicate that osmotic cues in the form of altering water and other solutes flux play a significant role in cellular morphogenesis and migration [8,9]. To respond to these osmotic cues all living cells require efficient regulation of these rapid water and solutes movements; however, the lipid bilayer membranes that encapsulate all living cells and several subcellular organelles are greatly impermeable to water and other polar solutes. Therefore, to overcome these indispensable biological barriers all living cells are obligated to employ membrane transporter(s) that will allow rapid and regulated movement of water and other non-polar solutes in and out of the cells and/or cellular compartments in response to extra- and/or intracellular signals. This puzzle baffled the scientific community for several decades until Peter Agre and co-workers demonstrated a channel-forming, integral membrane protein that facilitates water transport [10,11]. As the regulated movement of water is fundamental to all life process later studies reported the ubiquitous presence of the members of this channel protein [12]. These members are collectively called aquaporins (AQP). AQPs belong to MIPs (Major Intrinsic Protein), a super-family of integral membrane proteins. These small integral membrane channels can be functionally classified into two sub-groups, water specific transporters (orthodox aquaporins), and aquaglyceroporins, which allow transport of small-uncharged polar solutes such as glycerol, metalloids and urea [13,14]. The physiological roles of AQPs are more extensive: (i) several recent studies have suggested that some members of orthodox AQP are permeable to a variety of substrates including gases [15], ions [16], and (ii) some members of aquaglyceroporins are adventitious transporters of trivalent metalloids such as antimonite (SbIII) and arsenite (AsIII) [17]. Structurally, most aquaporins share a common topology of six trans-membrane helices and two half-helices that fold into the center of the channel forming an hour-glass structure [18].Physiological functions and regulation of AQPs have been extensively studied in human and several other model systems. By far the most extensively studied parasitic protozoan AQP is from Plasmodium. Plasmodium expresses a single AQP. AQPs from several species of Plasmodium share a high degree of homology with each other, mammalian AQPs and glycerol facilitator (GlpF) of Escherichia coli. Plasmodium AQPs are permeable to water, glycerol, urea, and several other polyols. Although this AQP is not essential for the parasite survival, glycerol transport through this channel plays a significant role during gametocytes development inside the red blood cells [18]. Several aquaporins have been identified in trypanosomatid parasites, from three in Trypanosoma brucei, [19,20] to four in Trypanosoma cruzi [20,21] to five in Leishmania [14,20]. When compared to E. coli and mammalian aquaporins, parasite AQPs are generally more robust water transporters. It appears that most protozoan parasites aquaglyceroporins are bifunctional and conduct both water and glycerol at reasonable rates [18].High potential chemotherapy targets can be found at the parasite-host interface [20,22]. Integral membrane proteins such as AQPs play an important role on the parasitic organism adaptations and survival during all of its life cycle stages, even more so at the parasite host interface. Accordingly, AQPs could be attractive drug targets and/or mediators of specific drugs such as arsenic and antimony [20]. In P. falciparum glycolysis-related metabolites such as methylglyoxal and dihydroxyacetone inhibited proliferation at 200 μM and 3 mM, respectively [23]. Although the levels of sensitivity against these compounds are far from ideal for clinical studies, these show promise that cytotoxic AQP substrates can be used for treating parasitic infections. Metalloids, such as arsenic and antimony, permeability of AQPs have also been exploited to treat several human diseases for more than a century. Arsenical and antimonial compounds are still being used as the primary line of treatment against trypanosomiasis and leishmaniasis [24,25].After the discovery of the first AQP by Peter Agre in 1992, knowledge about human and other mammalian AQPs has expanded enormously within 20 years. However, the physiological significance of parasitic protozoan, especially trypanosomatid AQPs and their role in drug response are still in its infancy. Therefore, in this review, we discuss the current understanding and roles of kinetoplastid AQPs in trypanosomatid parasites with respect to physiology and drug response.In order to thrive, leishmaniasis needs to complete a triad of complex interactions between the Leishmania parasites, the sand fly vector, and the mammalian host. Female sand flies of the genus Phlebotumus (Old World) and Lutzomiya (New World) are the most important vectors responsible for the transmission of the disease. Leishmania parasites change their morphology depending on their stage in life cycle. These distinctive morphologies are known as promastigotes and amastigotes. Promastigotes are extracellular and characterized for their slender spindle like body with an anterior flagellum, and reside in the sand fly gut. Promastigotes proliferate by binary fission. Once promastigotes are phagocytized by mammalian macrophages they are transformed into amastigotes, which appear as oval-shaped and aflagellated structures that reside inside phagolysosomes. Amastigotes, which also proliferate by binary fission, finally burst out of the infected cell to infect fresh macrophages and perpetuate the cycle. The life cycle of Leishmania starts when the sand fly ingests an infected blood meal from an infected host. After being ingested by the fly, amastigotes transform into promastigotes. Rapid morphological transformation and development of the parasite inside the vector is prompted due to the change in conditions (such as, increased pH and decreased temperature) between the mammalian host and the sand fly midgut. Initially, within the peritrophic vesicle amastigotes transform into weakly motile and dividing forms with a short flagellum at the anterior end of the parasite, known as procyclic promastigotes [26]. About 48 to 72 hours later, procyclics differentiate into non-dividing and highly motile nectomonad promastigotes that escape the peritrophic vesicle and move into the midgut lumen. Later at the anterior end of the midgut, nectomonads differentiate into proliferating leptomonads. Finally, leptomonads differentiate into two morphologically distinct forms: non-motile haptomonads promastigotes and highly motile with long flagellum metacyclic promastigotes, which are infective to mammalian hosts [27,28,29]. In its next blood meal the sand fly will inoculate the infectious metacyclis into the mammalian host. Transmission of cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL) caused by L. infantum and L. chagasi can be both zoonotic or anthroponotic whereas only anthroponotic transmission has been reported for VL caused by L. donovani [30].The disease caused by Leishmania is called leishmaniasis. The disease is endemic to 88 countries in five continents. The majority of cases are reported from countries in the tropics and subtropics. Depending on the species the spectrum of disease can range from self-healing but disfiguring cutaneous leishmaniasis (CL) to more debilitating mucocutaneous leishmaniasis (MCL) to potentially fatal visceral leishmaniasis (VL). CL is caused by several species; in the Old World L. major and L. tropica are primarily responsible whereas in the New World L. mexicana, L. braziliensis, L. panamensis and L. amazonensis are the primary causative agents. MCL is endemic to the New World and caused principally by L. braziliensis. VL is reported to be caused by L. donovani (Asia and Africa), L. infantum (Europe) and L. chagasi (New World) [30,31,32]. Even though disease incidence is difficult to accurately assess, an estimate of 10 to 12 million people are infected worldwide with 350 million people at risk for transmission, and an annual approximate of 2 million new cases (about 1.5 million CL/MCL and rest VL) and over 50,000 deaths [33]. Additionally, current emergence of Leishmania/HIV co-infection is imposing a real threat in various parts of the world [34]. In CL parasites are confined to the dermal macrophages of the inoculation site(s) and inflict localized ulcerating but painless lesion(s). However, depending on the host immunological status the infection by L. aethiopica (Old World) and L. amazonensis (New World) can spread over large areas to cause diffused CL (DCL). Clinical presentation of DCL resembles lepromatous leprosy. [35,36]. CL caused by L. braziliensis usually takes longer time to heal compared to CL caused by other species in the New World. More importantly, in 1 to 3% of L. braziliensis infected patients, parasite can metastasize into mucous membrane of the naso-pharynx to develop MCL. The infection is usually chronic and metastatic spread often causes severe mutilation of soft tissues and cartilages [36]. Presence of a symbiotic RNA virus in L. braziliensis genome was reported to be associated with severity of the clinical outcome of MCL [37]. In VL, also known as kala-azar, parasites infect macrophages of the visceral organs primarily spleen, liver and bone marrow. VL is almost always fatal if left untreated. Incubation period of VL can vary from few months to several years. Initial skin lesion at the inoculation site is not apparent in VL and the onset of the disease is usually gradual with nonspecific symptoms, such as, general feeling of illness. Later, fever may occur occasionally. Once the disease is established, fever may occur more regularly with double or triple peak each day. Hepatosplenomegaly and inguinal lymphadenopathy along with occasional abdominal pain or abdominal discomfort are common clinical signs at this stage of the disease. At more advanced stages darkening of the skin color may happen [36,38]. Post-kala azar dermal leishmaniasis (PKDL) is a sequel of VL where the causative agent is L. donovani. PKDL can happen in up to 20% cases in Indian subcontinent and >50% in Sudan after clinical cure of VL. In PKDL, the parasites become dermotropic and infect dermal macrophages. In India, PKDL may appear after years to decades of clinical cure of VL whereas in Sudan it appears within few months of cure. Except for cosmetic unattractiveness, PKDL usually does not cause any clinical complication or physiological discomfort. Consequently, many patients do not seek any treatment. This is an important factor in the epidemiological point of view as PKDL patients are potential human reservoir for the anthroponotic transmission cycle of L. donovani [38].Organic pentavalent antimonial compounds, stibogluconate (Pentostam) and meglumine antimoniate (Glucantime) have been the first line treatment against all forms of leishmaniasis for more than six decades. However, the clinical efficacies of these drugs are currently being challenged by the emergence of acquired resistance [39]. Indian subcontinent is a major contributor of VL. At present, more than 60% of Indian VL patients are unresponsive against antimonial treatment [40]. Pentavalent antimonials [Sb(V)] are pro-drugs and they are reduced to trivalent antimony [Sb(III)]—the active form of the drug (Figure 1) [41]. Whether the reduction of pentavalent to trivalent compound takes place either in the macrophages, in the parasite or in both is still unknown [42]. Entry mechanisms of antimonials into macrophages and phagolysosomes are yet to be identified. Nevertheless, Leishmania aquaglyceroporin 1 (LmAQP1), is the first Sb(III) facilitator identified in Leishmania (Figure 1) [14]. The second line of treatments includes amphotericin B, Alkyl-lysophospholipids (ALP) such as miltefosine and edelfosine, and pentamidine. Although miltefosine was originally developed as an antineoplastic drug, it also demonstrated significant antileishmanial activity. Miltefosine is the first oral antileishmanial drug approved in India. It has been successfully used against VL, including antimony unresponsive cases in India. However, adverse gastrointestinal, hepatic and renal side effects have been observed. In addition, contraindications with women in childbearing age along with high cost are major limiting factors. Even though clinical resistance to miltefosine has yet to be reported, it has been observed that after 9 to 12 months of successful treatment there is patient relapse, whether this is because of reinfection or developed resistance by the parasite it is yet to be studied [43]. Amphotericin B deoxycholate (AmpB) is currently the first line of treatment against antimony unresponsive VL in India. Unfortunately, as in miltefosine, adverse side effects are major limiting factors of AmpB treatment. Although it has been shown that lipid formulations of amphotericin B are highly promising in respect to cure rate and toxicity, high cost of these lipid formulations is an impediment for mass scale use in the endemic countries such as India. More second line options, such as, allopurinol, atovaquone, fluconazole, paromomycin, and sitamaquine are in various stages of clinical trials [40]. Diagrammatic representation of proposed antimonial transport mechanism in a Leishmania infected macrophage. Pentavalent Antimony [Sb(V)] is transported into the macrophage, reduced to trivalent antimony [Sb(III)], and consequently transported into the phagolysosome. Finally, LmAQP1 facilitates the transport of Sb(III) into the parasite cytoplasm.To date, LmAQP1 is the only reported Sb(III) transporter in Leishmania. Disruption of one of the two LmAQP1 alleles in Leishmania major resulted into a 10-fold increase in resistance to Sb(III) [44]. This has been corroborated by downregulation of LmAQP1 mRNA levels in clinical isolates of drug resistant L. donovani [45,46]. On the other hand, over expression of LmAQP1 makes the wild type parasites >100 fold more sensitive to Sb(III). More importantly, overexpression of LmAQP1 can reverse the phenotype in drug resistant isolates [47]. Taken together, LmAQP1 plays a significant role for the successful treatment of leishmaniasis using antimonials. Other factors such as levels of trypanothione, antimonite reductase and antimony-TSH conjugate exporter also play an important role in generation of drug resistance phenotype [48]. The L. major genome encodes five aquaporins: LmAQP1, LmAQPα, LmAQPβ, LmAQPγ, and LmAQPδ. While LmAQP1 shows strong similarity to bacterial aquaglyceroporin GlpF and human AQP9 the other L. major aquaporins (LmAQP α-δ) are closer to plant aquaporins [18]. LmAQP1 is an avid water transporter, and the conduction capacity of this aquaglyceroporin is 65% of that of human AQP1. In contrast to Plasmodium and Trypanosome AQPs, which are inhibited by mercurials, LmAQP1 is a mercurial independent channel. LmAQP1 also helps in the conduction of glycerol, glyceraldehydes, dihydroxyacetone (DHA) and sugar alcohols. Expression of LmAQP1 is limited to the flagellum of promastigotes whereas in amastigotes it is localized to the flagellar pocket, the rudimentary flagellum and the contractile vacuoles [47]. Furthermore, LmAQP1 plays a major role in volume regulation and osmotaxis, two important factors to overcome the osmotic challenges the parasite faces during its life cycle [47]. Therefore, it is evident that LmAQP1 plays an important role in Leishmania physiology and drug resistance. At physiological pH, the trivalent metalloids behave as molecular mimics of glycerol, and are conducted through aquaglyceroporin channels. The extracellular loop C of LmAQP1, which connects the adjacent transmembrane helices 4 and 5 of the protein, plays an important role in solute selectivity and function of the channel. In the absence of crystal structure, our structure-function studies have identified the critical residues of loop C for channel selectivity and function of LmAQP1. We identified that glutamate152 of loop C is responsible to discriminate between metalloids and glycerol [49]. Alanine 163 in loop C resides near the pore mouth and it is critical for the channel function, as alteration of this alanine may inactivate the channel function [50]. Expression of LmAQP1 in Leishmania is highly regulated. In absence of definitive promoters, Leishmania regulates its genes at post-transcriptional and/or post-translational levels. Recently, we reported that mitogen activated protein kinase 2 (MPK2) positively regulates the stability of LmAQP1 by phosphorylation at threonine 197. Dephosphorylation made LmAQP1 more susceptible to degradation. Similarly, an altered MPK2 that was incapable of phosphoryl transfer also destabilized LmAQP1 (Figure 2). Interestingly, phosphorylation at Thr197 also affected the localization of LmAQP1 and (Figure 2) altered the drug sensitivity and osmoregulatory activity of the parasite [51]. Altogether, LmAQP1 seems to play a major role in the ability of the parasite to cope with the osmotic challenges presented by the vector and host. Downregulation of LmAQP1 is also responsible for antimonial resistance. Hence it is tempting to propose that agonists and antagonists of LmAQP1 may be used to alter drug resistance and/or for transmission intervention respectively. Immunofluorescence of L. donovani promastigotes transfected with either wild type or altered LmAQP1 and/or LmMPK2. A) overexpression of LmAQP1, B) co-expression of LmAQP1 and MPK2, C) overexpression of T197A LmAQP1, D) co-expression of LmAQP1 and MPK2 K42A. Cells were stained for LmAQP1 (green), α-tubulin (red); and DNA (blue). The three fluorescence images were finally merged.Human African Trypanosomiasis (HAT) also known as sleeping sickness is caused by Trypanosoma brucei.This vector-borne disease is endemic to the sub-Saharan Africa. The life cycle of T. brucei is complex and divided into insect vector stage and mammalian host stage. During blood feeding, an infected tsetse fly inoculates metacyclic trypomastigotes into the skin of the host. From the skin, parasites enter the bloodstream via lymphatic system and transform into elongated bloodstream trypomastigotes, which multiply by binary fission and can later invade central nervous system (CNS). Trypomastigotes are highly pleomorphic; they can range from slender bodied with long flagellum about 33 µm in length to as short as 14 µm, stumpy and without free flagella. The stumpy forms with a rudimentary free flagella are non-dividing and cannot be transformed back to slender free flagellated form within mammalian hosts. When a fly feeds on an infected individual, only the stumpy non-free flagellated forms are transformed into dividing procyclic trypomastigote forms in the insect midgut. After approximately two weeks, procyclics migrate to the salivary glands. In the salivary glands, procyclics get attached to the epithelial cells and transform into dividing epimastigotes. After 2 to 5 days, some of the epimastigotes give rise to non-dividing stumpy metacyclic trypomastigotes, which are infective to mammals. The parasite takes about 3 weeks to complete the developmental cycle within the fly, and once infected the fly remains infective for the rest of its life [36].HAT is eventually fatal if left untreated. Most of the cases are reported from poor and remote rural areas. HAT endemic zone has a strong correlation with the distribution of its vector, the tsetse fly (Glossina spp), which is endemic to the 37 sub-Saharan African countries. Approximately 60 million people are at risk but only about 15% are under surveillance [52]. The disease ranks at 7th in the list 17 most neglected tropical diseases [53].T. brucei has three morphologically identical sub-species- T. b. rhodesiense (East African or rhodesian HAT), T b. gambiense (West African or gambian HAT) and T brucei brucei (Animal African trypanosomiasis) [36]. The first two sub species can infect humans while T b. brucei is not infective to humans. Depending on the growth phase of the parasite and the infested organs, the clinical symptoms of HAT are divided into two distinct and successive phases. At the beginning, growth of the parasite is restricted to the hemolymphatic system while at later stages parasites infiltrate into the CNS. The first phase symptoms are more acute and characterized by febrile lymphadenopathy and nonspecific hepatosplenomegaly and skin rashes. The second phase is more chronic, and patients develop meningoencephalitis with headaches and extensive neurological changes culminating into severe sleep disorders resembling narcolepsy, convulsions, and semi-coma eventually leading to death. The disease progression caused by T b. gambiense and T b. rhodesiense is not similar. Gambian HAT is characterized by a chronic progressive course with clear demarcation between phases and an average duration of around 3 years. On the contrary, rhodesian HAT is considered to be ‘acute’ as it represents with acute septico-pyaemia-like febrile illness and little phase demarcation that leads to fatality within months [36,52,54,55].Treatment choices against HAT depend on the subspecies involved and the stage of the disease. At the hemolymphatic phase of infection pentamidine, an aromatic diamidine, and suramin, a colourless, polysulphonated symmetrical naphthalene derivative, are the drugs of choice [56]. However diagnosis is often late, especially in case of gambian HAT, and by the time of diagnosis the parasite has advanced to the CNS. HAT with CNS involvement is harder to treat because treatment choices are often limited by (i) inefficient penetrability across the blood brain barrier (BBB) and (ii) toxicity. Melarsoprol, a trivalent arsenical, and eflornithine, an ornithine analogue, or nifurtimox-eflornthine combination can reaches therapeutic levels inside the CNS [54]. Pentamidine is being used to treat HAT since 1930s and it is still the first choice of drug to treat the hemolymphatic phase of gambian HAT. A daily or alternate day deep intramuscular injection of 4 mg kg−1 body weight for total 7 to 10 injection is the recommended dosage regimen [57,58]. However the drug is ineffective against CNS stage of HAT because (i) serum binding and tissue retention reduce the availability of free drug to cross the BBB and (ii) even if a low amount of pentamidine cross the BBB, it is taken out quickly by P-glycoprotein and multidrug resistance-associated proteins [57]. Pentamidine can be selectively accumulated inside the parasite cells up to millimolar levels. It primarily accumulates in the mitochondria followed by the nucleus and the acidocalcisomes. As a diamidine, pentamidine has high affinity for nucleic acid and it disrupts the mitochondrial genome especially the kinetoplast. High amount of pentamidine accumulation in the mitochondria also disrupts the mitochondrial membrane potential [57]. Use of pentavalent organic arsenicals as anti-trypanosomal agents dated back to 1900s. Less toxic melarsoprol with trivalent arsenic was introduced during 1940s. Since then it has been used constantly to treat HAT especially the late stage infection. Lipid solubility of melarsoprol formulation makes it permeable to BBB. It has been established that As(III) has strong affinity for glycerol-3-phosphate dehydrogenase, inhibit glycolytic enzymes and form a stable complex with trypanothione to form MelT thereby disrupting the energy and redox metabolism of the cells [4,56,59]. Although this drug is equally effective against both gambian and rhodesian late stage HAT, it is no longer the first line of treatment against gambian HAT primarily because of its toxicity, and reports of unresponsive cases due to drug resistance. However, melarsoprol is the only available option to treat CNS stage of rhodesian HAT [4]. Although the current incidence rate of HAT is promisingly decreasing [3], limited options for treatment along with prolonged use of same anti-trypanosomal agents have generated alarming levels (20%–30% of cases in some areas) of melarsoprol unresponsiveness [57] and thus it could potentially revert back to a new epidemic form of HAT.Only 3 AQPs have been identified and characterized in T. brucei namely TbAQP1, TbAQP2 and TbAQP3 [19,20]. TbAQP1 is localized in the flagellar membrane whereas TbAQP3 is localized in the pelicular membrane independent of the developmental stages [60,61]. Interestingly, TbAQP2 is confined to flagellar pocket in bloodstream forms and it is redistributed over the pelicular membrane in insect stage forms [61]. All characterized AQPs from T. brucei are aquaglyceroporins, they transport water along with other small uncharged molecules including glycerol, urea and trivalent metalloids [19,62]. Water and glycerol transport capacities for all TbAQPs are similar. It is presumed that as the blood stream form of T. brucei thrives under constant conditions in blood, it is never exposed to the osmotic challenges; however, when the parasites pass through renal medulla or are ingested by tsetse fly they face rapid changes in environmental osmolarity. Similarly, transmission from fly to mammals is also a massive osmotic insult [21,61]. Depending on the developmental stages T. brucei differentially regulates the abundance and localization of different AQPs [61]. Taken together, it is tempting to speculate that TbAQPs play critical roles to cope with these osmotic bottlenecks. Likewise, at the advance stage of T. brucei infection when parasite migrates to CNS, differential osmolite concentrations of blood and CSF (cerebrospinal fluid), especially low concentrations of glucose [63] glycerol [64] and urea [65] might send osmological cues via AQP for the navigation from blood to CSF. AQP plays significant roles in directional movements of cells as well as determining the cell shapes [12]. Several post-translational modifications of AQPs, such as, phosphorylation via protein kinases and altered cyclic adenosine monophosphate (cAMP) levels are known to modulate AQP localization thereby aiding their functionality and regulate osmotactic movements and cell shape [66,67,68,69]. T. brucei also experiences differential MPK activities and cAMP levels throughout their life cycle. For instance, TbMPK2 is necessary for developmental progression within the fly whereas TbMPK5 down regulation and high level of cAMP promote blood stream stumpy form formation [70,71,72]. Differential localization of different TbAQPs at different lifecycle stages [60,61] may be related to these kinase mediated phosphorylation events. Additionally, transcript levels of different TbAQPs modulated depending upon the developmental stages of the parasite [60,61]. Taken together, it is highly likely that TbAQPs play significant roles (i) in directional movement of parasite from hind gut to salivary gland within the vector, (ii) changing the shapes depending on the developmental stages (iii) to cope with the osmotic insult, and (iv) in maintaining the glycerol homeostasis to avoid inhibition of glycolytic reaction.Evidence of arsenical (atoxyl) unresponsive cases of HAT were reported back in the early 1900s by Paul Ehrlich who hypothesized that changes in specific ‘chemioreceptors’ could confer resistance. More recent studies have validated this hypothesis as alterations of several cell surface transporters could generate drug resistant phenotype [57,59]. Several physiologically important surface transporters could adventitiously facilitate drug uptake. In HAT, arsenical and diamidine resistant parasites are reported to be cross-resistant and have shown reduced uptakes of both drugs. Later studies have revealed that purine transporter 2 (P2) facilitates the uptake of both arsenical and diamidine explaining the cross resistant phenotype (Figure 3). Uptake of both arsenicals and pentamidine are competitively inhibited by adenosine, the natural substrate of P2 (Figure 3). P2 is reported to be defective in both laboratory raised and clinical isolates of drug resistant parasites. However, the degree of resistance to pentamidine in a P2 null mutant of T. brucei was significantly lower when compared to both laboratory raised and clinical isolates of drug resistant parasites. More importantly, pentamidine or arsenical uptake by P2 null mutant was not inhibited in the presence of adenosine suggesting the presence of an additional high capacity transporter(s) for the drugs [57,59]. Recently, Alsford et al. (2012) identified the roles of TbAQP2 and TbAQP3 in pentamidine and melarsoprol cross-resistance by using high-throughput RNAi library screening method. TbAQP2/TbAQP3 null mutant cells are more than 2 fold and 15 fold resistant against melarsoprol and pentamidine respectively (Figure 3) [73]. Further studies revealed that TbAQP2 plays the pivotal role towards pentamidine and melarsoprol resistance. However, TbAQP2 does not play any role towards resistance against other arsenicals (such as, sphenylarsine oxide) or diamidines (berenil, phenanthridine trypanocides, isometamidium and ethidium) [59,61]. These suggest the existence of additional transporters. The role of TbAQP2 in pentamidine and melarsoprol cross-resistance was further substantiated by the discovery of altered TbAQP2, both in laboratory raised and in several clinical resistant isolates. In resistant isolates TbAQP2 locus was found to become either chimeric with TbAQP3 where >120 base pairs of 3’ end of TbAQP2 was replaced by the nucleotide sequence of TbAQP3 3’ end with concomitant loss of TbAQP3 alleles or deleted from the genome [61,74]. Melarsoprol and Pentamidine uptake mechanisms of T. brucei. T. brucei AQP2 and P2 facilitate the uptake of both melarsoprol and pentamidine. Adenosine competitively inhibit pentamidine uptake by P2. The HA1-3 (P type H+ ATPase) might aid pentamidine uptake by providing a proton motive force to TbAQP2.Dihydroxyacetone (DHA), a natural substrate of TbAQPs, can be used as an anti-trypanosomal agent. All 3 TbAQPs are highly permeable to DHA. DHA can be used as a carbon source by phosphorylating DHA to DHA phosphate (DHAP) using DHA kinase (DHAK). Later, DHAP can enter the glycolysis pathway. However, as DHAK gene is absent in T. brucei genome, they are unable to use DHA as a carbon source. Consequently, when T. brucei cells are exposed to DHA they can accumulate very high levels of DHA in the cytoplasm. High level of DHA causes cell cycle arrest at G2/M phase followed by autophagic cell death. Although IC50 of DHA is rather high (~1 mM) for a drug candidate, it can serve as a potential starting point for the development of new analogs [75,76].The life cycle of T.cruzi is complex and involves several developmental stages in its vector, a large sanguinivorous reduviid insect belonging to the subfamily of Triatominae, and in its hosts, about 150 species of wild and domestic animals including human. During the blood meal, the infected insect vector releases metacyclic trypomastigotes on the skin along with feces and urine. Once metacyclic trypomastigotes break inside the skin through the bite wound, they start invading the cells of local reticuloendothelial system and connective tissues, and differentiate into aflagellated dividing amastigotes inside the cells. After the host cell reaches its capacity, amastigotes transform back into trypomastigotes by growing flagella followed by lysis of the host cell [5,77]. The trypomastigotes are non-dividing but highly motile. Through the hemolymphatic system, they migrate to distant organs and primarily infect heart muscle, skeletal and smooth muscles, and ganglion cells to repeat the amastigote-trypomastigote cycle. Trypomastigotes have the capacity to invade any nucleated cell to form amastigotes. Circulating trypomastigotes are also infective to insect vectors. Along with the blood meal, trypomastigotes enter into the insect gut, and are transformed into proliferating epimastigotes. Later, epimastigotes move further down the gut to the rectum. At this stage of development, epimastigotes attach themselves to the rectal wall. Attachment of epimastigotes to the rectal wall induces metacyclogenesis. During the next blood meal the vectordefecates on the mammalian skin of the mammalian host, The metacyclic trypomastigotes in the feces enter the new host through the breaks on the skin [77,78]. Triatoma infestans, Rhodnius prolixus, and T. dimidiata are by far the most important vectors with regard to human Chagas disease. Triatomine ontogeny contains five nymphal stages followed by adults. All the developmental stages including adults are blood sucking, thus potentially can harbor and transmit T.cruzi. The disease can also be transmitted non-vectorially through blood transfusion, transplantation, transplacenta and even orally through the ingestion of contaminated food and drinks [36,79].Chagas disease, also known as American trypanosomiasis, continues to affect human civilization since ancient times in many Latin American countries [80]. It is currently estimated that about 7 to 8 million people worldwide (primarily Latin America) are infected with Chagas disease [81] resulting into 10,000 to 14,000 deaths per year [77]. Presence of multiple vectors and reservoirs along with asymptomatic infection make the epidemiology of the disease more complex. Chagas disease is usually a lifelong disease [82]. The progression of the disease can be divided into three clinical phases: an acute phase and an intermediate phase followed by a chronic phase. The acute phase develops about 1 to 2 weeks after exposure to the pathogen. Acute phase infection is usually asymptomatic especially in adults. However, approximately 1% of patients develop nonspecific symptoms like fever, headache, anorexia, malaise, myalgia, joint pain, etc., with or without painful erythematous swelling called chagoma around the site of inoculation. Parasitemia is usually high in acute phase; trypomastigotes can be visible in blood smear. Every nucleated cell can be potentially invaded by the pathogen in the acute phase. After about 8 to 10 weeks, the majority of the patients start to develop antibodies against a variety of T. cruzi antigens. As a result, the parasitemia subsides significantly and spontaneously resolve the symptoms, but some patients may remain infected for years to decades without any symptoms. However, approximately 5%–10% of symptomatic cases can be fatal due to severe myocarditis, meningoencephalitis or both. Diminishing parasitemia leads to progression of the disease into the intermediate phase. In the intermediate phase, parasites are barely visible in the blood. Chronic phase of infection can develop in approximately 30% of the patients, and most of them develop cardiomyopathy. Along with the heart disease, some patients may develop digestive system abnormalities, such as, megaesophagus and/or megacolon. The host immune status along with tissue damage inflicted during the acute phase of infection are believed to be the primary reason for chronic phase symptoms. In immunocompromised patients, Chagas disease may reactivate to develop more severe clinical symptoms [5,36,83].Several drugs have been tried to treat the Chagas disease but effective treatment options are limited to only two drugs namely benznidazole and nifurtimox. Benznidazole and nifurtimox were introduced more than 4 decades ago. However, they are effective only against the acute phase of infection and contraindicated during pregnancy, and in patients with severe renal or hepatic inefficiency. The situation is further complicated by adverse side effects, such as, skin rashes, nausea, and kidney and liver failure [5,36,54]. Additionally, long treatment periods and high cost of the drugs promote treatment drop out which could potentially develop into drug resistance. An in vitro study reported the link between T. cruzi type I nitroreductase (NTR I) activity and cross-resistance against nufurtimox and benznidazole [84]. Another study proposed the possible association of NADPH-dependent cytochrome P450 reductase B (CPR B) and drug detoxification that could lead to unresponsiveness [85]. Recently, Campos et al (2013) reported that higher expression of P-glycoprotein efflux pump leads to drug resistance in T. cruzi [86].Like other trypanosomatid parasites T. cruzi faces huge osmological fluctuations during its life cycle. The nature of the feeding cycle drives, the osmolarity of gut and the rectal content of triatomine bugs. Gut osmolarity varies between 300–970 mOsm whereas feces osmolarity ranges from 370 to 760 mOsm. On the contrary, osmolarity of mammalian blood is approximately 300 mOsm [87]. As described earlier, AQPs are responsible to cope with these insults. Till date four AQP homologs namely, TcAQP and β-γ have been identified in T. cruzi genome [18]. Among them, only TcAQP has been characterized so far. TcAQP is an orthodox aquaporin that transports only water and is sensitive to HgCl2 and AgNO3 inhibition. However, water conductance capacity of TcAQP is poor when compared to other parasitic AQPs. TcAQP expresses throughout all developmental stages of the parasite and is localized in the acidocalcisomes and contractile vacuole complex adjacent to the flagellar pocket of the parasites [21]. Acidocalcisomes are membrane bound acidic organelles that store calcium [88]. Acidocalciosomes are also present in some other unicellular organisms, such as, apicomplexan parasites (Plasmodium and Toxoplasma) [89], green algae Chlamydomonas reinhardtii [90], slime mold Dictyostelium discoideum [91], and agrobacterium Agrobacterium tumefaciens [92]. Acidocalcisomes and plant tonoplast have several functional similarities including osmoregulation [93]. Contractile vacuole is an established organelle involved in osmoregulation in protozoa. The basic structural plan of the contractile vacuole system is very similar in all protozoa. It consists of two parts: a central vacuole, or bladder, and a surrounding network of tubules and vesicles called spongiome. In T. cruzi acidocalcisomes and contractile vacuole system work together in a sequential manner to equip the parasite to face the hypoosmotic challenge during its transmission from vector to mammals. During hypoosmotic shock, a hypothetical adenyl cyclase is activated to form cAMP. Elevated cAMP level promotes fusion of acidocalcisomes with the contractile vacuole and translocation of an aquaporin to the contractile vacuoles, which aid them in water accumulation. Then, the water-loaded vesicles fuse with the flagellar pocket membrane to secrete the water (Figure 4) [7]. There is no report of association between any T. cruzi AQP and drug response. However, as hyposmotic shock is a major challenge for the parasite during transmission from the vector to the mammalian host, targeting osmoregulation systems for new drug development may be a feasible approach.Osmoregulation mechanism of T. cruzi. Volume increase due to hypo-osmotic stress triggers the production of cAMP. Elevated cAMP levels induce protein kinase activity that promotes fusion of acidocalcisomes (AC) with the contractile vacuoles (CV) and amino acid release. The fusion translocates the TcAQP from AC to CV and this is believed to aid the contractile vacuole in water sequestration followed by release into the flagellar pocket. AA: amino acids, PolyP: polyphosphate, Pi: inorganic phosphate.In the absence of effective vaccines against any trypanosomatid diseases, chemotherapy takes the center stage to combat them. However, increasing incidence of drug unresponsiveness, due to decades of use of the same or similar drugs, has created a major impediment. Second line choices are too limited, highly toxic and/or excessively expensive for these neglected diseases of the developing world. We are in dire need of cheaper and less toxic new drugs. Identification of parasite specific targets is critical for the development of new agents. Within two decades of discovery, significant advancement has been achieved with regard to mammalian AQPs. However, knowledge about AQPs from other living systems especially in parasitic protozoa is lagging far behind. Importantly, preliminary knowledge showed subtle but promising structural and functional differences between parasitic protozoan AQPs and their host counterparts. Parasite AQPs are necessary for the successful perpetuation of the diseases caused by them. In conclusion, further exploration of parasite AQP structures and functions including their regulatory mechanisms will eventually reveal their real potential for novel chemotherapeutic approaches and/or transmission intervention(s). We acknowledge the startup funds from HWCOM, FIU to RM. MS is a Biomedical Research Initiative Fellow and JFO is a McNair Fellow at FIU for 2013.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Mitochondrial diseases, which altogether represent not so rare diseases, can be due to mutations either in the nuclear or mitochondrial genomes. Several model organisms or cell lines are usually employed to understand the mechanisms underlying diseases, yeast being one of them. However, in the case of mutations within the mitochondrial genome, yeast is a major model because it is a facultative aerobe and its mitochondrial genome can be genetically engineered and reintroduced in vivo. In this short review, I will describe how these properties can be exploited to mimic mitochondrial pathogenic mutations, as well as their limits. In particular; pathological mutations of tRNA, cytb, and ATPase genes have been successfully modeled. It is essential to stress that what has been discovered with yeast (molecular mechanisms underlying the diseases, nuclear correcting genes, import of tRNA into mitochondria or compounds from drug screening) has been successfully transferred to human patient lines, paving the way for future therapies.Mitochondria are the vital center of cell function. In addition to their primary function, i.e., as the power plant of the cell and controler of the cell redox balance, mitochondria are involved in many essentiel cellular processes. They mediate apoptosis via the permeability transition pore, are involved in many catabolic and anabolic pathways such as beta-oxidation, heme biosynthesis, steroidogenesis, amino acid metabolism, assembly of Fe/S clusters and gluconeogenesis. They also control stress responses, in particular the oxidative stress response via reactive oxygen species (ROS) and calcium Ca2+ storage (rewiewed in [1] and references therein). They play a central role in aging [2] and are subject to mitophagy, the pathway that mediates the selective elimination of dysfunctional or unwanted mitochondria and is critical to proper cellular functions [3].Without a proper mitochondrial structure, the cell is not viable, and mitochondrial dysfunctions underly many types of human diseases, even though the causal relation between mitochondrial functions and these multifactorial diseases is still open to debate [4].Mitochondria are associated with diseases such as diabetes [5], cardiac [6] and vascular [7] diseases, cancer [8,9], and also neurodegenerative diseases. A disorder of endoplasmatic reticulum (ER)-mitochondrial communication is now suspected to be the basis of Alzheimer’s disease [10]. Junctions between the ER and mitochondria are indeed essential to the exchange of metabolites such as lipids and Ca2+ and play a role in mitochondrial dynamics [11]. Mitochondria are also highly suspected to be involved in Parkinson’s [12] and Huntington's diseases [13].Because of these numerous pathways and interconnected cellular functions, many genes are involved, and mutations in these genes may lead to the various diseases mentioned. Almost all these genes are encoded by the nuclear genome, and only a few, coding for some components of the respiratory chain, tRNAs and rRNAs, are encoded by the mitochondrial genome. In humans, this amounts to a total of 37 genes [14]. Despite the very small number of genes involved, pathologies due to mitochondrially-encoded mutations cannot be considered as rare diseases since they are found in approximately one in 200 individuals, with clinical expression in 1:5000 to 1:10,000 individuals [15,16].In this short review, I will focus only on mitochondrially-encoded mutations since they exhibit very peculiar properties compared to nuclearly-encoded mutations. Reviews on mitochondrial diseases in general can be found in [17] for historical aspects, [18] for bioenergetics, [19] for nucleo-mitochondrial intergenomic cross-talk as well as in [20] and [21] for the role played by yeast.The mitochondrial system presents some specificities which are briefly summarized below: (i) The mutation rate of mitochondrial DNA is significantly higher than that of nuclear DNA; (ii) the cell contains many mtDNA molecules, and mtDNA does not follow Mendelian rules of inheritance but is maternally inherited. In the case of deletions and many mtDNA point mutations, a mixture of wild-type and mutated molecules co-exist in the cell, a situation which is called heteroplasmy. Their respective proportions may vary in the progeny and in different tissues, the symptoms appearing only when a high proportion of mutated molecules is reached, called the “threshold”; (iii) since some tissues (heart, brain and muscle, for example) are more energy-dependent than others, one expects more deleterious effects in these tissues; (iv) the relation between genotype and phenotype is not straightforward, with one mutation leading to different syndromes in different individuals (even within the same family) and the same syndrome being due to different mutations in different genes; (v) since the nuclear and mitochondrial genomes interact genetically for mitochondrial function, the variation of nuclear background among individuals must also be considered. More details on these caracteristics can be found in [16] and references therein.This complexity is far from being understood. Mitochondrial involvement in cell programs which control oogenesis and differentiation, as well as the basic mechanisms which distribute mtDNA molecules to cell progeny such as the bootleneck that takes place during oogenesis, remain to be uncovered (reviewed in [22]). These caracteristics do not facilitate genetic counseling or the quite frequent establishment of a mutation as being mitochondrial nor do they cause a disease.Because of the complexity of the human organism and the difficulties associated with direct experimentation, it is necessary to make studies and analyses in model organisms. As will be seen, there is no perfect model system.Mammals come to mind as the first choice. Mouse models seem well adapted since they are relatively simple mammals, which can be easily manipulated by many genetic and molecular biology techniques, although they are labor-intensive. They offer several advantages because mice and humans display quite similar gene contents, comparable types of internal organs and physiological processes.However, for a long time, establishment of mice with pathogenic mutant mtDNA was impeded by the impossibility to reintroduce an in vitro engineered form of a mitochondrial gene into mitochondria. Progress has been made with the mutator mouse which carries a form of gamma DNA polymerase devoid of proofreading [23], a strategy based on the detailed analysis of the yeast mitochondrial polymerase [24,25]. These mice generate mitochondrial genome mutations at high frequency, among which the desired mutations can be searched for, if they exist. Alternatively, the impressive mito-mice system (reintroduction of mitochondria with a somatic mtDNA mutation into mouse zygotes [26]) permits precise mutations to be created, but is still a long and costly procedure. In addition, one should add that there is no guarantee that the resulting mice will reflect the human phenotype [23,27,28,29]. The precise reasons for such situations are unclear but the prime suspect might well be the nuclear background, a parameter, which reappears in different aspects of these diseases (see [30,31] and Section 3). Several reviews cover these important points in more detail [32,33,34,35].An alternative mammalian system such as patient cell lines, is commonly used, but cell lines which are immortalized are not physiological and do not reproduce the characteristics of differentiated cells in the body. Even primary cell lines from patients, usually derived from fibroblasts or myoblasts, which are not necessarily the affected tissues, do not retain all characteristics [36]. Analyzing the same mutation, discrepancies appear among results obtained in different cell lines (see for example the case of the A3243G mutation of tRNALeu(UUC) [37,38]).Cybrids, which fuse an enucleated cell carrying a suspected pathological mitochondrial mutation with cells lacking mitochondrial DNA, suffer from the same limitations as cultured cells in general. However, they have been essential for the process of uncovering whether the mitochondrial genome is responsible for a given mitochondrial disease and for the study of its effects [39].Several non-mammalian organisms have also been exploited. The interest in studying mitochondrial functions in the worm C. elegans, which is easy to manipulate and has a very simple RNAi gene knock-down protocol by feeding, has considerably increased in the past years. Mitochondrial dysfunction confers phenotypes related to longevity, sterility, developmental larval arrest in L3 or ethidium bromide sensitivity ([40,41,42,43] reviewed in [44]). A mitochondrial deletion exists which has been used to study heteroplasmy [45], and it has been shown that, while the proportion of heteroplasmy is variable from one animal to another, the proportion of heteroplasmy in the whole population is constant (60%) during a hundred generations. The molecular mechanism is not known, and this study only reflects what has been found with a specific deletion, isolated by chance. The situation is quite similar in the fly D. melanogaster. As a genetic model system it offers many advantages, and has been widely used, in particular to study neurodegenerative disorders. Mutants in nuclear-encoded mitochondrial genes (ribosomal proteins, some respiratory complex components) have been identified as “bang”-sensitive or deficient in eye-development (reviewed in [44]). One interesting mitochondrial-encoded mutation in the ATP6 subunit has, however, been identified as a suppressor of the SesB mutant (localized in the mitochondrial adenine nucleotide translocator or ANT) and mimics pathological features seen in Leigh syndrome [46]. No specific mitochondrial DNA mutations have been described otherwise, except for a large-scale deletion in the mitochondrial DNA of the fly D. subobscura, which was obtained by chance. As was the case for the mitochondrial deletion of the nematode, this discovery helped to analyze heteroplasmy and gene expression, but was not direcly relevant to known human mitochondrial pathologies [47].Yeast (unless mentioned otherwise, this means the budding yeast Saccharomyces cerevisiae) has been extensively used for understanding human diseases. As a simple and unicellular microrganism, it may seem quite different from humans and their diseases, but several important properties have made yeast a pivotal organism for many human studies and, as will be seen in Section 2.3.2, in particular for mitochondrial diseases: 
+          
+            
+			(i)
+              Simplicity, rapidity and low cost.
+            
+            
+			(ii)
+              Highly developed genetics with a diverse and well-adapted toolbox. Thousands of mutations exist in practically every nuclear gene of the genome, and any new mutation can be created in vitro and reintroduced into the genome thanks to the rich collection of tools, which has been developed.
+            
+            
+			(iii)
+              A wealth of functional studies organized into an efficient database. When the complete genome was published [48], we had some functional knowledge of about 30% of the roughly 5800 genes; the figure now reaches practically 85%. All information obtained is centralized in the Saccharomyces Genome Database or SGD, which is easy to use and kept up to date [49].
+            
+            
+			(iv)
+              Protein sequence conservation and heterologous complementation. All the properties I have described can be usefully exploited because bioinformatic analyses have revealed a high conservation thoughout eucaryotic evolution for many genes involved in basic cellular functions. As a consequence, we have gone a long way since the pioneering work of [50] on heterologous complementation of a yeast (S. pombe) mutation by a human gene. Recent orthology studies now show that nearly 1000 yeast genes are associated with human diseases [51], and in many cases the mammalian ortholog is functional and complements the yeast deletion mutant.
+            
+            
+			(v)
+              The “omics” approaches. Not only has the thorough annotation of the S. cerevisiae genome, performed by the yeast biologists themselves, served as basis for annotation of many other organisms, but the global approaches which have been developped to study regulatory networks, protein and gene interaction networks have served as pilots for applications to other organisms. This was also the case for computational analyses and the development of systems biology.
+            
+          
+		  Simplicity, rapidity and low cost.Highly developed genetics with a diverse and well-adapted toolbox. Thousands of mutations exist in practically every nuclear gene of the genome, and any new mutation can be created in vitro and reintroduced into the genome thanks to the rich collection of tools, which has been developed.A wealth of functional studies organized into an efficient database. When the complete genome was published [48], we had some functional knowledge of about 30% of the roughly 5800 genes; the figure now reaches practically 85%. All information obtained is centralized in the Saccharomyces Genome Database or SGD, which is easy to use and kept up to date [49].Protein sequence conservation and heterologous complementation. All the properties I have described can be usefully exploited because bioinformatic analyses have revealed a high conservation thoughout eucaryotic evolution for many genes involved in basic cellular functions. As a consequence, we have gone a long way since the pioneering work of [50] on heterologous complementation of a yeast (S. pombe) mutation by a human gene. Recent orthology studies now show that nearly 1000 yeast genes are associated with human diseases [51], and in many cases the mammalian ortholog is functional and complements the yeast deletion mutant.The “omics” approaches. Not only has the thorough annotation of the S. cerevisiae genome, performed by the yeast biologists themselves, served as basis for annotation of many other organisms, but the global approaches which have been developped to study regulatory networks, protein and gene interaction networks have served as pilots for applications to other organisms. This was also the case for computational analyses and the development of systems biology.More information can be obtained from two reviews, both by Botstein and Fink [52,53], written 23 years apart. They put the possible use of yeast as a reference experimental organism in perspective. This time lapse is large enough to follow the impressive evolution of knowledge, concepts and techniques developped in yeast, “an experimental organism for the 21st century biology” [53]. There are several specific properties which make this organism invaluable for mitochondrial research: (i) S. cerevisiae is a facultative anaerobe and can grow by fermentation when devoid of any mitochondrial genetic information; this means that any mutation which affects mitochondrial function and would be essential in a strict aerobe can be maintained and analyzed on fermentable medium (usually glucose). (ii) There is a very strong conservation, from yeast to humans, of the genes encoded within the mitochondrial genome, i.e., components of the respiratory chain, tRNA and rRNA genes. There is only one exception, which is the absence of complex I. (iii) It is the only organism, together with the algae C. reinhardii, in which mitochondria can be transformed. This means that all techniques relative to reverse genetics can also be applied to the mitochondrial genome; mutations can be created in vitro and reintroduced at will in vivo, at their proper site, in the mitochondrial DNA. Originally described by two groups [54,55], the biolistic transformation (shooting microprojectiles layered with recombinant mitochondrial DNA at high velocity into yeast cells) has been efficiently developed to the point of now being a routine technique [56]. This approach is greatly facilitated by the very high homologous recombination frequency of the mitochondrial genome, which allows efficient integration. With this technique, many kinds of modification can be introduced and, in particular, mutations which precisely mimic the human pathological mutations (Section 3). (iv) Finally, we should mention that in contrast to what is observed in other organisms, S. cerevisiae is homoplasmic, which means that in a mutated cell, all mtDNA molecules possess the same mutation; the phenotype is therefore independent of any threshold effect, and the presence or absence of respiratory growth defects in cells carrying a presumed pathological mutation provides strong evidence for the classification as pathological or not in humans.Human mutations found in patients have been reported in all genes (producing proteins or RNA) encoded by the mitochondrial genome, and some of them have been confirmed to be causative of the disease; more information can be found in MITOMAP [57].The biolistic transformation has a low yield since it kills most of the cells, which means that positive selection has to be applied. This is done in two steps: The first one selects the surviving cells and the second the mitochondrial transformants among them (usually around one in 1,000 survivors). The first pathological mutations were created in the COXI and COX3 mitochondrial genes (coding for Subunit I and Subunit III of cytochrome oxydase, respectively). The I280T and M273T COX1 mutations have been observed in hematopoeitic cells from patients suffering from acquired idopathic sideroblastic anemia [58], but their biochemical effect is unclear. In yeast, these mutations caused similar effects to those reported in humans (mild effects on respiration) and the biochemical study deepened the analysis and allowed to propose a biochemical hypothesis. A short deletion in COX3 was also designed in yeast and had as strong an effect as in humans [59].Several CYT b mutations have also been created with biolistic transformation. Many of them were focused on modeling regions of the mammalian Qo site in yeast cyt b or on further understanding the differential efficacy of Qo site inhibitors on mammalian and pathogen bc1 complex [60,61,62,63,64]. However, pathological human mutation equivalents were also constructed and analyzed ([65,66,67,68] and references therein). In humans, their biochemical properties have not been seriously investigated and their effects have mostly been deduced from what is know of the enzyme structural properties. Interestingly, a detailed comparative analysis of the molecular effects of the G15699C mutation (Lys319Pro in yeast) has been reported [65]. In humans, it showed a dramatic loss of steady state levels of Complex III and a strong decrease in enzyme activity. In yeast, it showed that the mutation hindered the assembly of the complex but that the assembled complex was stable. The catalytic activity of the enzyme was decreased (as in humans) and a lower Km for quinol substrates, and thus a slower rate of electron transfer from the Q0 site, was observed.Complex I does not exist in S. cerevisiae and therefore cannot be manipulated the same way despite the fact that there are several well established pathological mutations in the mitochondrial subunits. Since the green alga Chlamydomonas reinhardii, which possesses a Complex I rather similar to the human one, is the other organism for which mitochondrial transformation is possible, it should be possible to develop it into a model system for Complex I. A recent attempt has been made in this direction with one pathological ND4 mutation (L158P) introduced into the mtDNA of the algae [69]. This mutation has an impact on Complex I activity and leads to a clear in vivo phenotype, as respiration in the dark is impaired.The work done on ATPase subunit 6 and tRNAs (which represent in fact quite a high proportion of the pathological mutations [70]) is more specifically focused on mitochondrial diseases. Five pathological mitochondrial mutations in the ATPase 6 subunit have been mimicked in yeast. T8993G [71], T8993C [72], T9176G [73] and T9176C [74] have been found in patients presenting a NARP (neuropathy ataxia retinitis pigmentosa) or MILS (maternally-inherited Leigh’s Syndrome) syndromes. These mutations, in conserved regions of subunit a, all impair the ATP synthase. Examination of the same mutations in yeast has shown that these mutations have a similar impact on the ATPase as they have on the human ones, i.e., a decrease in mitochondrial ATP synthesis rate, confirming yeast to be a useful model for molecular and biochemical studies. In addition, because the biochemical studies have been thoroughly performed in yeast, some new information, not yet obtained in humans, has been uncovered (review in [75]). For exemple, while in humans the T8993G mutation presents a decrease in ATP synthesis, there are still debates about the reason for this. Contradictory experimental results have proposed this decrease to be due either to defects in ATPase assembly/stability or the functioning of the enzyme proton channel. In yeast, it has clearly been shown that the enzyme is correctly assembled but that the reverse functioning of ATP synthase (proton translocation out of mitochondria) is significantly compromised.This new information gathered from yeast experiments was quite evident for the fifth mutation, T8851C. This mutation is associated with a bilateral striatal lesion, a rare neurological disorder with largely unknown biochemical consequences [76]. The analysis of the same mutation in yeast revealed a functional impairment of the ATP synthase, seemingly at the level of proton translocation. As the assembly/stability of the ATP synthase is not affected, a functional impairment of the enzyme must be responsible for the respiratory growth deficiency. The authors indeed showed that there was a very low F1-mediated ATP hydrolytic activity. Considering the high level of sequence conservation between the proteins, the block of proton translocation is probably the cause of this neurological disease [77]. Finally tRNA mutations mimicking human ones have been constructed, mostly in tRNALeu(UUR), which is an observed hot spot for mutations. In this case, the screening is more complex: (i) No positive screeening is possible, and this entails searching for rare phenotypically defective colonies among normal ones; (ii) mutations of the mitochondrial translation apparatus lead to a very rapid loss of mtDNA (rho°) so that the interesting point mutation—with a respiratory deficient phenotype—is hidden among a high frequency of rho°—which presents the same respiratory deficient phenotype. Although ATPase 6 mutations also cause a high frequency of rho°, the ATPase 6 studies have taken advantage of a clever genetic tool (expression of an allotropic form of the ARG8m expressed into mitochondria, which requires functional mitochondrial translation [78]). This is unfortunately not amenable to studies of mt-tRNA mutations.To demonstrate that is was feasible to generate specific tRNA mutations, our lab successfully recreated a known tRNA mutation by biolistic transformation [79]. With this possibility at hand, three MELAS (Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode) mutations in mt tRNALeu(UUR)—3243G, C3256T and T3291C—were created in yeast through systematic tRNALeu gene sequencing of cells with a respiratory deficient phenotype and were shown to have very serious phenotypic consequences [80]. The yeast cells carrying those mutations were all unable to grow on respiratory substrates, and a large proportion of them lost their mitochondrial genome. A variety of pathological mutations covering different phenotypes in humans were then generated, their presence being screened by ACRS (artificially created restriction site) independently of the presumed phenotype. They exhibited different types of phenotypes: tRNALeu(UUR) T3250C is present in a family with mt myopathy (MM/CPEO, mitochondrial myopathy/chronic progressive external ophtalmoplegia; [81]) and A3260AG is associated with adult onset maternally inherited myopathy and cardiomyoapthy (MMC patients [82,83]. As opposed to the MELAS yeast mutations previously described which could not grow on respiratory substrates, these two mutants grew slowly and reached a lower plateau than the wild-type strain. Accordingly, they showed a significant decrease of O2 consumption, with a more limited effect on the T3250C equivalent mutation. In contrast, yeast cells carrying the G3249C mutation (with the severe Kearns-Sayre syndrome in humans) are as drastically affected as the MELAS mutation type (unpublished). Two other mutations, the tRNALys G8328A [84] found in a case of sporadic encephalomyopathy and the tRNAVal C1624T (Leigh syndrome and encephalopathy; [85]) both presented a conditional growth defective phenotype (heat-sensitivity) in yeast. From this study, it is apparent that very severe syndromes such as MELAS or Kearns-Sayre, produce very severe defects in yeast resulting in complete absence of growth on respiratory medium, while other mutations leading to less severe phenotypes in humans (CPEO, MM, encephalopathy) have milder effects when mimicked in yeast, with different degrees paralleling the degrees observed in humans [86]. In the same publication, the nuclear context was checked very carefully, taking advantage of a yeast mutation (kar1-1, [87]), which allows the placement of the same mitochondrial genome in different nuclear backgrounds. This study indeed revealed that the same mitochondrial mutation had a more or less severe phenotype according to the nuclear background in which it was placed, but that the rank in phenotypic effect of the different mutations was always the same in a given background, confirming previous results [88].The molecular basis of the various respiratory defects was investigated. The structure, relative amount and aminoacylation of the tRNAs were checked, and it was found that the defects reflected what was observed in human cells [89], thus validating yeast once again as a tool to study mitochondrial disease. This system, although valuable, is limited by the fact that while there is a rather good sequence conservation between humans and yeast, not all human mutations can be mimicked in yeast. In order to circumvent this problem, my group attempted to replace the yeast tRNA by its human counterpart. To determine if it was feasible, we first introduced the human tRNALeu(UUR) into an otherwise wild-type mitochondrial genome. In such a context, we could examine the biochemical functions of the human tRNA (tRNA maturation and aminoacylation) in cells which grow normally since they have the wild-type yeast tRNA. All properties were found to be normal. In a second step, we placed the wild-type human tRNALeu(UUR) in the presence of one of the pathological mutations (with a temperature-conditional phenotype) and observed that the human gene could support yeast cell growth at the defective temperature. The same experiment was performed with the human tRNA Asp which could also functionally replace a mutated version of the yeast tRNA Asp. These results show that the human tRNAs can functionally replace their yeast counterparts and open the way to the possibility of creating and investigating any human mit tRNA mutation in the future [90].In conclusion, modeling mutations in yeast has been informative; it allowed to either deepen or elucidate some biochemical mechanisms (as for several ATPase mutations), bring arguments in favor of pathogenicity in humans (such as in the apparent contradiction of the tRNAVal C1624T mutation phenotypic effects observed in the mother and her offspring [85]) or understand how to compensate the defective phenotype (see Section 4).Among possible therapies against these deadly diseases, one immediately thinks of new chemical drugs or peptides, which could be delivered to the human body. As far as cell or gene therapies are concerned, the first difficulties lie with the fact that the “good gene” cannot be imported as such into the mitochondria since no nucleic acids are imported. We either have to find nuclear genes, which can correct the mitochondrial mutation since they are imported naturally (the geneticist calls them “suppressor”) or find a way to develop an import system for nucleic acids into mitochondria. For all these situations, yeast has been extremely useful in investigating these different approaches, taking advantage of the mutations, which have been modeled.Creating pathological human mutations in yeast indeed provides a wealth of information on the molecular basis of these mutations, but the real strength of the yeast model is the power of its genetics. Starting with mutations, which have a defective phenotype on respiratory substrates, it is quite easy to select for secondary mutations, which can alleviate this phenotype. Since mitochondrial diseases involve two players (the nuclear genome cross-talking with the mitochondrial one) these secondary mutations (or suppressors) could a priori be localized in either of the two genetic compartments; in yeast, it is very easy to discriminate the two categories, in particular because one can lose the mitochondrial genome at will. Alternatively, one can manipulate the gene dosage and search for wild type genes, which could correct the initial mutation when their gene dosage is increased (overexpression). Both approaches have been successfully used.In the case of cyt b mutations, many suppressors have been obtained and most were secondary mutations in the CYT b gene itself. However, some extragenic nuclear suppressors were also obtained and turned out to be mutations of the Iron-Sulfur protein. They provide important information on the relationship between structure and function of this protein and its interaction with Complex III [66,91].Direct suppressors of the NARP mutations have never been searched for. However, a gene has been identified which, when overexpressed, induces a metabolic bypass of the defective ATPase; ODC1 (coding for the oxodicarboxylate carrier) is able to correct the growth deficiency of some ATPase mutants as long as a few functional ATPases are still present [92]. It seems that under such conditions, the ODC1 gene can improve the respiratory capacity of the yeast NARP T8993G mutation [75].In the case of the tRNA mutations, the strategy was more direct. Correction by gene overexpression was deliberately searched for, with the rationale that overexpressing a wild-type gene should be transposable to other systems, in particular to humans. The TUF1 gene, encoding the mitochondrial translation elongation factor EF-Tu, was initially isolated for its ability to correct, when overexpressed, the defective phenotype of some randomly obtained tRNA mutations during a large library screening [93]. It was therefore the first gene tested on pathological mutations, and it was found to restore all the phenotypic defects of the yeast mutant equivalent to C3256T [80]. It was later shown that TUF1 corrected many different pathological tRNA mutations. The same type of experiments was performed with the cognate tRNA synthetase, which was also shown to be efficient when overexpressed [88]. More recently, new correcting gene candidates have been identified, and their effect remains to be caracterized [94]. It is interesting to note that serious arguments exist that support the fact that these suppression effects are not linked to the catalytic activity of these two proteins in protein synthesis but more probably to a chaperone effect [86,94]. Their correcting effects are also dependent upon the nuclear genetic background, perhaps in relation to the intrisic level of the correcting gene naturally present within the cell [88,95].At this point, it is important to evaluate whether modifying the expression in nuclear genes can correct mitochondrial defects in human cells, which was the initial reason behind this screen. In 2008, based on the yeast data, Sasarman and coworkers [38] tested the effect of EF-Tu (and EFG2) in patient human tissues and observed that there was some correcting effect. As in yeast, the same effect also existed for the cognate synthetases [96,97,98].Since a point mutation in mtLeuRS that strongly reduces the catalytic activity did not impair the correcting ability [86] and since the effect is not restricted to the cognate synthetase [99], the region of the aaRS involved in this potential chaperone-like interaction with tRNA was searched for. It turned out that a C-terminal region of 66 aminoacids in yeast (67 in humans) had full suppressing activity, and moreover that this region was necessary and sufficient to rescue the defective phenotype of human equivalent mutations in yeast mt-tRNA Leu, mt-tRNA Val and mt-tRNA Ile [100]. It remained to be seen if such peptides had the same corrective effect on patient cell lines. This has proven to be the case based on the recent finding [101] that three human mt-aaRS (mt-ValRS, mt-LeuRS and mt-IleRS) are all able to suppress, in human transmitochondrial cybrid cells, the mitochondrial functional defects associated with pathogenic homoplasmic mutations in the mt-tRNA Ile gene. This effect was observed independent of the strength of the original phenotype. This suppression effect, as in yeast, can be restricted to the non-catalytic carboxy-terminal domain. Reciprocally, one specific synthetase (leucyl tRNA synthetase) was able to partially rescue defects due to mutations in non-cognate tRNAs, and this effect was restricted to the C-terminal peptide which can enter mitochondria and interact with the same spectrum of mt-tRNA [102]. We now know that, besides well-described protein-import pathways into mitochondria, RNA import also exists as a quasi-ubiquitous process. Situations and mechanisms vary widely between organisms (reviews in [103,104,105,106,107]) but the import concerns only small non-coding RNA and mostly tRNAs. Once again, studies in yeast were the starting point in improving the understanding of the phenomenon and developing new strategies.The presence of a cytoplasmic tRNALys(CUU)—also called tRK1—within the mitochondria was found more than thirty years ago [107,108] but not understood at that point in time since the mitochondrion encodes its own tRNALys(UUU)—called tRK3—and was proposed to be able to decode both AAA and AAG codons. It is only very recently that the role of this supposedly redundant tRNA was understood. tRK1 is imported into mitochondria in several steps. First, it is charged by the cytosolic LysRS, it is then recognized by one of the two isoforms of enolase, a glycolytic enzyme, and the complex then moves to the mitochondrial surface where the charged tRK1 is taken up by the pre-mitochondrial LysRS [109,110], and more specifically by its N-terminal domain [111]. This basic knowledge was exploited in an elegant way to show that tRK1 is essential for mitochondrial protein synthesis at high temperatures. Under such conditions, tRK3 is not modified properly and cannot recognize mRNAs containing an AAG codon, a function then ensured by tRK1 [111]. From this work and studies run on the tRNA determinants for aminoacylation and import [112] the following questions immediately arose: Is it possible to manipulate these determinants to import cytosolic tRNAs with altered aminoacylation identities? Could these engineered tRNAs be functionally active to correct diseases as explained below?It was first shown in yeast that tRK1(CUU) changed into tRK1(CAU) could charge methionine, be imported into isolated mitochondria and incorporated into mtDNA encoded proteins. This in vitro approach was followed by an in vivo test in which a COX2 amber mutation was suppressed by modified (both for aminoacylation and import properties) tRK2(CUA). It was also shown that, in organello, an imported modified yeast tRNA was functional on the human mitochondrial translation apparatus [113]. These important results were then transposed to patient human cell lines and the imported tRNA was shown to restore mitochondrial function in transmitochondrial cybrid cells bearing the MERFF mutation [114]. More recently, the defective phenotype of a patient cell line carrying a MELAS mutation was partially rescued by an importable tRNAsLys with Leucine aminoacylation identity [115].This very basic research demonstrated that specifically designed tRNAs can address human mitochondria in vivo, be active in mitochondrial translation, and (partially) compensate the defective phenotype of human patient cell lines with potential applications for therapy.While no “natural” tRNA import has been found in mammalian mitochondria, several small RNAs are probably imported (5S RNA [116]; the RNA moiety of RNAseP [117] and the mitochondrial RNA processing endonuclease [118]). More recent works now aims to develop possible applications. The appropriate selection of aptamers [119] and the understanding of the role of human tRNA synthetase in human tRNA import [120] have allowed progress to be made. Synthetically engineered modified oligonucleotides carrying a nucleotide sequence complementary to the mutated region can be targeted at human mitochondria in vivo and change the heteroplasmy ratio [121]. While other systems are now being studied (based for example on the Leishmania RNA imported complex—or RIC complex—which enters human cells by a caveolin-1 dependent pathway [122]), the work undertaken with yeast has pioneered and stimulated the topic of RNA import into mitochondria. Alternatively to the genetic approach, one can also attempt to identify drugs, which could alleviate the defects induced by the mutation. Because mitochondrial defects impair the proper functioning of the mitochondrial respiratory chain, one might expect that some chemicals, which act upon respiration functions, might relieve the symptoms. The positive effect of idebenone (a derivative of Coenzyme Q10) on patients with Coenzyme Q10 deficiency was first described [123]. This product was also successfully used later to cure the cardiomyopathy, which often develops in Friedriech Ataxia patients [124]. Other metabolic products have also been tested. The possibilities of treating the metabolic disorders of mitochondrial diseases were based on the following rationales: “(i) removal of noxious metabolites such as dichloroacetate; (ii) administration of electron acceptors; (iii) administration of vitamins and cofactors; (iv) administration of oxygen radicals scavengers; (v) increase of mitochondrial biogenesis with reverastrol; (vi) increase of autophagy activity with lithium”. They have been reviewed in [125].Based on this, a systematic screen of metabolic products able to improve the viability of MELAS models was done [126] using yeasts as the primary test substrate. These studies convincingly showed that supplementation with riboflavin (vitamin B2) or coenzyme Q10 effectively counteracted the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. Despite these encouraging results, no attempt has been made to apply this knowledge to large-scale drug screening, in part because of the lack of appropriate models. The possibility to mimic, in yeast, mitochondrial pathological mutations (see above) and the versatility, cheap price and fast growth associated with the important knowledge accumulated on S. cerevisiae over the years, should make this organism particularly suitable for a first step in large screening. It is only recently that such an approach has been described [127]. The authors screened about 12,000 chemical compounds in a yeast strain where the FMC1 gene had been deleted. In this mutant, there are much fewer assembled ATP synthase complexes than in a wild-type strain, whereas the ones that are assembled are fully functional. The ∆fmc1 strain does not grow on glycerol [128]. Presumed active compounds were then tested on the yeast models of atp6-NARP mutants described in Section 3. Three compounds, chlorhexidine, oleic acid, and dihydrolipoic acid, were identified, which turned out to also be effective in a human cybrid-based model of NARP. This work, although it did not reveal completely unexpected compounds, is important, as it constitutes a proof of principle that yeast can be efficiently used for large-scale screening of new compounds active on ATPase deficiencies. In addition, once the drug is identified, it can itself become an efficient tool to understand the mechanism of action, opening ways to new information about cell pathways, as exemplified by prion studies [129,130].Recently, large-scale screening has been undertaken to correct defective mutations of the mitochondrial gamma-polymerase (encoded by the MIP gene in yeast). While this gene is nuclear-encoded, its effects impact the mitochondrial DNA and any drug, which would act upon its defective forms, is of great importance. Screening different mip mutants, six unforeseen compounds answered the criteria; three of these are now being further analyzed [131].Despite the strong reductive aspects of using a small unicellular eucaryote to undestand some dysfunctions of the complex human organism, yeast has proven invaluable for a better understanding of mitochondrial diseases. Basic research progress has been made to understand the molecular mechanisms which underly the proper function of mitochondria in the cell. This in turn has allowed further steps, which have been validated later in human patient cell lines and could help with possible therapies. One can bet that we have not yet heard the last of yeast!The author wishes to thank the AFM (French Association against Myopathies) for their continuous support throughout the years. She is grateful for the pleasant and efficient partnership in laboratory work with YF Zhou, the many stimulating discussions with the Roma partners (L. Frontali, S. Francisci and their teams). Many thanks also go to Agnès Delahodde and Cecile Fairhead for discussions and critical reading of the manuscript.The author declares no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The role of epigenetics in human disease has become an area of increased research interest. Collaborative efforts from scientists and clinicians have led to a better understanding of the molecular mechanisms by which epigenetic regulation is involved in the pathogenesis of many human diseases. Several neurological and non-neurological disorders are associated with mutations in genes that encode for epigenetic factors. One of the most studied proteins that impacts human disease and is associated with deregulation of epigenetic processes is Methyl CpG binding protein 2 (MeCP2). MeCP2 is an epigenetic regulator that modulates gene expression by translating epigenetic DNA methylation marks into appropriate cellular responses. In order to highlight the importance of epigenetics to development and disease, we will discuss how MeCP2 emerges as a key epigenetic player in human neurodevelopmental, neurological, and non-neurological disorders. We will review our current knowledge on MeCP2-related diseases, including Rett Syndrome, Angelman Syndrome, Fetal Alcohol Spectrum Disorder, Hirschsprung disease, and Cancer. Additionally, we will briefly discuss about the existing MeCP2 animal models that have been generated for a better understanding of how MeCP2 impacts certain human diseases.Methyl CpG binding protein 2 (MeCP2) was first identified and characterized as a DNA binding protein that specifically binds to methyl-CpG dinucleotides [1]. The MECP2 gene is localized on the X-chromosome and contains complex regulatory elements that control its precise expression levels [2,3,4,5]. In both human and mouse, the MECP2/Mecp2 gene consists of four exons encoding for two different protein isoforms, MeCP2E1 and MeCP2E2 [6,7]. The two Mecp2/MeCP2 isoforms show differential temporal and brain region-specific differences in their distribution [7], and we previously showed that MeCP2E1 isoform is similarly distributed compared to total MeCP2 in murine brain cells [8]. The main functional protein domains of MeCP2 include the methyl binding domain (MBD), the transcriptional repression domain (TRD), the C-terminal domain (CTD), and the inter domain (ID) [9,10]. The MBD facilitates binding to methylated CpG dinucleotides and the preference for adjacent A/T-rich motifs [9,11]. It is also capable of binding to non-methylated DNA sequences such as the four-way DNA junctions [12,13]. The TRD domain mediates the transcriptional repressor role of MeCP2, and interacts with co-repressor complexes, such as c-Ski, mSin3A, HDAC1, and HDAC2 [5,9,14,15]. The CTD is suggested to be important for MeCP2 function and its chromatin binding activities [12]. Inter domain interactions significantly impacts MeCP2 structure and increases the stability of the protein [16,17].MeCP2 is a nuclear protein that is mainly localized to methylated pericentromeric heterochromatin, referred to as chromocenters [18]. MeCP2 is widely expressed in several tissues, and is suggested to mediate transcriptional regulation through association with 5-methyl cytosine (5mC) and 5-hydroxymethlcytosine (5hmC), the two major types of DNA modifications [19,20,21]. MeCP2 binding to 5mC is associated with repressive functions through its interaction with co-repressor complexes, while binding to 5hmC is suggested to facilitate gene expression through the organization of dynamic chromatin [15,22,23,24]. Research studies on MeCP2 have shown diverse functions of this protein including transcriptional repression, transcriptional activation [25,26,27], RNA splicing [28,29], long range chromatin remodeling [30,31], modulation of chromatin architecture, and maintenance of DNA methylation [32]. This diversity in the function of MeCP2 underscores its role in many disorders.MeCP2 is a multifunctional epigenetic regulator that is involved in transcriptional regulation as well as modulating chromatin structure [17,33]. Epigenetics control gene expression without altering the corresponding DNA sequences and impact development and disease [21,34,35,36]. Epigenetic mechanisms control the expression of many neurodevelopmentally important genes through chromatin remodeling, histone modifications and DNA methylation [37,38,39,40,41]. The prevailing view of MeCP2 as an epigenetic modulator is related to its ability to associate with different epigenetic marks (either through direct DNA binding or recruiting other transcription factors). Thus, MeCP2 acts as an ‘epigenetic reader’ that contributes in the establishment of functional states of chromatin structure, a process that is fundamental for normal cellular function. However, it is still not fully understood whether MeCP2 acts as a genome-wide epigenetic regulator or as a gene-specific transcriptional modulator, and evidence exists in favor of both mechanisms [15,42].MeCP2 protein is highly expressed in the brain compared to other tissues [18,43,44]. The protein expression pattern within the brain follows a defined pattern, with early appearing structures, such as brainstem and thalamus, expressing the protein before more rostral structures, such as the cortex [18,43]. Temporally, MeCP2 protein expression profile is low at birth, and increases dramatically at specific time periods coinciding with the process of neuronal maturation and synaptogenesis in different parts of the brain [43,45]. Cell type-specific studies have revealed that MeCP2 levels are highest in mature neurons, while astrocytes and immature neurons express lower levels of MeCP2 [8,46,47]. The increased levels of MeCP2 expression in mature neurons are maintained throughout adulthood, implying a requirement for postmitotic neuronal function [47,48,49]. In neurons, MeCP2 is involved in neuronal maturation, dendrite formation, and synaptic functions [15,49,50]. Studies of mouse models lacking Mecp2 expression in neurons further demonstrate the critical role of MeCP2 for normal brain function, especially with regard to synaptic modulation and maintenance [18,51]. Most MeCP2 mutations are de novo and can be grouped into three general categories; severe loss-of-function mutations, mild loss-of-function mutations and a broad group of duplications and other non-coding mutations [52,53]. Each category of mutation is associated with (a) subset(s) of neurological symptoms. Genotype-phenotype analyses have shown that there is no direct and simple correlation with the type of observed mutations and the resulting disease phenotypes. The difficulty in attributing a particular type of mutation to a specific phenotype might be in part due to the pattern of X-chromosome inactivation (XCI) [54,55]. Overall, MeCP2-related diseases that are associated with protein dysfunction are mainly characterized by cognitive impairment and intellectual disabilities [33]. However, dysregulation of the protein are also frequently observed in cases of inherited disorders and autoimmune diseases such as systemic lupus erythematosus (SLE) [56]. In this review, we will primarily focus on human diseases that are associated with MeCP2 dysfunction, and will aim to highlight the role of MeCP2 in neurological/neuropsychiatric and non-neurological disorders. In addition, animal models that have enabled a better understanding of the mechanism of MeCP2 action will be discussed.Rett Syndrome (RTT) is a progressive X-linked neurological disorder that mainly affects young girls with an incidence rate of 1:10,000-1:15,000 [15,57]. RTT is, perhaps, the most common cause of mental retardation in females [58,59]. Although it was initially thought to occur exclusively in females, males have also been identified with classic RTT [60]. In approximately 90% of RTT cases, the disease is due to MECP2 gene mutations [61]. MECP2 mutations that cause classical RTT in females typically result in neonatal encephalopathy and death in the first year of life in males. However, similar MECP2 mutations may result in RTT phenotypes in males with Kleinfelter syndrome (47, XXY), or somatic mosaicism [62,63].There is the classic RTT as well as atypical forms of RTT that deviate from the classical clinical presentation. Classic RTT is noticed primarily during infancy and can be divided into four stages that reflect the characteristic abnormalities displayed in RTT patients [64]. The first stage takes place after a period of normal development, during the first 6-18 months after birth, when developmental progression ceases and acquisition of new skills is delayed. General motor performance such as crawling, sitting, and walking are also severely impaired at this stage [58,65,66]. The second stage of RTT starts at approximately one to four years, with developmental stagnation accompanied by general growth retardation, loss of purposeful hand movements and speech, tongue protrusion, abnormal facial expression, weight loss, and gait ataxia/apraxia [58,64,67]. Autonomic dysfunction such as irregular breathing patterns, forced expulsion of air and saliva, and apnea can also be observed at this stage [68]. The duration of this stage is from weeks to approximately a year. The third RTT stage is regarded as a relatively ‘quiet’ period as stabilization of some of the symptoms occurs. However, neuromotor regression and stereotypic hand movements still persists [58,64,67]. A defining feature at this stage is the occurrence of seizures, which ranges from easily controlled to intractable epilepsy [69]. The duration of this stage is usually from years to decades [67]. The last RTT stage takes place from ages 5-15 years and beyond. Motor deterioration continues and results in loss of mobility and dependence on wheel chair. Additional abnormalities include dystonia, severe constipation, oropharyngeal dysfunction, and cardiac abnormalities [68]. As patients become older they often develop Parkinson’s-like features [64,68]. The duration of this stage varies with individuals, and usually lasts for decades.Atypical forms of RTT deviate from classic RTT with respect to age of disease onset, clinical profile and severity of symptoms. These forms of RTT occur due to skewing of XCI, and may range from milder forms to more severe manifestations than classic RTT [70,71]. Mild variants of RTT are characterized by a later age of onset, typically occurring between one to three years of age and display mild stereotypic movements and neurologic symptoms. The preserved speech or the Zappella variant is a mild variant of RTT characterized by the ability of patients to speak a few words. Patients with this variant have a normal head size and are usually overweight [72]. The more severe variants include the congenital form (also known as the Rolando variant) that lack the early period of normal development, and a form of classical RTT with early onset seizures before the age of six months (also known as the Hanefeld variant) [73,74]. Mutations in cyclin-dependent kinase-like 5 (CDKL5) are associated with the early-onset seizure variant form of RTT, while mutations in forkhead box protein G1 (FOXG1) are associated with congenital RTT Syndrome variant [75,76].Magnetic resonance imaging (MRI) and autopsy examination have shown that major morphological abnormalities detected in the central nervous system of RTT patients include an overall decrease in the size of the brain and of individual neurons [77]. The reduction in brain size is distributed throughout the brain and affects both white, and to a greater extent the grey matter in different brain regions [78,79]. Cortical and cerebellar degeneration have also been demonstrated to progressively occur with increasing age in RTT patients [80].MECP2 duplication syndrome was predicted by the observation that mice engineered to overexpress MECP2 develop a progressive neurological disorder including stereotyped and repetitive movements, epilepsy, spasticity, hypoactivity, and early death [81,82]. This gain of function mutation occurs mostly in males [83]. Males with this disorder present clinical features, such as infantile hypotonia, severe to profound mental retardation, poor speech development, recurrent infections, epilepsy, and progressive spasticity [84,85]. The incidence rate of MECP2 duplication disorder is estimated to be 1% of unexplained X-linked diseases and most of the reported cases are inherited [86]. However, de novo cases have also been documented [87]. There is considerable clinical overlap in patients with classic RTT and MECP2 duplication disorder specifically in behavioral phenotypes, such as stereotypic hand/body movements, anxiety, and social avoidance [88]. However, in contrast to RTT, immunodeficiency is observed in patients with MECP2 duplication disorder. It remains unclear if this phenotype occurs due to secondary effects from increased dosage of MeCP2 protein [89]. Approximately 40% of males with MECP2 duplication reported so far have died before their 25th birthday, usually from respiratory infections [90].MECP2 mutations that cause RTT have also been reported in cases of Angelman Syndrome (AS) [91,92,93]. Angelman Syndrome is primarily caused by mutations or imprinting errors of the UBE3A gene located on chromosome 15, and is characterized by intellectual disability; severe speech impairment and gait ataxia. Considerable phenotypic overlap exists between AS and RTT; however they differ with respect to timing of symptom onset. Angelman Syndrome has an earlier onset and patients are characterized with low hypotonicity at birth [91]. X-linked mental retardation (XLMR) is a genetic disorder arising from mutations or duplication of genes across the X chromosome, including the MECP2 gene. MECP2 point mutations have been identified in up to 2% of individuals with XLMR and duplications of the gene are also implicated in approximately 1% to 2% of XLMR cases [86,94]. MECP2 mutations that cause RTT or severe neonatal encephalopathy are not identified in XLMR patients and vice versa [86,95]. In addition, the molecular lesions underlying MECP2 duplications that result in XLMR are different from those observed in MECP2 duplication syndrome [96]. Males with XLMR show phenotypes, such as severe intellectual disability, speech impairment, and motor abnormalities, whereas females display mild intellectual disability or are unaffected [86].Males with a normal karyotype and a mutation in the MECP2 gene present with a distinct clinical condition, and severe neonatal encephalopathy (SNE) [97]. Severe neonatal encephalopathy is a disorder characterized by a static encephalopathy, severe developmental delays and respiratory abnormalities. The mutations are usually inherited from mothers with favorable XCI skewing that display mild/no RTT symptoms. Males with SNE often die within the first years of their life due to autonomic dysfunction [95,98]. Some MECP2 mutations that do not cause RTT in females have also been implicated in moderate to profound mental retardation, deficits in language and motor skills, obesity, autistic features, and psychiatric disorders in males [54].Autism and RTT are similar developmental disorders that belong to the spectrum of autism disorders classified as pervasive developmental disorders, or Autism Spectrum Disorders (ASD) [99]. Although they are similar developmental disorders, autism differs from RTT with respect to its genetic basis. While RTT is caused by MECP2 mutations, the genetic basis of autism is not fully clear and is proposed to involve multiple genes [100]. Mutations in the MECP2 regulatory elements (resulting in decreased expression of the protein) are commonly associated with autism [101]. Reduced expression of MeCP2 protein has been shown to occur frequently in the frontal cortex of autistic patients and is correlated with increased MECP2 promoter DNA methylation [102]. The silencing of autism-related genes through promoter DNA hypermethylation is commonly associated with autism, and drugs that can demethylate promoters might be useful in activating these genes [103,104]. In a recent study, we show that reduced DNA demethylation at the Mecp2 promoter and intron 1 regulatory elements treated with Decitabine is associated with increased Mecp2 expression. Our results provide insight on use of such drugs for future therapeutic interventions of autism [105]. Moreover, MECP2 mutations that are associated with classic RTT have been identified in a number of autistic females who do not meet the diagnostic criteria for RTT [100]. This makes it difficult to determine if a MECP2 mutation that is associated with autism diagnosis is a different disorder from RTT, or if both disorders are simply different representations on a spectrum associated with MECP2 mutations. MECP2 mutations have also been reported in patients with mild cognitive and motor difficulties and early-onset schizophrenia [106].Prenatal exposure to alcohol is associated with adverse effects on neurodevelopment, and results in a set of severe neurodevelopmental disorders known as fetal alcohol spectrum disorders (FASD) [107,108]. The incidence of FASD is estimated to be as high as 1 in 100 births and this disorder commonly manifests as cognitive and intellectual disabilities [108]. Accumulating evidence-implicating MeCP2 in FASD pathogenesis further reinforces the critical role of MeCP2 for central nervous system function. Several studies have demonstrated aberrant expression levels of MeCP2 in rodent FASD models, and this is suggested to be an important epigenetic determinant in FASD [109,110]. For example, prenatal exposure to ethanol has been demonstrated to significantly decrease MeCP2 expression in both prefrontal cortex and striatum of rodent offspring [109]. Research in animal models suggests that the global epigenetic changes due to ethanol are related to variations in the levels, duration as well as time of exposure [108,111]. Further supporting the potential role of MeCP2 in FASD, RTT-causing mutations have been reported in a FASD patient [112]. MeCP2 has been also shown to modulate the alcohol intake and sensitivity to alcohol, demonstrating the role of MeCP2 in alcoholism [113]. Huntington's disease (HD) is a progressive neurodegenerative disorder that affects muscle coordination and results in cognitive decline and psychiatric disorders [114,115]. It is one of several tri-nucleotide repeat disorders caused by the length of a repeated section of a gene exceeding a normal range. Expansion of a CAG triplet repeat stretch within the Huntingtin gene (HTT) results in a mutant form of the protein, which gradually damages brain cells [114,116]. Transcriptional dysregulation has been suggested to play major roles in HD pathology, and it was recently demonstrated that the huntingtin protein (Htt) directly interacts with MeCP2 in mouse and cellular models of HD. Aberrant interactions between Htt and MeCP2 is suggested to contribute to aberrant transcription in Huntington’s disease by regulating brain-derived neurotrophic factor (BDNF) levels [117]. The implication of MeCP2 in such diverse range of neurological disorders necessitates a complete understanding of its relationship to brain development and function, as well as its interaction with other epigenetic factors that mediate dysregulation of normal epigenetic program of the brain. Apart from to its role in neurological disorders, MeCP2 has also been shown to play a role in many cancers such as breast, colorectal, lung, liver, and prostate cancer [118,119,120,121,122,123]. MeCP2 role in cancer is related to the epigenetic regulation of cancer-related genes, particularly mechanisms that involve hypermethylation of gene promoters [118]. The growth-promoting role of MeCP2 in prostate cancer cells has been demonstrated previously, where it was shown to control mechanisms, such as cell proliferation and apoptosis [119]. In gastric cancer, the depth of invasion has been shown to be associated with MeCP2 protein levels. In gastric carcinoma cells, microRNA miR-212 was shown to suppress translation of MECP2 transcripts, which in turn resulted in reduced depth of cellular invasion [124]. In addition, MeCP2 has been linked to other cancers, such as myeloma [125], hematological malignancies [126], ductal carcinomas [127], and cervical cancers [128]. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that affects multiple organs. The disease predominantly affects females, with a female to male ratio that ranges from 4.3–13.6 to 1 [56,129]. Although the cause of SLE is still not clear, evidence supports an important role for abnormal T cell DNA methylation in the pathogenesis of SLE, as methylation sensitive genes show increased expression in T cells of SLE patients [130]. In active SLE T-cells, the expression of DNA methyltransferase 1 (DNMT1), the main enzyme that maintains DNA methylation during cell division, is reduced, resulting in promoter hypomethylation of these methylation-sensitive genes [56,131,132]. MeCP2 is suggested to play an important role in this process as it is critical for the epigenetic regulation of methylation-sensitive genes, and genetic polymorphisms in MECP2 have also been identified in patients with SLE [56,133,134]. Moreover, MeCP2 associates with DNMT1, the association of which is required to maintain DNA methylation [32]. The association between MeCP2, DNMT, and methylation-sensitive genes suggests an important role for epigenetic regulation in the pathogenesis of SLE and other autoimmune diseases.Rheumatoid arthritis (RA) is a systemic autoimmune disease that results in chronic inflammation and destruction of many tissues and organs, primarily flexible synovial joints [135,136,137]. It is characterized by the presence of auto antibodies, which may be detected in the blood long before the onset of disease [138,139]. Increasing evidence suggests that DNA methylation and histone modifications regulate the progression of Rheumatoid arthritis. Interestingly, it has been shown that MeCP2 expression levels were up-regulated in rodent models of RA, and it is hypothesized that the increased MeCP2 protein levels play a role in the pathogenesis of RA through the canonical Wnt pathway [140,141].Hirschsprung’s disease (HSCR) is a congenital disorder of the colon characterized by the absence of certain nerve cells known as ganglion cells [142,143]. The incidence of HSCR is about 1:2000–5000, with males being affected 4 times more frequently than females [144]. The lack of ganglion cells is associated with impaired craniocaudal migration of neural crest cells, and results in severe constipation or intestinal obstruction [145,146]. Defects in the differentiation of neuroblasts into ganglion cells may also contribute to the disorder [142]. A recent study implicating MeCP2 in the pathogenesis of HSCR suggests that aberrant reduced levels of MeCP2 may play an important role in suppressing the proliferative ability of cells in patients with Hirschsprung’s disease [147]. Table 1 represents a list of MeCP2-associated diseases and the genders that are mostly affected.Human diseases associated with Methyl CpG binding protein 2 (MeCP2) and gender mostly affected. Several animal models have been generated in order to better understand the molecular mechanisms, progression and pathology of disorders that are associated with MeCP2 dysfunction. These include mostly rodent models (particularly mouse models) as well as zebrafish and Drosophila models. Different strategies are often employed to alter the expression and function of MeCP2 in these animal models. Mecp2 knockout mice have been generated using the Cre recombinase-loxP system. These mice appear normal until approximately three to four weeks of age, when they begin to exhibit behavioral phenotypes such as unusual gait, hindlimb clasping, seizures, tremors, anxiety, learning and memory deficits, and irregular breathing. Brain neuropathology observed in Mecp2 knockout mice is similar to that observed in RTT individuals. Mecp2 knockout mice have smaller densely packed neurons, reduced dendritic spine density, deficits in axonal fasciculation and reduced number of mature synapses. Mecp2 knockout mice die at approximately 10 weeks of life [150,151,152]. The reactivation of Mecp2 in the brains of Mecp2 null mice rescued many RTT-phenotypes seen in the null mice, including delayed disease progression, increased life span, deceased mortality, and restored neurological functions [153,154]. Mecp2 mutant mice differ from the above-mentioned knockout mice as they express a truncated/mutant MeCP2 protein. One of such mutant mice is the Jaenisch strain (Mecp2tm1.1jae), generated by deletion of exon 3 of the Mecp2 gene. Jaenisch mice display behavioral phenotypes similar to the Mecp2 knockout mice, although the phenotype is slightly milder and the lifespan is longer (12 weeks) [82,155]. Another Mecp2 mutant mouse model has been generated that expresses a protein truncated at amino acid 308 (Mecp2308). It is assumed that the truncated MeCP2 protein produced in these mice has residual unknown functions. Mecp2308 mice appear normal until approximately six weeks of age when they develop similar but milder neurological phenotype as Mecp2 knockout mice [156]. Brain-specific deletion of Mecp2 in neural precursor cells, beginning at embryonic day 12 (Nestin-cre line), results in mice that display phenotypes similar to Mecp2 knockout mice [150,155]. This suggests that MeCP2 dysfunction in the CNS is sufficient to cause phenotypes observed in RTT. Selective loss of Mecp2 in hypothalamic and amygdala neurons (Sim1-cre line) results in mice that display similar abnormal physiological stress response as that observed in brain-specific conditional mice. These mice were also obese and aggressive, suggesting a role for MeCP2 in regulating social and feeding behaviors [157]. Deletion of Mecp2 in other parts of the brain such as in brainstem neurons (TH-cre line) significantly affects locomotor activity, with no effect on social interaction, breathing patterns, learning and memory [158,159].Mice overexpressing human MECP2 at approximately twice the endogenous levels exhibit delayed neurological symptoms at approximately 10 weeks. These symptoms include enhanced motor learning and synaptic plasticity in the hippocampus. However at 20 weeks, these mice develop seizures and become hypoactive and the majority of them die by approximately one year of age [81]. Increased MeCP2 expression in other transgenic lines also results in motor abnormalities and neurological symptoms [160]. These transgenic mice mimic MECP2 duplication syndrome that is observed in humans, and reinforces the notion of a critical requirement for precise dosage of MeCP2 protein.A mouse model of Rett syndrome was generated by introducing a premature STOP codon at the amino acid position 168, and resulted in a truncated MeCP2 protein (Mecp2R168X). These mice display neurological phenotypes such as hind limb clasping and breathing irregularities similar to other mouse models [161]. Another RTT mouse model is the A140V Mecp2 mutant mice (Mecp2A140V). This MeCP2 mutation has also been described in cases of X-linked mental retardation and manic-depressive behaviors. A140V mutant mice produce a mutant MeCP2 protein that lacks the ability to bind to Alpha thalassemia/mental retardation syndrome X-linked (ATRX) protein. These mice have an apparently normal life span, and lack specific phenotypes that are displayed in other mouse models including seizures, tremors, and breathing irregularities. However they show increased cellular packing and reduced dendrite branching similar to what is observed in autopsy brains from RTT individuals [162]. MeCP2 partners with ATRX and is involved in the silencing of imprinted genes in brain [163]. Recently, a Mecp2e1-deficient mouse model was developed with a point mutation in exon 1 changing translational start site of the first exon from “ATG” into “TTG”. These mice show many phenotypes observed in Mecp2 null mice including hindlimb clasping, forelimb stereotypy, and excessive grooming followed by death within 7 to 31 weeks [164]. In order to demonstrate the functions of neuronal activity-dependent phosphorylation at S80, S421 and S424, two knock-in mice models were generated; Mecp2S80A and Mecp2S421A;S424A abolishing the phosphorylation at S80 and S421 sites. The Mecp2S80A mice showed reduced locomotion while opposite locomotors behaviors were seen in Mecp2S421A;S424A, providing insights on the functions of S80 phosphorylation in resting neurons and S421 in active neurons [165]. A rat model of Rett Syndrome was also generated with reduced Mecp2 expression in the brain. This RTT rodent model revealed reduced expression of Bdnf, with no significant phenotypes that mimic RTT [166]. Other RTT animal models include the Drosophila and zebrafish models. The Drosophila RTT model was generated by overexpressing known RTT mutations such as R294X and R106W, which resulted in locomotary dysfunction [167]. The recently reported zebrafish model of RTT was generated by introducing a C to T transition-mutation at position 187 of the Mecp2 coding sequence. This resulted in a nonsense mutation and a truncation of the MeCP2 protein at the position 63 (Mecp2Q63*). RTT zebrafish model display altered motor behaviors, although the phenotype is weaker in comparison to other Mecp2-deficient animal models. In contrast to MeCP2-null mouse models, Mecp2-null zebrafish are viable and fertile [168].Other than animal models, in vitro cellular models of Mecp2 dysfunction have also been established in order to better understand the how the effects of MeCP2 deficiency impairs normal brain function. Loss of Mecp2 from cultured neuronal cell populations obtained from embryonic or postnatal Mecp2-deficient mice indicate biochemical and morphological abnormalities similar to Mecp2 null animals [49,159,169]. Moreover, other studies have revealed putative roles of MeCP2 in astrocytes and microglia despite the low levels of MeCP2 in these cell types. Mecp2-deficient astrocytes and microglia have been demonstrated to produce aberrant levels of soluble factors, such as glutamate, which inhibit dendrite branching from co-cultured neurons in vitro [47,48,170]. In addition, loss of Mecp2 from astrocytes has been demonstrated to affect astrocytic gap junction function, thereby resulting in their failure to adequately support dendritic development [48]. Table 2 presents an overview of animal models that are developed to study the molecular function of MeCP2.Epigenetic regulation of gene expression is fundamental for proper organism development and function. MeCP2 is a multifunctional epigenetic regulator and the malleability in its function underscores its role in many human diseases. Since its discovery, significant progress has been made to understand its dynamic molecular properties, and increasing evidence reveals its central position in many neurological, neurodevelopmental, neuropsychiatric and non-neurological disorders. Despite tremendous progress in understanding the molecular mechanisms by which dysregulation of MeCP2 expression and function results in these disorders, we are still far from translating this knowledge towards effective therapeutic approaches. The availability of excellent animal models promises, not only hope, but also a better strategy to overcome the challenge of translational research. It is very likely that the number of diseases associated with MeCP2 dysfunction will grow rapidly in near future, thus, a better understanding of how MeCP2 functions in complex regulatory networks will pave the way for the discovery of better disease biomarkers as well as novel targets for treatments.Animal models of MeCP2 dysfunction. Angelman SyndromeAutism Spectrum DisordersAlpha Thalassemia/Mental Retardation Syndrome X-linkedBrain Derived Neurotrophic FactorCyclin-Dependent Kinase-Like 5C-terminal DomainDNA Methyl TransferaseFetal Alcohol Spectrum DisordersForkhead Box Protein G1Huntington's DiseaseHirschsprung’s DiseaseHuntingtin GeneHuntingtin ProteinMethyl Binding DomainMethyl CpG Binding ProteinHuman MECP2 GeneMouse Mecp2 GenemicroRNARheumatoid ArthritisRett SyndromeSystemic Lupus ErythematosusTranscription Repression DomainX Chromosome InactivationX-linked Mental Retardation5-methylcytosine5-hyrdoxymethylcytosineWe apologize that many excellent papers are not included in this paper due to space limitation. The authors would like to thank members of the Rastegar laboratory (specially Vichithra RB Liyanage) for helpful discussions. The research of M. Rastegar is supported by funds from the Scottish Rites Charitable Foundation of Canada (SRCFC, Grant 10110), Natural Sciences and Engineering Research Council of Canada (NSERC Discovery Grant 372405-2009), Canadian Institute of Health Research (CIHR) Grants TEC-128094 and CEN-132383, as well as Health Sciences Centre Foundation (HSCF). CD Ezeonwuka was a recipient of MHRC-UMGF studentship award.The authors declare no conflict of interest. 

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The renal collecting duct is the nephron segment where the final urine content of acid equivalents and inorganic ions are determined. The role of two different cell types present in this nephron segment has been determined many years ago: principal cells that express the epithelial sodium channel ENaC and aquaporin 2, regulate electrolyte reabsorption, while intercalated cells, which express acid-base transporters and vacuolar H+-ATPase, maintain an apropriate acid-base balance. Recent evidence challenges this historical view. Rather than having independent and non-overlapping functions, the two cell types in the collecting duct appear to functionally cooperate to regulate acid-base and volume homeostasis via complex paracrine and endocrine interplay. This review summarizes these recent findings.The western diet generates an excess of protons that would dramatically alter intracellular and plasma pH if not strictly regulated. Plasma bicarbonate (on average 24 mM) buffers the acids generated from our metabolism. As it is freely filtered by the renal glomerulus, bicarbonate must be efficiently reabsorbed to maintain blood levels. This occurs via the action of two main organs: the lungs through expulsion of volatile carbon dioxide, and the kidneys by reabsorbing filtered bicarbonate back into the blood and by de novo generating bicarbonate ions.Filtered bicarbonate is predominantly reabsorbed from the proximal convoluted tubule (Figure 1) (at least 80%) by the concomitant action of cytosolic and luminal carbonic anhydrases, the apical sodium-proton exchanger 3 and the basolateral sodium-bicarbonate cotransporter NBC1 [1,2]. The remaining bicarbonate is reabsorbed from the thick ascending limb of the loop of Henle, the distal nephron, specifically the distal convoluted tubule & the connecting tubule, and finally the cortical and both the outer and inner medullary collecting duct [3,4]. As the last segment of the nephron where bicarbonate reabsorption and urine acidification can be fine-tuned, this final segment plays an essential role in one’s acid-base balance, as highlighted by patients who develop distal renal tubular acidosis (see Section 7). In this latter segment of the nephron, two cell types co-exist: principal cells and intercalated cells, whose respective roles have historically been thought to be independent. Principal cells are involved in sodium and water reabsorption via the apical epitelial sodium channel ENaC and aquaporin 2, respectively. Intercalated cells participate in acid-base homeostasis. In this review, we have focused on the function of intercalated cells as recent findings challenge the role historically attributed to these cells as exclusively involved in acid-base homeostasis. Here, we review the long-established function of intercalated cells in acid-base regulation and we then summarize the emerging role of these cells in chloride and sodium homeostasis. We also review their amazing plasticity to inter-convert from one type to another, and their involvement in human pathologies.Nephron anatomy model including the fractional delivery of bicarbonate to the various nephron segments. The fractional delivery (FD) values of bicarbonate to the various sites are only indicative, as they vary with an individual’s diet [5]. PCT: proximal convoluted tubule, TDL: thin descending limb, LH: loop of Henle, MTAL: medullary thick ascending limb, CTAL: cortical thick ascending limb, DCT: distal convoluted tubule, CNT: connecting tubule, CCD: cortical collecting duct, OMCD: outer medullary collecting duct, IMCD: inner medullary collecting duct.Kidney cortical collecting duct (CCD) contains at least two different cell types: principal cells that transport water, Na+ and K+, and intercalated cells, which mediate acid-base transport [6] (Figure 2). In mouse, rat and rabbit connecting tubule (CNT), CCD and outer medullary connecting duct (OMCD), intercalated cells represent approximately 40% of all cells [7,8,9,10,11,12]. The respective percentages of type-A, type-B and non-A, non-B intercalated cells vary between nephron segments and species as shown in Table 1. Intercalated cells express high amounts of cytosolic carbonic anhydrase II and are interspersed among the principal cells of the distal convoluted tubule (DCT), connecting tubule (CNT), and collecting duct [9,11,13]. They are distinguished from other cell types by their positive staining for the vacuolar H+-ATPase and carbonic anhydrase II (CAII), by their dark cytoplasm and high mitochondrial density [10,11]. Three kinds of morphologically and immunologically different intercalated cells have been identified in mouse kidney and named type-A, type-B, and non-A, non-B intercalated cells [9,13].Percentages of intercalated cells in various distal nephron segments in mouse, rat and rabbit. These rough percentages were assessed using electron microscopy and immunohistochemistry. The variations are likely due to diet differences and therefore may not reflect the situation in humans. CNT, connecting tubule, CCD, cortical collecting duct, OMCD, outer medullary collecting duct, IMCD, inner medullary collecting duct. These numbers are from [12,13,15,16,17].Type-A intercalated cells have a columnar shape with apical microprojections and tubulovesicular structures under the apical surface. These cells express the kidney anion exchanger 1 (the kidney isoform of band 3) at their basolateral membrane and vacuolar H+-ATPase is expressed at the apical side (Figure 2). Type-A intercalated cells are characterized by a robust apical endocytosis that regulates the amount of vacuolar H+-ATPase under acid load conditions [6,10].Almost mirroring the type-A intercalated cells, type-B intercalated cells display the opposite polarity, with the bicarbonate exchanger pendrin located at the apical membrane and vacuolar H+-ATPase at the basolateral membrane (Figure 2). This opposite polarity not only reflects a re-location of key proteins to opposite membranes but also the expression of different bicarbonate transporters. With their squamous shape, type-B intercalated cells have a smooth apical surface, an organelle-free zone below the apical membrane, and cytoplasmic vesicles dispersed throughout the cells. Additionally, these cells bind peanut agglutinin, in contrast to type-A intercalated cells [9,13,14]. Specific binding of type-B intercalated cells to peanut agglutinin has become a useful tool to study intercalated cells remodeling under acidic conditions as detailed in Section 5.Model illustrating the collecting duct cell types. Type-A intercalated cells secrete protons into the lumen through the apical vacuolar H+-ATPase proton pump and the H+/K+-ATPase, and reabsorb HCO3− in exchangefor Cl− through the basolateral kidney by carbonic anhydrase II (CAII). Additionally, these cells express two potential chloride/bicarbonate exchangers, the basolateral SLC26A7 and the apical SLC26A11. The Na+-K+-2Cl− cotransporter (NKCC1) is also expressed at the basolateral membrane of these cells, along with the chloride channel ClC-Kb. Type-B intercalated cells secrete HCO3− into the lumen through apical pendrin and protons are effluxed into the blood through the basolateral vacuolar H+-ATPase. These cells also express the ClC-Kb chloride channel and SLC26A11 at their basolateral membrane. At the apical membrane, pendrin’s function is coupled to that of SLC4A8 to promote sodium and chloride reabsorption. Principal cells regulate ion homeostasis by expressing the epithelial sodium channel (ENaC), a K+/Cl− cotransporter (KCC), the water channel aquaporin 2 (AQP2) and the renal outer medullary potassium (ROMK) channel in their apical plasma membrane, and the sodium/potassium ATPase at the basolateral membrane. So far, no basolateral chloride channel has been identified in these cells.Non-A, non-B intercalated cells are mainly located in the CNT and the proximal collecting tubule. These large cells express both pendrin and vacuolar H+-ATPase at their apical membrane, and display a large number of mitochondria and intracellular vesicles [13]. The function of non-A, non-B intercalated cells is not known. The presence of apical vacuolar H+-ATPase and bicarbonate transporter suggests that these cells could be involved in either proton or bicarbonate secretion or that they are an intermediate cell-type undergoing interconversion as detailed below [9,13].Intercalated cells are enriched in mitochondria, and express proteins involved in transport of proton equivalents such as vacuolar H+-ATPase, carbonic anhydrase II and bicarbonate transporters [1,18]. These characteristics suggest a role for these cells in acid-base regulation [19].With their apical vacuolar H+-ATPase and basolateral kidney anion exchanger 1 (Figure 2), type-A intercalated cells, were implicated early on in active proton secretion into the tubular lumen. Carbon dioxide enters the cells where carbonic anhydrase II catalyzes its hydration to produce one bicarbonate ion and a proton. The proton is pumped across the apical membrane via the vacuolar H+-ATPase and the bicarbonate ion is transported across the basolateral membrane by the kidney anion exchanger 1 protein [1,20]. Therefore, the function of type-A intercalated cells results in both net reabsorption of bicarbonate and concomitant proton secretion into the urine.Type-B intercalated cells are thought to mirror type-A intercalated cells as they have almost the opposite polarity. Also rich in carbonic anhydrase II, these cells instead reabsorb protons via the basolateral vacuolar H+-ATPase and excrete bicarbonate into the urine via the apical bicarbonate transporter pendrin [21]. As menioned earlier, the function of non-A, non-B IC remains obscure.Over the years, the molecular identity of proteins involved in these opposite physiological functions has become clearer. For example, at the basolateral membrane of type-A intercalated cells, SLC26A7 appears to have a role in bicarbonate reabsorption linked to that of kidney anion exchanger 1 [22]. SLC26A7 acts as a pH-sensitive chloride channel which mediates Cl−/HCO3− exchange by recycling Cl− and reabsorbing bicarbonate [23]. Deletion of SLC26A7 results in distal renal tubular acidosis [24]. At the apical membrane of type-B intercalated cells, expression of the bicarbonate transporter SLC4A8 functionally complements that of pendrin (SLC26A4) as outlined below [25]. Water movement across the apical membrane of type-B intercalated cells may be facilitated by expression of aquaporin 5 [26]. Interestingly, a recent report demonstrated that while the Na+/K+- ATPase is required to energize principal cells of the collecting duct, the vacuolar H+-ATPase rather than the Na+/K+- ATPase, appears to be essential in maintaining cell volume and resting membrane potential in intercalated cells [27]. In fact, expression of the Na+/K+- ATPase in intercalated cells remains controversial [8,28,29,30,31].In agreement with the role of intercalated cells in regulation of systemic acid-base balance, these cells are equipped with a range of tools that allows them to sense and respond to changes in acid-base balance. Among the many sensors spread over the nephron, some are specifically active in intercalated cells. Soluble adenylyl cyclase (sAC) is directly activated by CO2 and bicarbonate concentrations and its activity is also modulated by calcium [32,33]. In type-A and type-B intercalated cells, activated sAC it produces a sAC-dependent rise in cAMP resulting in translocation of vacuolar H+-ATPase to the apical membrane, and further proton pumping to the lumen [34]. Another potential pH sensor is the alkali sensor insulin receptor-related receptor (InsR-RR), which is expressed in type-B intercalated cells and non-A, non-B intercalated cells [35]. During alkalosis, InsR-RR is activated by alkaline pH both in vitro and in vivo and is thought to stimulate secretion of bicarbonate by type-B intercalated cells [36]. Finally, a number of channels such as the renal outer medulary potassium channel ROMK and some aquaporins have their activities modulated by intracellular or extracellular proton concentrations [37].With the identification of pendrin as the apical chloride/bicarbonate exchanger in type-B intercalated cells ten years ago [38], a new functional role emerged for intercalated cells. The traditional view of principal cells being predominantly involved in sodium and potassium exchange and intercalated cells being responsible for acid or base secretion, has been challenged by evidence demonstrating that the different collecting duct cell types functionally overlap. Principal cells apically express the epithelial sodium channel ENaC, which regulates sodium reabsorption in a vasopressin- and aldosterone-dependent pathway. Chloride reabsorption was thought to occur predominantly via a passive, paracellular pathway with a smaller transcellular component. The transcellular pathway was assumed to occur via apical chloride/bicarbonate exchange and a basolateral chloride channel expressed in type-B-intercalated cells [39]. The group of Susan Wall first demonstrated that on a NaCl restricted diet, slc26a4 knockout mice were hypotensive and failed to reabsorb chloride, in contrast to wild-type animals [40]. Further, over-expression of pendrin in intercalated cells resulted in chloride-sensitive hypertension [41]. In agreement with these previous findings, pendrin function was found to be up-regulated by angiotensin II and aldosterone [42,43]. Thus, these studies provided a molecular pathway for the coordinated reabsorption of water and sodium ions by principal cells and chloride ions via type-B intercalated cells.Additionally, significant electroneutral and thiazide-sensitive sodium reabsorption was found in type-B intercalated cells [44], which persisted in mice lacking alpha ENaC [45]. This finding supported the presence of an alternative sodium reabsorption pathway in the collecting duct. Further work from Drs. Eladari and Chambrey’s groups identified this other molecular pathway to occur through the sodium-driven chloride/bicarbonate exchanger NDCBE/SLC4A8, which works in tandem with pendrin at the apical membrane of type-B intercalated cells to mediate net reabsorption of sodium and chloride [25,46]. The sodium acumulated intracellularly via the concomitant activity of apical SLC4A8 and pendrin was shown to exit the basolateral membrane of type-B intercalated cells through AE4 (slc4a9) protein [27]. Thus, according to these new findings, intercalated cells are not only required to regulate bicarbonate excretion in the urine but are also involved in transcellular reabsorption of sodium and chloride ions. These novel findings reconcile the findings from Sebastian and colleagues who observed that some patients with distal renal tubular acidosis have an urinary loss of sodium and chloride that is sustained even upon correction of the acidosis [47].A fascinating aspect of intercalated cells is that they can convert from one type to another depending on acid-base status. More than twenty years ago, studies showed that feeding rabbits an acid diet for 20 hours resulted in a four-fold decrease in the number of peanut agglutinin binding cells without altering the total number of intercalated cells, supporting internalization and degradation of the peanut agglutinin binding sites [14]. These studies infer that a change in diet could result in conversion of type-B intercalated cells to type-A intercalated cells, a phenomenon called “plasticity of epithelial polarity” [6,14]. To study the molecular basis of epithelial intercalated cells’ plasticity, an immortalized cell line of type-B intercalated cells was generated [48]. Primary type-B intercalated cells were isolated from rabbit kidneys and transfected with a plasmid encoding a temperature-sensitive mutant of the large T antigen of SV40. This allowed the continuous division of the cells at permissive temperature to form a monolayer of type-B intercalated cells. These immortalized cells demonstrated multiple characteristics of type-B intercalated cells: negative staining for the basolateral chloride/bicarbonate exchanger, positive staining for the vacuolar H+-ATPase at the basolateral membrane and binding the peanut lectin at the apical membrane.When the immortalized type-B intercalated cells were seeded at low density and allowed to become confluent, they were found to retain characteristics of type-B intercalated cells. However, when plated at high density, they converted to acid-secreting cells within a few hours, a characteristic of type-A intercalated cells [49]. Van Adelsberg and colleagues determined that at high density, these cells produced an extracellular matrix (ECM) protein, later identified as hensin (see below) [50], which induces reversion of polarity of the chloride/bicarbonate exchanger location from apical to the basolateral membrane. Later studies identified that the reversion of polarity is in fact likely due to down-regulation of apical pendrin [21] and synthesis of basolateral kidney anion exchanger 1. This conversion of type-B to type-A intercalated cells also induced robust apical endocytosis and alteration in cell morphology, as they became more columnar like type-A intercalated cells.A large multi-domain protein called hensin (“change in shape” in Japanese) or DMBT1 was purified from the extracellular matrix and found to induce the epithelial cell conversion process when secreted as a multimeric protein [51]. Antibodies for hensin prevented this effect, indicating that hensin is necessary for conversion of polarity. In fact, soluble hensin was synthesized by both low density and high density type-B intercalated cells, but only hensin secreted by high density type-B intercalated cells was observed in the extracellular matrix. Sucrose density gradient analysis of soluble hensin showed that low density cells secrete monomeric hensin, and high density cells secrete higher order multimers. High density cells caused aggregation of monomeric hensin, suggesting that the multimerization resulted from surface events in the high density cells [51].To confirm that type-B intercalated cells are progenitors of type-A intercalated cells, mice lacking hensin in the intercalated cell lineage were generated [50]. Deletion of hensin did not affect the percentage of intercalated cells from the total number of cells in the CCD, relative to principal cells [50]. However, immunological studies revealed that all the cortical intercalated cells in the mutant mice were type-B intercalated cells, with apical pendrin and basolateral or diffuse/bipolar vacuolar H+-ATPase expression as compared to only 30% of intercalated cells being type-B in the wild type mice. In the medulla of the knockout mice, a new cell type was identified, which resembled the cortical type-B intercalated cells in ultrastructure, but did not express pendrin. The presence of type-B intercalated cells exclusively and the absence of type-A acid secreting cells in the collecting duct of the hensin knockout mice resulted in continuous bicarbonate secretion, which caused metabolic acidosis and complete distal renal tubular acidosis (see below) [50].Additional work dissected the role of hensin polymerization on intercalated cell conversion. Acidosis induces hensin polymerization and deposition, a process that involves β1 integrin [50]. Deletion of β1 integrin expression from intercalated cells caused a phenotype that was identical to the deletion of hensin, consistent with hensin and integrin being important functional partners for intercalated cell conversion. Additionally, hensin requires the interaction with galectin 3 to polymerize in the extracellular matrix and promote the conversion of the cells [52]. The peptidyl prolyl cis-trans isomerase activity of cyclophilin A was also found to be necessary to initiate the hensin polymerization process [53]. When cyclophilin A was blocked by the cyclosporin A immunosuppressant, it prevented hensin polymerization and caused distal renal tubular acidosis (see below).The renal collecting tubule contains two different cell types, intercalated cells and principal cells. This pattern of differentiation in the collecting duct may be driven by a process called “lateral inhibition”, which involves various key regulating processes requiring the Notch signaling pathway and Foxi1 transcription factors.Lateral inhibition is a process in which a cluster of cells differentiate through a specific signal, such as the activation of the Notch surface receptor, while neighbor cells remain “deaf” to this signal and retain their undifferentiated state [54]. Notch signaling was indeed found to play a key role in renal collecting duct development. Deletion of Notch signaling from the mouse collecting duct increased the number of intercalated cells, whereas overexpression of active Notch intracellular domain in the collecting duct resulted in the absence of intercalated cells, and the only cell type present was principal cells [55]. Active Notch signaling seems to promote the ureteric bud cells to develop into principal cells, whereas inactive Notch signaling promotes the formation of intercalated cells.Forkhead 1 (Foxi1) also plays an important role in renal development, as it regulates expression of pendrin, a protein expressed in both the inner ear and in type-B intercalated cells of the kidney. Foxi1 is expressed in the distal nephron and in the endolymphatic duct/sac epithelium of the inner ear in mice at embryonic stage E16 and E11.5, respectively [56]. Mice lacking the Foxi1 gene are deaf, have disturbed balance and do not express pendrin, suggesting that Foxi1 could be an upstream regulator of Pendrin. Foxi1 null mice produce an alkaline urine compared with WT mice, a symptom resembling distal renal tubular acidosis [57]. Northern blots and immunohistochemistry experiments on kidneys of Foxi1 null mice revealed the absence of kidney anion exchanger 1, pendrin and anion exchanger 4 [58]. In fact, examination of the collecting duct epithelium of Foxi1 mice revealed the presence of a single cell type that was positive for both principal and intercalated cell markers [57]. Although Atp6v1b1 mRNA expression was similar to that of WT mice, the vacuolar H+-ATPase protein product was not detected in Foxi1 null kidneys due to inefficient translation or rapid protein degradation. A more recent study showed that the lack of Foxi1 expression in mice causes the absence of A (Atp6v1a), B1 (Atp6v1b), E2 (Atp6v1e2), and a4 (Atp6v0a4) subunits that normally form the vacuolar H+-ATPase in three different epithelia (inner ear, kidney and epididymis) [59]. Thus, Foxi1 and Notch signaling appear to be two upstream regulators of intercalated cell formation.The literature also contains reports demonstrating the conversion of intercalated cells into principal cells. Culture of isolated and sorted type-B intercalated cells can result in the appearance of principal cell characteristics, such as amiloride-sensitive sodium reabsorption and ability to secrete potassium [60]. Lithium, a commonly prescribed therapy for bipolar mood disorder, causes nephrogenic diabetes insispidus (NDI) in 50% of the patients [61]. Lithium-induced NDI results from a down-regulation of aquaporin-2 expression in principal cells [62], originating from lithium-induced arrest of principal cells division [63]. Interestingly, after discontinuation of lithium treatment, a transient cell type has been identified that shares both principal and intercalated cell characteristics, including vacuolar-H+-ATPase and aquaporin 2 expression [64]. Thus, intercalated cells are extraordinarily plastic as they can not only convert from one type to another, but they can also convert into principal cells.Evidence of functional cooperation between the various intercalated cell types raises one central question: how is this inter-cellular cooperation regulated? As intercalated cells are involved in salt homeostasis, mineralocorticoid hormones regulate total or plasma membrane expression levels of a number of key intercalated cell proteins. For example, total pendrin expression and transport activity are increased by aldosterone [65], via a nitric oxide and cAMP dependent mechanism [66]. Further, the effects of angiotensin II and aldosterone on collecting duct acidification have also been reported in early studies [67,68,69,70,71,72]. Angiotensin II and aldosterone stimulate vacuolar H+-ATPase activity by increasing its abundance in the plasma membrane [73], via the action of G protein, phospholipase C, protein kinase C and extracellular signal-regulated kinase (ERK) 1/2 proteins [74,75]. Aldosterone also acts in a parallel pathway through cyclic AMP and protein kinase A [75]. In the Madin-Darby canine kidney (MDCK).C11 model cell line that resembles intercalated cells, the prorenin receptor, which is an accessory protein of the vacuolar H+-ATPase, plays an important role in regulating ATPase activity [76]. Finally, in the kidney medulla, aldosterone or high dietary sodium bicarbonate increases AE1 expression levels [77]. As aldosterone also regulates plasma membrane abundance of the epithelial sodium channel ENaC in neighbouring principal cells [78], both angiotensin and aldosterone act on both principal cells and intercalated cells. A molecular mechanism regulating intercalated cell signaling was recently proposed by Shibata and colleagues. They demonstrated that a single phosphorylation at serine 843 in the mineralocorticoid receptor expressed in type-A and type-B intercalated cells prevents ligand binding to the receptor and is able to block aldosterone signaling and chloride reabsorption [79]. Dephosphorylation, likely via protein phosphatase 1, increases surface expression of electrolyte flux mediators, predominantly in type-B intercalated cells.In addition to the effects of aldosterone and angiotensin II on the function of proteins expressed in intercalated and principal cells, a number of paracrine signaling pathways are emerging and have been extensively reviewed in [80]. One such mechanism occurs via purinergic signaling. Mice deficient in the P2Y2 purinergic receptor have sodium imbalance and hypertension [81]. Release of the purinergic receptor substrate, ATP, by intercalated cells was proposed to occur via connexin Cx30 proteins [82] and may alter potassium channels either at the apical side of principal cells or intercalated cells while inhibiting sodium and water reabsorption [83,84]. Further, Gueutin and colleagues recently demonstrated that ATP-triggered prostaglandin E2 release by type-B-intercalated cells participates in hydroelectric imbalance as observed in dRTA patients where impairment of ENaC activity occurs in neighboring principal cells [46]. Other modulators of water and salt reabsorption include the G protein coupled receptor OXGR1, which is apically located in type-B and non-A, non-B intercalated cells. This receptor is sensitive to local accumulation of dicarboxylate such as alpha ketoglutarate that can locally accumulate in the distal nephron [85,86]. Another modulator is the vasodilator bradykinin, which can bind to its B2 receptors in the basal side of collecting duct cells [87,88]. Therefore, there is a growing body of evidence supporting that intercalated cells are not only involved in acid-base homeostasis but also in electrolyte balance as reviewed in [89].The abnormal functioning of intercalated cells causes a variety of diseases that mostly affect acid-base balance with mild impairment in electrolyte homeostasis.Diseases associated with abnormal intercalated cell function.Plasma pH must be tightly regulated between 7.38 and 7.42 in order to maintain normal cell function. A decrease in plasma bicarbonate concentration causes a drop in plasma pH and this condition is known as metabolic acidosis [20,104]. As mentioned earlier, the renal proximal tubule plays an essential role in bicarbonate reabsorption. Upon synthesis of protons and bicarbonate by the cytosolic carbonic anhydrase II, the basolateral sodium/bicarbonate exchanger NBCe1 (encoded by SLC4A4 gene) mediates reabsorption of filtered bicarbonate, while protons are secreted into the pro-urine via the apical sodium/proton exchanger, NHE3 [1,105]. In the distal nephron, intercalated cells mediate the fine-tuning of bicarbonate reabsorption, by reabsorbing the remaining filtered bicarbonate through basolateral kidney anion exchanger 1 and excreting protons via the apical vacuolar H+-ATPase. Distal renal tubular acidosis (dRTA) is characterized by the inability of type-A intercalated cells to excrete acids into the urine (Table 1). As proton excretion is coupled to bicarbonate reabsorption, a decrease in protons excreted into the urine in the distal nephron results in a decrease in plasma bicarbonate concentration. Interestingly, dRTA patients also develop a mild electrolyte imbalance with abnormal urinary excretion of sodium and chloride [47].Distal renal tubular acidosis can arise from genetic or immune defects as well as a side effect of drugs affecting type-A intercalated cells. Familial dRTA can be autosomal dominantly and recessively inherited [90]. Loss-of-function mutations in the SLC4A1 gene lead to abnormal functioning of the kidney anion exchanger 1 causing either autosomal dominant or recessive dRTA. This abnormal functioning results in defective bicarbonate reabsorption through the basolateral side of type-A intercalated cells. Mutations in the B1 subunit of the cytosolic V1 ATPase complex or the a4 subunit of the V0 transmembrane pore complex of the vacuolar H+-ATPase cause recessive dRTA associated with deafness. Mutations in carbonic anhydrase II lead to a mixture of recessive distal and proximal RTA, which is often associated with autosomal recessive osteopetrosis (increased bone density), growth failure, mental retardation and hearing impairement [106].The molecular basis for recessive versus dominant familial dRTA caused by mutated anion exchanger 1 has been clarified recently. Heterologous expression of the kidney anion exchanger 1 in Madin-Darby canine kidney (MDCK) cells shows that it is functional and localizes to the basolateral membrane, reflecting the location of this exchanger in native cells [107,108,109]. In these cells and in human embryonic kidney 293 cells, expression of dominant or recessive dRTA mutants of the anion exchanger 1 demonstrates mis-trafficking of the mutant, varying from intracellular retention in the endoplasmic reticulum or the Golgi to apical mis-localization [107,108,109,110]. Co-expression of intracellularly retained dominant dRTA mutants of the kidney anion exchanger 1 with wild-type (WT) kidney anion exchanger 1 retained the WT protein intracellulary [107,111,112]. In contrast, when co-expressed with a recessive dRTA mutant of the anion exchanger 1, the WT protein rescued cell surface trafficking of the mutant.Distal RTA can also originate from autoimmune disorders such as systemic lupus erythematosus and Sjogren syndrome [113], and from chronic liver diseases [114], but the pathogenicity of these causes of acquired dRTA are much less well understood.Patients with either a mutated a4 or B1 subunit of the vacuolar-type H+-ATPase have complete dRTA, hearing loss, hypokalemia and dehydration [115,116]. Mice lacking the B1 subunit of the vacuolar-type H+-ATPase have abnormal urinary acidification upon acid challenge but do not develop spontaneous acidosis and had normal hearing [93,94]. This mild phenotype was caused by the compensatory over-expression of alternate B2 subunit of the ATPase [117]. Knockout of the vacuolar H+-ATPase a4 subunit resulted in a more severe phenotype in two independent mouse models [96,97]. In both animal lines, knocking out the a4 subunit resulted in severe deafness due to expanded cochlear and endolymphatic ducts [118], impaired sense of smell [97,119] and spontaneous metabolic acidosis. The a4 knockout mice also developed hypocitraturia and early nephrocalcinosis [97]. Furthermore, development of dRTA was accompanied by an unsuspected proximal tubule defect, specifically low molecular weight proteinuria and phosphaturia [96]. Interestingly, proximal tubular cells of the knockout mice showed abnormal endocytic traficking and accumulation of lysosomal materials. As this phenotype resembles that of anion exchanger pendrin knockout mice, it was proposed that pendrin and the proton pump not only cooperate in intercalated cells of the kidney but also in the inner ear for endolymph homeostasis.Twenty years ago, two mouse models knocked out for the anion exchanger 1 were generated [120,121]. These mice, which were either lacking the erythrocyte anion exchanger 1 or both erythrocyte and renal anion exchangers 1, had a similar phenotype: 85% of the newborn mice died within two weeks after birth and the surviving animals developed severe anemia due to the premature degradation of their red blood cells. Characterization of potential renal defects was not described at that time. Ten years later, Stehberger and colleagues reported that the mice lacking the anion exchanger 1 have characteristics of complete dRTA and express many features of the human disease [91]. Mice deficient in the anion exchanger 1 displayed a urinary concentrating defect with increased serum osmolarity, decreased urine osmolarity and consequently increased urine output leading to dehydration. The urinary concentrating malfunction correlated with altered expression and localization of aquaporin-2 water channels in principal cells, thus highlighting again that principal and intercalated cell functions are inter-dependent. These studies showed that the anion exchanger 1 plays an important role in maintaining acid-base homeostasis by distal regeneration of bicarbonate in mouse and human kidneys.Interestingly, Sun and colleagues observed that in AE1 knockout mice, a 4-day acid load increased Slc26a7mRNA and protein levels in both the outer and inner medulla [22]. In agreement with this finding, genetic ablation of SLC26A7 in mouse intercalated cells induced a metabolic acidosis and alkaline urine pH, characteristics of distal renal tubular acidosis [24]. As AE1 knockout mice develop dRTA, increased expression of Slc26a7 cannot compensate for the absence of AE1 and vice-versa.Carbonic anhydrase II is very abundantly expressed in proximal tubular cells, intercalated cells and in osteoclasts. Patients with a deficiency in carbonic anhydrase II develop intracranial calcifications and mental retardation in addition to renal tubular acidosis and osteopetrosis [98,99,100]. Mice deficient for carbonic anhydrase II develop renal tubular acidosis similar to that of humans deficient in this protein; however osteopetrosis and cranial calcification were not observed in the murine model [101]. These mice displayed a marked down-regulation of proteins involved in acid-base homeostasis in intercalated cells such as SLC26A7, pendrin and AE1 but no change in aquaporin 2 expression was observed [122].Genetic ablation of pendrin and SLC4A8 confirmed their role in acid-base balance as well as electrolyte homeostasis (Figure 2). Mutated pendrin causes Pendred syndrome, which is characterized by sensorineural deafness and abnormal organification of iodide in the thyroid [102]. Mutations on the gene encoding pendrin rarely cause impaired acid-base balance under ambient conditions [123,124]. However, the lack of renal pendrin expression resulted in a lower bicarbonate excretion than in WT mice, after administration of the aldosterone analogue deoxycorticosterone, and in a severe metabolic alkalosis, characterized by impaired renal ability to properly excrete an excess of bicarbonate in the urine [38]. In addition, sodium and chloride restriction increased urine output, enhanced intravascular volume depletion and hypotension [40]. This is partially due to downregulation of epithelial sodium channel (ENaC) expression that lowers its capacity to preserve sodium [125]. In the apical membrane of the distal convoluted tubule, the thiazide-sensitive sodium/chloride cotransporter NCC compensates for the genetic ablation of pendrin or carbonic anhydrase II [126,127]. Additionally, genetic disruption of the SLC4A8 protein abolished the thiazide-sensitive, amiloride-resistant sodium and chloride reabsorption by type-B intercalated cells [25], confirming the role of this protein in electrolyte reabsorption.Multiple animal models lacking subunits of the vacuolar H+-ATPase have been generated. Mice lacking the B1 subunit display a mild metabolic acidosis [94] but deletion of the a4 subunit has a more dramatic effect on urine acidification [96], confirming the essential role of this protein in proton secretion into the urine. Recent data support that vacuolar H+-ATPase activity is more important than that of the sodium/potassium ATPase in maintaining cell volume and resting membrane potential in intercalated cells [27].Although the functional role of principal and intercalated cells has been established a long time ago, recent advances highlight that cells in the heterogenous collecting duct have not yet revealed all their secrets. While type-A intercalated cells seem to remain predominantly involved in acid-base homeostasis by pumping protons into the urine and reabsorbing bicarbonate, type-B intercalated cells appear to have a dual role in both regulating acid-base homeostasis and sodium-chloride reabsorption. Thus, principal cells are not the only ones in this nephron segment involved in water and electrolyte reabsorption. It would be surprising if, interspersed within principal and type-B intercalated cells, the role of type–A intercalated cells was restricted uniquely to acid-base balance. Like type-B intercalated cells, type-A intercalated cells are also equipped with proteins that could potentially affect volume homeostasis. For instance, some evidence point to a role of the kidney anion exchanger 1 in type-A intercalated cells in regulation of electrolyte reabsorption, as single nucleotide polymorphisms within SLC4A1 have been associated with the development of hypertension [128].Many exciting questions remain to be answered. For example, the collecting duct reabsorbs chloride via both a transcellular and a paracellular pathway, but the role and regulation of the chloride paracellular reabsorption in this segment of the nephron remains unclear, as does the impact of this transcellular regulation on paracellular fluxes. Activity of the tight junction protein claudin-4 that is expressed in the collecting duct, is modulated via phosphorylation by aldosterone [129], supporting that paracellular flux of chloride may also be hormonally regulated. Additionally, a number of other molecules that could potentially influence acid-base homeostasis, such as SLC4A4 or SLC26A11 have been identified in these cells but whether they influence either electrolyte or acid-base homeostasis remains ill-defined. Overall, it appears that the up-coming years will potentially provide exciting novel functional roles for all these molecular determinants that will complete our functional picture of these heterogenous cell types in the distal nephron.The laboratories of E. Cordat and R.T. Alexander are supported by the Kidney Foundation of Canada and the Canadian Institutes of Health Research (CIHR). RT Alexander is a recipient of an Alberta Innovates Health Solutions Clinical Investigator Award and a CIHR Clinician Scientist Award. E Cordat is supported by a KRESCENT New Investigator Award. We thank the researchers in the bicarbonate transport and renal intercalated cell fields for making this review possible and apologize for any citations we have omitted.E. Almomani, S. Kaur, RT Alexander and E Cordat all participated in designing, writing and editing of the review.The authors have no conflicts of interest to declare.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).There is evidence that the economic crisis in Greece has substantially affected patients and health care services, with chronic patients forming a particularly vulnerable group. The aim of this study was to investigate whether and in what way the current economic environment has affected patients with selected chronic conditions. A cross sectional study was carried out with a sample size of 1200 patients suffering from hypertension, diabetes and chronic obstructive pulmonary disease (COPD). Following a large family income decrease (35.4%) in the last 3 years, chronic patients reported decreased spending for various expenditure categories in order to maintain their ability to finance their health care needs. Among the disease groups studied, statistically significant differences were found for self-rated heath (SRH), out-of pocket health expenditures, health services utilization and the perceived need for physician services. Although need for physician visits for issues related to the chronic condition has largely been reported as met, this was achieved by increased out-of-pocket expenditures and large family budget cuts for essential household goods and services. Austerity measures and reduction of public health expenditure by the state appear to have led to high private expenditures and to de jure or de facto insurance coverage loss for primary care services.Following the international economic downturn, Greece entered a severe financial crisis in 2008 which threatened the country’s economic stability [1]. In response to this, a series of austerity measures were introduced in order to reduce deficit and improve the county’s credibility to the international capital markets [2].Health care was one of the main sectors targeted in order to reduce public spending and achieve future primary surplus. According to officially published data, public spending has been reduced by 23.1% between 2009 and 2011 [3]. According to our own estimations, public spending has been further decreased in 2014, which corresponds to an overall decrease of 28.6% compared to 2009.It is noted that, even before the economic crisis, public share (69.7%) of total health expenditure was relatively small. The remaining 30.3% was mostly covered by out-of pocket payments and, to a lesser extent, by private health insurance, which accounted for approximately 6% of total private spending [3]. In 2011, public share was decreased to 68.3%, and consequently households’ expenditures on health rose to 31.7% [3].Under the economic crisis, disposable income is continuously decreasing, unemployment increased by more than 200% compared to 2008, while in 2011 more than 23% of the population was living below the poverty threshold [4]. Hence, households’ ability to finance health care has been significantly reduced which raises major concerns regarding access to health care.Furthermore, there is evidence to suggest that the current economic conditions have largely affected population health. The economic crisis has been found to affect self-reported health status [5], and the incidence of acute myocardial infractions [6], major depression [7] and suicide attempts [8].Chronic diseases represent a substantial share in total burden of disease and consequently absorb significant health care resources. Thus, the aim of this study was to explore the impact of crisis on health and health expenditures’ management of patients suffering from hypertension, diabetes and chronic obstructive pulmonary disease, as well as the effect on health services utilization.A cross sectional study was carried out with a sample size of 1200 patients suffering from diabetes type II, hypertension and chronic obstructive pulmonary disease (COPD). These diseases were chosen on grounds of epidemiology and disease burden [9], as well as their socioeconomic impact on the Greek healthcare system [10,11,12]. The sample size was determined according to the prevalence of each condition. Each disease group sample was estimated at 400 patients which is the minimum sample size (385) for population size above 600,000 when expected response distribution is set to 50%, acceptable margin error to 5%, and confidence interval to 95%. It is noted that each chronic patients group under study has a higher than 600,000 prevalence in Greece.Patients were approached through their treating physician. Specifically, 120 physicians were randomly selected through the official list of the National Medical Association of Greece, based on specialty and geographical distribution. Starting on a predefined day, among his/her consecutive patient visits, each physician selected 10 patients based on the set of 10 random numbers between 1 and 20 which had been given to him by the study authors. Selected patients were informed about the objectives of the study and provided with the patient consent form. If the random-number-patient selected had any co-morbidity related to the other three conditions under study, he/she was excluded and the next patient was recruited by the physician. Fieldwork was carried out during February 2013.All patients who gave their consent to participate in the study were contacted via telephone and were interviewed on the basis of a structured questionnaire. Participants answered a series of closed and open type questions, including age, gender, total monthly family income (as an ordinal variable, Table 1), monthly family income decrease since 2010 (measured as a continuous variable), self-rated health status (measured on a scale 0–100), extent to which they had decreased spending for various expenditure categories including specific health care services (on a 4-point scale ranging from “not at all” to “very much”), and extent to which they had decreased out-of-pocket expenditures for physician visits, pharmaceuticals, lab tests and nursing home care. Health care utilization was assessed for the 6 months’ period prior to the study. Participants were asked about number of times they had visited a physician or they had been admitted to a hospital for issues related or not to their chronic condition, as well as the number of times they had felt the need to visit a physician for an issue related to their condition or another health issue.Sample size and patients’ distribution by sociodemographic characteristics and disease category.Analysis of variance (ANOVA) followed by multiple t-tests and Bonferroni correction, as well as a series of one sample t-tests and paired t-tests were performed in order to investigate mean differences between groups and the statistical significance of the means calculated for the variables described above. All analyses have been performed using STATA 2009 software.Of all 1200 patients who participated in the study, 400 suffered from hypertension, 400 from diabetes and 400 from COPD (Table 1). The various patient groups differed significantly as far as age was concerned (p = 0.001) but there were no differences in relation to gender (p = 0.158) and income (p = 0.653). In relation to age, hypertensive and diabetic patients tended to be older than 60 years of age at a higher proportion than COPD patients.The average reported family income per month was 1083.30 € (95% CI: 1052.10€, 1114.60 €), which was not found to differ significantly among disease groups (p = 0.164) (Table 2). The average 3-year income loss (since 2010 when austerity measures were introduced) was 592.70 € (95% CI: 551.30 €, 634.10 €), which corresponded to a 35.4% total 3-year decrease. However, it appears that austerity measures had a uniform effect on the disease groups as the average 3-year income loss did not differ significantly among them (p = 0.657).Patients were further asked to what extent they had decreased spending on various expenditure categories. The vast majority of them stated that they had decreased spending for all expenditures categories except for health (Table 3). Specifically, of the patients for whom the various expenditures categories were applicable or were reported that represented a need, 56.4% stated that they had not decreased spending for health. However, 20.2% reported that they had decreased health care expenditures much and very much. Similar extent of decreased spending reported 53.9% of patients regarding education, and 47.7% regarding food and nutrition. Nonetheless, concerning other household expenditures, more than 90% stated that they had reduced expenditures for personal care and cosmetics, gifts, personal footwear, personal clothing, entertainment and trips. Of the patients, 88.1% claimed their expenditure for housing and accommodation had decreased since 2010 while the respective figure for expenditure concerning every day transport and telecommunication was 76.6% and 76.1%, respectively.Regarding health care expenditures, respondents were asked how much they had reduced out-of-pocket expenditure for physician visits, lab tests, their chronic disease drugs, complementary drugs, and nursing home care. Of those who did have to spend for or used the aforementioned services, 18.3% reported having decreased spending “much and very much” for physician visits, 16.5% for lab tests, 6.7% for chronic disease drugs, 12.1% for complementary drugs, and 33.3% for nursing home care (Table 2). Expenditure reduction for the specific health care services did not vary significantly among disease groups.Impact of economic crisis on patients’ family income and out-of-pocket health care expenditures, by disease category.* not significant (p-value > 0.050).Distribution of patients reporting extent of decreased spending for various expenditure categories.When all disease groups were considered together, they reported having spent on average 76.60 € (95% CI: 66.50 €, 86.70 €) for physician visits, 14.30 € (95% CI: 4.20 €, 24.50 €) for hospital admissions, and 38.00 € (95% CI: 35.60 €, 40.5 €) for their monthly pharmaceuticals during the last 6 months (Table 2).For primary care physician visits, average out-of-pocket expenditure differed significantly among patients suffering from the conditions investigated (p = 0.009). It was reported to be 56.70 € for diabetic patients, 77.10 € for hypertensive patients, and 94.90 € for patients suffering from COPD. The Bonferroni correction showed that the average amount reported to have been spent for primary care physicians visits differed statistically significantly only between the diabetes and the COPD group.For hospital admissions, average out-of-pocket expenditure was reported at 6.80 € for patients suffering from hypertension, 14.50 € for patients suffering from COPD and 21.40 € for patients suffering from diabetes. According to one sample t–tests, average reported out-of-pocket expenditure for hospitalizations for each of the groups can be assumed as zero, and according to ANOVA no statistically significant difference was found among disease groups (p = 0.519).Finally, according to ANOVA (p = 0.000) and the Bonferroni correction, average reported monthly pharmaceutical out-of-pocket expenditure appeared to differ at a statistically significant level between all disease groups. Specifically, the COPD group reported spending the largest amount for pharmaceutical treatment (51.00 €) followed by the diabetes (39.50 €) and the hypertension groups (24.00 €).In 2013, self-rated health (SRH) was reported to have a mean score of 62.2 (95% CI: 61.0, 63.4) on a scale of 0 to 100, for all patients (Table 4). In detail, hypertensive patients’ self-rated health score was reported to be 64.3 (95% CI: 62.2, 66.4), diabetic patients’ score was 61.9 (95% CI: 59.9, 63.9) and COPD patients’ score was 60.4 (95% CI: 58.4, 62.4). Health status differed significantly among the disease groups (p = 0.028) and in particular between the hypertension and the COPD group (p = 0.025). For 2012, SRH had been reported to be 64.8 (95% CI: 63.7, 66) for all patients, 67.4 (95% CI: 65.4, 69.4) for the hypertension group, 63.8 (95% CI: 61.9, 65.8) for the diabetes group and 63.4 (95% CI: 61.4, 65.5) for the COPD group. Similarly, according to ANOVA and Bonferroni correction, SRH for 2012 was found to differ significantly between hypertension and the COPD group (p = 0.019) and between the hypertension and the diabetes group (p = 0.043). The SRH deterioration between 2012 and 2013 was tested with paired sample t-test and was found to be statistically significant for all disease groups and for each group individually (p < 0.050). SRH deterioration did not differ significantly between disease groups (p = 0.215).On average, for all patients, the reported number of times they had visited a physician for an issue related to their chronic condition within 6 months prior to the survey was 2.49 (95% CI: 2.35, 2.64) (Table 4). COPD patients had visited a physician 2.87 times (95% CI: 2.59, 3.14) which is statistically significantly higher than the 2.35 average number of visits performed by hypertensive (p = 0.014) and diabetic patients (mean = 2.26, p = 0.002). As far as physician visits for a health issue not related to their chronic condition was concerned, for the overall study sample the average was 0.93 (95% CI: 0.82, 1.04) visits during the preceding 6 months. According to the ANOVA, there were no significant differences among the disease groups’ performed visits (p = 0.648). However, total visits for all reasons were found to differ significantly between the COPD group and the hypertension (p = 0.045) and diabetes group (p = 0.005) The number of hospital admissions in the 6-month period prior to the study for an issue related to patients’ chronic condition was on average 0.46 (95% CI: 0.36, 0.57) for the overall study sample, while for an issue not related to their chronic condition it was 0.31 (0.24, 0.37). Total number of admissions was reported to be on average 0.77 (95% CI: 0.64, 0.89) (Table 4). The ANOVA revealed that there were no statistically significant differences among disease groups regarding the average number of admissions for an issue related to the chronic condition (p = 0.050), neither for another health issue (p = 0.757) nor for all admission causes (p = 0.150).Finally, patients were asked to recall how many times, during the preceding 6 months, they had felt the need to visit a physician for their chronic condition or its complications, and for an issue not related to the chronic condition, irrespective of whether they had actually satisfied that need. On average, for all patients the need to visit a physician for an issue related to their condition was felt 2.29 times (95% CI: 2.04, 2.54) (Table 4). This need was felt 2.01 times (95% CI: 1.65, 2.36) by the hypertensive patients, 1.90 times (95% CI: 1.68, 2.13) by the diabetic patients and 2.93 times (95% CI: 2.32, 3.54) by the COPD patients. The only significant differences found were once again between the COPD and the hypertension group (p = 0.008) and COPD and the diabetes group (p = 0.002). According to Bonferroni correction, all other differences between the groups could be assumed as equal to zero. The average number of times all patients felt the need to visit a physician for a non-chronic condition related issue was reported at 1.09 (95% CI: 0.97, 1.21). This need did not differ statistically significantly among the disease groups (p = 0.721). In total, the number of times all patients felt the need for a primary care visit for all health issues was found to be 3.38 (95% CI: 3.07, 3.69). Similarly, this varied significantly between the COPD and the hypertension group (p = 0.021), and between the COPD and the diabetes group (p = 0.008).In order to investigate the possibility of unmet need, a new variable was developed representing the difference between the number of times patients felt the need for a primary care visit minus the number of the actual visits performed. The difference between perceived need and actual use of physician services related to patients’ chronic condition was found at −0.21 for all patients (Table 5). Based on one sample t-test, this difference was not found to be statistically significant (p = 0.083), indicating that for all study patients’ primary care needs were reported as being met. However, this was not the case for hypertensive (mean unmet need = −0.37, p = 0.027) and diabetic patients (mean unmet need = −0.36, p = 0.001) who appeared to utilize physician services more times than the reported perceived need. In contrast, the difference between perceived need and actual utilization for the patients suffering from COPD (p = 0.754) can be assumed equal to zero. However, despite that overall patients’ perceived need has been found as being met, according to descriptive statistics of the unmet need variable, 21.7% of all patients appeared to underutilize primary care services given their reported perceived need.Mean unmet need for the overall study sample for a health problem not related to their chronic disease was 0.13 which was found to be statistically significant (p-value = 0.000), indicating that need was not always satisfied when all patients were considered. When disease groups were examined separately, unmet need was observed for the diabetes (mean = 0.15, p = 0.001) and the COPD group (mean = 0.16, p = 0.007), while this was not the case for the hypertensive patients (mean = 0.08, p = 0.220) whose need appeared to be mostly satisfied. In total, the difference between perceived need for physician visits for all reasons and actual visits was found not to be significant for all patients (mean = −0.09, p = 0.518) and for each group separately (p > 0.050). This could indicate that, in total, patients’ need is met in most of the cases. Nevertheless, 22.77% of all patients appeared to underutilize primary care services. In particular, this is observed for 24.9% of hypertensive patients, 22.8% of diabetic patients and 20.7% of COPD patients.According to the analysis, economic crisis appears to have affected substantially patients’ available family income as, on average, patients’ income was reported to have been reduced by more than 30% since 2010 when the first Memorandum of Understanding (MoU) between the Greek Government and the European Commission, the European Central Bank and the International Monetary Fund was introduced. This income reduction did not vary significantly between the disease groups investigated. Decrease of monthly family income is not an unexpected finding given that between 2009 and 2013 in Greece, per capita Gross Domestic Product (GDP) decreased by 19.6% and per capita net national available income decreased by 27.3%. Unemployment increased by 162.3% between December 2009 [4] and December 2013.Health status and health care services utilization.Perceived need and actual use of physician services (Need-Use).* p-value < 0.005.As a result of the reported family available income decrease, more than 70% of patients reported that they had proceeded to large spending cuts for most of the expenditure categories. Although to a lesser extent, even expenditures for essential needs as accommodation/housing, food and education appeared to be significantly reduced. The effect of economic crises on diet and alcohol consumption behaviors has been reported previously [13]. Some decrease of accommodation expenditure is an expected finding given that property and rent prices declined significantly due to the crisis. The new index of apartment prices decreased by 28.5% in the years between 2009 and 2013 [14].Despite the fact that 1 out of 5 patients claimed that they were forced to substantially reduce spending for their health condition, health seemed as the last expenditure category which one would be willing to decrease spending for. Similar findings were reported in the Greek National Household Budget Survey of 2012 compared to 2011, where spending was decreased mainly for non-essential goods and services (over −15.0% for clothing-footwear, recreation and culture, restaurants and hotels, etc.), while for health the decrease was −8.6% [15]. Thus, our finding that out-of-pocket health expenditures were affected to a lesser extent than other goods categories is in line with the national findings for the general population and this applies particularly for chronic patients.This essentially points to the assumption that, in the current economic circumstances, budget cuts for all other-than-health goods and services probably support health care financing. Indeed, our findings at micro level are supported by the national figures for health expenditure, according to which the public share of health expenditure in Greece decreased from 69.7% in 2009 to 68.3% in 2011, while the private share increased from 30.3% in 2009 to 31.7% in 2011 [16].According to respondents, the greater spending reduction was directed to nursing home care services and to a lesser extent to physician services, lab tests and pharmaceuticals, which suggests that efforts were made in order to maintain the financing and subsequent utilization of services considered important. There is anecdotal evidence in the country that older people and chronic patients previously living in long term care facilities were taken back at family homes and are taken care of by families.For the 6-month period prior to the study, monthly out-of-pocket expenditures for primary care physicians, hospital admissions and pharmaceuticals appear to represent approximately 5% of the monthly family income for the overall study sample. Even though this seems a low figure, it has to be noted that out-of-pocket heath care expenditures were found to be statistically significantly higher for the COPD patients, corresponding to their reported higher need for and use of physician services.The investigation of the unmet need hypothesis revealed that during the 6-month period prior to the study, on average patients’ primary care need was actually being satisfied, especially so for services concerning their chronic condition and its complications. However, the analysis showed that on average all patients underutilized services for issues not related to their condition and this was particularly observed for the diabetes and COPD groups. Furthermore, although total need for primary care visits appeared to be largely met there was still a considerable minority (22.7%) who underutilized physician services given their need. In order to satisfy their need, patients reported spending 26.9 € per visit (95% CI: 22.6, 31.4) which means that they covered either the full physician cost or quite a large share of it.The finding that self-rated health deteriorated between 2012 and 2013 is supported by published literature on the negative effect of crises on health [5,13]. It is interesting that this deterioration was found unanimously for all chronic patient groups under study.Sample size estimation was based on the fact that each chronic patients’ group under study has a higher than 600,000 prevalence in Greece and did not use the specific prevalence of each disease. However, its size allowed the necessary statistical analysis to be carried out with power and p-value as originally set, and comprised of a two-stage random sampling technique.The questionnaire used was purpose made and was tested for face validity only. Still, it included a number of questions based on a previously used questionnaire in a national survey [5]. The previous 6-months period for assessing health care utilization was chosen in order to minimize recall bias.Concern was raised by one reviewer on the data concerning income. However, it has to be noted that the abrupt and steep decline of the income of the Greek population as a result of unemployment or specific legislation on salary and pensions cuts in both public and private sectors has been deeply and painfully experienced by the population, a fact which on the one hand minimizes recall bias and on the other is corroborated by national statistics on income decreases.The original study included an additional Alzheimer’s patient group. Following reviewers’ concerns, this patient group was excluded from the analysis given its significantly higher-age profile which would pose validity issues during its direct comparison with the other patient groups due to age-related comorbidities, and the potential disease and social support benefits which would buffer the crisis effect.Regarding study strengths, it is noted that almost all the main findings of the present study (which are based on micro level data) are supported by both other literature and relevant official statistics at macro level.Due to ageing, chronic patients are and will increasingly continue to form a significant proportion of the general population in Greece, and an especially important vulnerable to the economic crisis group. The present study has shown that health policy for chronic diseases in Greece merits a significant priority, particularly due to economic crisis. Equitable access to primary care, and effective prevention and chronic disease managed care schemes should be considered for development. Cuts of public share of health expenditure and cost-sharing tools for increasing efficiency of the health care system should be assessed with particular attention as to their potential impact on raising barriers to chronic care access.Although patients’ available income has substantially been reduced, patients make efforts so that essential health care services utilization is maintained. This is achieved by significant budget cuts for all household and personal goods and services, and by increased out-of-pocket spending to finance relevant health services. As previously shown, the cost per visit appears quite high. This cannot be justified in the Greek health care system, which claims to offer universal coverage through both pillars of social insurance and a state-funded health system. Economic crisis has led to a significant reduction of the public share of health expenditure, and obviously to the reduction of health services supplied by social insurance. Consequently, social insurance and government budget cuts actually put a burden on households’ budgets even though these have substantially been reduced.The picture drawn from the above leads to the conclusion that, at least as far as chronic patients are concerned, health insurance coverage for primary care services has de jure or de facto been lost to a large extent. Nevertheless, although self-reported health status has been deteriorating, amidst the economic crisis, chronic patients seem so far to manage to keep the level of care needed by prioritizing their health over other living standard needs. However, the question arising is for how long chronic patients will be able to finance their health and to satisfy their needs given that the macroeconomic environment in Greece is not expected to improve in the near future and health care reforms are mostly oriented to comply with the imposed targets for expenditure cuts.The authors would like to thank the editor and the reviewers for their important and constructive comments.The authors declare no conflict of interest.The study was supported by Novartis Hellas.AS and JK formed the research question and initiated the study. AS contributed to the statistical analysis. All authors contributed to the interpretation of the results and to the writing of the manuscript.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Recently we described skin tumors driven by skin-specific expression of Zmiz1 and here we define keratoacanthoma pathobiology in this mouse model. Similar to human keratoacanthoma development, we were able to segregate murine keratoacanthomas into three developmental phases: growth, maturation, and regression. These tumors had areas with cellular atypia, high mitotic rate, and minor local invasion in the growth phase, but with development they transitioned to maturation and regression phases with evidence of resolution. The early aggressive appearance could easily be misdiagnosed as a malignant change if the natural pathobiology was not well-defined in the model. To corroborate these findings in the Zmiz1 model, we examined squamous skin tumors from another tumor study in aging mice, and these tumors followed a similar biological progression. Lastly, we were able to evaluate the utility of the model to assess immune cell infiltration (F4/80, B220 Granzyme B, CD3 cells, arginase-1) in the regression phase; however, because inflammation was present at all phases of development, a more comprehensive approach will be needed in future investigations. Our study of keratoacanthomas in selected murine models suggests that these squamous tumors can appear histologically aggressive during early development, but with time will enter a regression phase indicating a benign biology. Importantly, studies of squamous skin tumor models should be cautious in tumor diagnosis as the early growth distinction between malignant versus benign based solely on histopathology may not be easily discerned without longitudinal studies to confirm the tumor pathobiology.Squamous cell tumors of the skin can, at times, cause challenges in diagnostic histopathology to distinguish benign versus malignant (e.g., squamous cell carcinoma) behavior because of overlapping morphologic features and markers, as well as inability to follow the natural course of disease in human subjects [1]. Many of these diagnostic challenges can also extend into the evaluation of cancer in animal models. However, the diagnostic relevance of a histopathological “signature” in a tumor from an animal model should be clarified through validation studies following the natural progression of these tumors [2].Keratoacanthomas (KAs) are skin tumors classically characterized as crateriform masses filled with abundant keratin material and lined by proliferative stratified squamous epithelium [3,4]. In humans they tend to occur on areas of the skin exposed to ultraviolet light, such as the face, hands, and forearms [5]. These are first clinically recognized as small raised masses that can enlarge fairly quickly over the course of months and then most often regress with complete disappearance by two to nine months [4].Three distinct phases of KA growth have been described both clinically and histologically in humans: growth, maturation and regression [4,6]. In the growth and maturation phases, KAs can display local invasion along with cellular pleomorphism and proliferation, which can make them challenging to differentiate from squamous cell carcinoma (SCC) [1]. Moreover, in some cases, KAs have been reported to be a low grade malignancy or transform to SCC, further confusing the distinction between malignant and benign diseases [4,7]. As a result, KAs have a history of controversy regarding their biology and clinical relevance. Similar issues can confound animal models of cancer and their diagnoses. In this study of murine KAs, we wanted to validate tumor biology and develop standardized morphologic definitions for the different phases of tumor development for future translational investigations.We studied cutaneous squamous tumors from two different genetically engineered mouse models. Zmiz1Δ1-185;K14-Cre double transgenic mice (Zmiz1) generated KAs as previously described [8]. We also studied tumors from a Sleeping Beauty (SB) transposon mutagenesis screen performed in Rag-2-deficient mice (B6.129S6-Rag2tm1Fwa N12; or Rag2−/−, Taconic, Hudson, NY, USA) and heterozygotes (RAG2+/−) (both abbreviated “Rag-SB”). [9]. Squamous skin proliferations were excluded from this study if they had (1) reactive acanthosis or (2) overt squamous cell carcinoma that lacked evidence of originating from a KA-like lesion. All animal use in this study was approved by the University of Iowa Animal Care and Use Committee (IACUC). Animals were euthanized when morbidity or tumor burden reached a predefined threshold as approved by the UI-IACUC, thus the ability to follow all tumors through regression was not feasible and therefore only approximately 14% of tumors were within the regression phase at the time of the study’s endpoint.Mice from the Zmiz1 (n = 33) and Rag-SB (n = 42) groups were examined for predilection of tumor development. In both studies, comprehensive necropsies and histopathological analyses were performed [8,9]. Distribution of KAs was recorded and grouped according to regional anatomic sites (Table 1).Distribution of keratoacanthomas(KA) tumors on Zimz1 and RAG-SB mice expressed as a percentage of the total and, in parentheses, the total number identified.Skin tumors from the Zimz1 and Rag-SB studies described above were excised at necropsy and immersion fixed in 10% neutral buffered formalin. Pilot studies suggested that deep margins were the principal sites to evaluate for invasive tendencies in the murine KAs. Accordingly, tumor excision with lateral (~2 mm) and deep margins (~3 mm) were targeted. Complete necropsies were performed on all animals and importantly, draining lymph nodes and lung samples were evaluated and did not show evidence of metastasis. Following fixation (approximately 3–5 days) tissues were routinely processed, paraffin-embedded, sectioned at 4 µm and stained with hematoxylin and eosin (HE). For this study, murine tumors were histopathologically examined by two board-certified veterinary pathologists. Based on KA literature, tumors were evaluated for morphologic features that paralleled the phases of KA development in humans (Table 2).KAs from Zmiz1 (n = 90) and Rag-SB cohorts (n = 47) were selected for this study based on size (broad range of small to large tumors) and quality of the histologic preparation (appropriate sectioning to be able to see tumor margins, etc.) to give a broad repertoire for examination. Each tumor was evaluated and scored after mutual agreement of two pathologists and by following the general principles of histopathologic scoring of data [10]. Each tumor was given a clinical “KA score” related to the KA development phase as defined in Table 2. Those tumors in the “growth” phase were given a score of “1”, those in the “maturation” phase were given a score of “2” and those in the “regression” phase were given a score of “3”. The tumors were also scored based on the estimated percentage of tumor mass that was composed of keratin (“keratin score”) using a scale from “0” (0%–10% of tumor filled by keratin), “1” (11%–20% of tumor filled by keratin), and so on to “10” (91%–100% of tumor filled by keratin). Finally, KA “diameter” (surface edge to surface edge of tumor) in millimeters (mm) was measured for all of the tumors. These scores were evaluated and linear regression statistical analysis was performed using GraphPad Prism software.Phases of murine KA development.Proliferative epidermis with 
+				
+                Anisokaryosis
+				Anisocytosis
+				Numerous mitotic figures
+				Monomorphic/homogenous atypical cells that do not tend to differentiate into normal epidermal layers 
+				Multifocal local invasion of panniculus adipose/muscle 
+				AnisokaryosisAnisocytosisNumerous mitotic figuresMonomorphic/homogenous atypical cells that do not tend to differentiate into normal epidermal layers Multifocal local invasion of panniculus adipose/muscle The epithelium commonly forms tongues/cords with scant to variable keratin that is more lamellar and orthokeratoticBegins a subtile transition to differentiation of the epidermis with rare keratohyaline granulesSimilar to Growth phase with
+				
+                “Glassy” keratinocytes
+				Evidence of parakeratotic keratin
+				Ghost cells
+				Keratohyalin granules 
+				All consistent with increased differentiation into epidermal layers. 
+				“Glassy” keratinocytesEvidence of parakeratotic keratinGhost cellsKeratohyalin granules 
+				All consistent with increased differentiation into epidermal layers. All consistent with increased differentiation into epidermal layers. The thickness of the epithelial tongues/cords becomes thinner than in growth phase.Rarely multifocal local invasion of panniculus adipose/muscleSlight overall thinning of the epidermis compared to others within this phaseSlight flattening of the epidermal tongues/cords compared to others within this phaseFewer “glassy” keratinocytes to be foundLoss of tongues/cords/trabeculaeProgressive thinning of the epitheliumThe keratin core becomes hollowed out and filled with keratin Oftentimes acanthotic epidermisFibrosis of the subjacent dermisAs an initial validation to define infiltrating inflammatory cells we selected one tumor in the regression phase from each genotype to do immunohistochemistry (n = 3 KAs total). Inflammatory cell markers (CD3, B220, F4/80, Arginase-1, Nos2, and granzyme-B) were assessed and these were performed on the formalin-fixed paraffin-embedded samples as summarized in Table 3. All immunohistochemical staining was performed manually using peroxidase methods and Dako Envision systems (Glostrup, Denmark).Primary antibodies and their commercially available sources, catalog numbers, dilutions and specific antigen retrieval conditions utilized in the study.Tumors had been collected from Zimz1 mice that ranged in age from 2–12 months (mean 3.6 months); while the range of ages of Rag-SB mice was 6.5–15.5 months (mean 11.6 months). Grossly, KAs from both mouse models progressively developed as focal raised skin tumors. While KAs were generally found on any skin surface, there was a predilection for the back, head, and limbs in Zimz1 mice and the back and chest in Rag-SB mice (Table 1). These raised nodular lesions appeared as tan to white keratin-filled craters surrounded by raised and moderately erythematous skin margins (Figure 1); very similar to the gross appearance of human KAs [4]. Classical histological descriptions of KAs are characterized by a distinct central crater filled with abundant keratin material (Figure 2). We were then able to histologically classify the KAs into three distinct growth phases.The inception of KAs typically occurred as a subepidermal solid to crateriform mass originating from or near the follicular epithelium (Figure 3A) which shortly thereafter involves the surface epithelium to create an exophytic mass. The growth phase was characterized by tumors with a thickened wall of acanthotic keratinocytes, nominal to moderate orthokeratosis (Figure 4A) and mixed inflammation (Figure 4B, Table 2). The tumor-associated inflammation in Zimz1 and Rag+/−SB mice was composed primarily of scattered to patchy aggregates of lymphocytes and macrophages with fewer neutrophils, eosinophils and rare plasma cells. The RAG−/−SB mice lacked T and B cells (data not shown) which is expected as these mice lack mature lymphocytes [13]. The lining keratinocytes had patchy to coalescing fields of pleomorphism with anisocytosis, anisokaryosis and multiple mitoses per high powered field (Figure 4C), which has also been described in human KAs [1]. The deep portion of the mass had cords and trabeculae of neoplastic cells that sometimes showed minor invasion the adipose and skeletal muscle tissues of the panniculus (Figure 3B), but deep invasion and metastasis was not observed.Gross appearance of a mature keratoacanthoma from a Zimz1 mouse. The circular tumor is composed of a central core of keratin (asterisk) surrounded by raised skin margins.Classic example of a Keratoacanthoma (KA) from a Zmiz1 mouse. Low magnification photomicrograph showing the crateriform appearance of the KA filled with keratin (asterisk) and bordered by proliferative epithelium. Bar = 500 µm. Overview of keratoacanthoma (KA) biology. (A) The inception of KA tumors (arrows) occurs in the dermis arising near (in Figure 3A serial sections were not available to confirm continuity with follicle) or from the follicular epithelium. Note: in this tissue section the epidermis is acanthotic (thickened) immediately overlying the tumor with mild dermal inflammation. (B) During the “growth” (extending into the “maturation”) phases of KA development, the deep proliferating squamous epithelium can form cellular cords or tongues (arrows) that invade adjacent deeper structures including adipose or skeletal muscle (M) tissues in the panniculus. This example of tumor invasiveness can lead to misdiagnosis of malignant biology. (C) In the regression phase, the KA lining epithelium becomes progressively thinner with apoptotic bodies (arrows). As the KA crateriform structure is obscured in regression, the tumor “flattens” into epidermal like structure with smaller mass-like structures often filled by lamellar keratin (asterisk) that resemble benign follicular cysts. Bars = 100 µm.KAs in the mature phase had a distinct central crater that was packed with abundant keratin material (Figure 4D, Table 2). This central crater was lined by an irregular rim of thickened epithelium with multifocal regions of “glassy cells” which are squamous cells with a large amount of very pale eosinophilic cytoplasm and are considered a key diagnostic feature in human KAs [4] (Figure 4E,F). In these murine KAs, glassy cells were less prominent than as those described for human KAs at this stage [4,14]. Inflammation was generally similar to that seen in the growth phase. Mild to moderate dermal fibrosis was noted in some of the tumors in the mature phase. Perineural involvement, which has been documented to occur in human KAs at this stage [4] was not noted in any of the Zimz1 or Rag-SB KAs examined.Histopathologic features of keratoacanthomas from Zimz1 mice. Keratoacanthomas (KAs) were classified into three separate groups based on their morphology on HE section. Those in the “growth” phase (G) (A,B,C) had keratin and inflammation (B, arrows), and were lined by a proliferative layer of keratinocytes which were poorly defined into distinct epidermal layers with anisocytosis and anisokaryosis along with multiple mitotic figures (C, arrows). Those in the “maturation” phase (M) (D,E,F) had a central crater of keratin (D, asterisk) and were lined by proliferative epithelium with patchy regions of “glassy cells” (E, F, arrows) and also had moderate dermal inflammation. Those in the regression phase (R) (G,H,I) had abundant keratin (G, asterisks) with noticeable thinning of the epithelial lining (H, I, arrow) and moderate dermal inflammation. A, D, G bars = 500 µm, B, E, F bars = 50 µm, C, F, I bars = 20 µm.In the regression phase of development KAs became less crateriform with the lining epithelium becoming increasingly thin (Figure 4G,H, Table 2). This thin epithelium resembled mild to moderately acanthotic epithelium and lacked glassy cells, pleomorphism, and mitotic figures seen in previous phases (Figure 4H,I). Portions of the resolving KA sometimes had a very thin epithelial (epidermis-like) lining surrounding lamellar keratin, morphologically resembling a follicular cyst (Figure 3C). Additional findings in these regressing KAs include mild to moderate fibrosis and in a few cases a moderate mixed inflammatory cell infiltrate that was more prominent than seen in other tumors. Clinically we were not able to study complete regression with return to normal skin morphology because of defined project endpoints requiring euthanasia. It is important to note, evidence of metastatic disease was absent in both models.We observed during the examination of KAs in this study that the largest KAs appeared to have the most abundant keratinization. Because of this, the tumors were scored for keratinization as a percentage of total tumor cross-sectional area, thus designated the “keratin score”. There was a linear correlation between the diameter of both Zimz1 and Rag-SB KAs to the keratin score indicating this initial observation was true (Figure 5A,D respectively). Similarly, there was a linear correlation between the keratin score and the KA score in both models (Figure 5B,E respectively). The KA score was not proportional to the overall diameter of the KA, which was not surprising as resolving KAs were often smaller than those in the maturation phase (Figure 5C,F respectively).Morphometry and histopathological scoring of KAs. KA diameter was measured and a keratin score was generated by visually determining the amount of keratin present within the KA crater using a score from 0-10. KA score (see Table 2) was also determined. KA diameter and keratin score had a linear correlation in the Zimz1 KAs (A) and RAG-SB KAs (D), as did the keratin score and KA score ((B,E), respectively). There was no correlation in KA score to KA diameter in both the Zimz1 (C) and RAG-SB (F).We evaluated the utility of detecting immune cell infiltration from 3 KAs in the regression phase. CD3 and fewer B220 immunoreactive cells were found at the peripheral edge of tumors (Figure 6A,B respectively). As mentioned previously, there was an absence of B220 and CD3 immunoreactive lymphocytes in the KAs from Rag2−/− SB mice (data not shown). Within the dermis in regressing tumors from both mouse models there were numerous F4/80 immunoreactive macrophages, which were the overwhelming cell type in most tumors (Figure 6C). Arginase-I immunostaining of macrophages, consistent with M2 phenotype, was scarce in all samples (data not shown). There were moderate numbers of Granzyme B immunoreactive cells, which includes CD8+ T cells and natural killer cells, within the dermis and were found primarily at the dermal/epidermal junction (Figure 6D), and also were present interdigitating between keratinocytes. Diffusely the keratinocytes themselves were weakly immunoreactive for NOS2 as has been described [15], but immune cells within the dermis were generally unstained for NOS2 (data not shown).Immunohistochemistry of regressing KAs. (A) CD3 immunoreactive cells were noted within the dermis and tumor keratinocyte layer (arrows). (B) B220 immunoreactivity was sparse (arrows). (C) F4/80 immunoreactivity was common throughout the subepithelial connective tissue of regressing KAs. (D) Granzyme B immunoreactive cells were scattered throughout the tumor stroma tissue (arrows). A, B, D bars = 20 µm. C, bar = 50 µm.In the current study, we were able to classify murine KAs into three distinct developmental phases similar to those described for human KAs and experimentally induced KAs in a rabbit model [16]. In humans, KAs are thought to originate from follicular epithelium [17] and this was consistent with our findings in the current study of mouse models. We were also able to study KA regression and immune cell infiltration in select cases and found the immune cell population to be similar to that described for human KAs [13,18]. While these points suggest that the biology of these murine KAs is comparable to humans, there were differences too. The prominent glassy cell layer described as characteristic in human KAs was present in mice and generally restricted to the maturation phase. These glassy cells were less prominent than described for humans [4], which could make diagnosis of murine KAs more challenging for those familiar with human KA morphology. While we cannot say that all murine models with KAs will mimic our findings, this study offers important considerations for future diagnostic efforts studying mouse skin cancer models. Importantly, we show murine KAs follow a developmental pattern similar to that described in human [4] and transgenic rabbit KAs [19] including a regression phase and the aggressive appearance noted in the early development phases did not result in malignancy. Our findings highlight the importance of understanding tumor biology in a new cancer model to ensure that the morphologic diagnosis is consistent and accurate. We wanted to assess the utility of detecting immune cell infiltration, specifically in regressing KAs. We were able to detect and localize within the tumors a variety of immune cells, suggesting immunohistochemical assessment would be potentially useful in clarifying the role(s) of immunologic-based regression in KAs. During routine assessment of HE stained section, we identified inflammation in all phases of KA development. Therefore, future studies evaluating immune cell infiltration associated with KA regression will need more rigorous approaches. For instance, each developmental phase should be compared for differences in immune cell infiltrates and/or immunohistochemical techniques should be complemented by more sensitive techniques such as flow cytometry to assess subtle shifts in immune cell populations. Interestingly, the immune cell infiltrates were generally seen at each phase of development (including an immunodeficient model) which raises questions about the role of the immune system in regulation of KA regression. Further, another hypothesis for KA regression which does not involve the immune system has been proposed for these follicular tumors. The idea is that growth and regression of these tumors may be under the regulation of factors that govern the normal hair cycle [20]. Future studies in the Zmiz1 model may be able useful to better discern the roles of immune cell infiltration and hair cycle factors in the regulation of regression in these tumors. In the human literature, there is controversy as to whether KAs are benign squamous proliferations that regress, or if they have potential to become malignant SCC [4,14]. Unfortunately, the distinction between KA and SCC using basic histopathology is not always obvious, and there is a lack of immunohistochemical stains or biomarkers that will distinguish the two tumor types [7]. There also do not appear to be any specific genetic aberrations that aid in diagnosis [21]. As such, KA are often treated clinically as low grade malignancies or potential SCC [14]. In the mouse models utilized in this current study, we did not see progression to SCC or malignant tumors arising from squamous proliferation [22], instead we observed KAs maturing through the three phases of development including regression. In Rag-SB mice we specifically studied KAs, although two skin tumors not included in this study were classified as SCC [9]. This certainly does not imply that KAs progress to SCC as these Rag-SB mice also developed many other types of malignancies [9] and there was not any evidence that these dermal SCC originated from KAs. Importantly, mouse KAs in the current study often had an aggressive appearance (areas of cellular atypia, proliferation and minor invasion) that could lead some observers to presumptively interpret it as SCC. Future studies may include other techniques (e.g., tumor transplantation studies, clonality, etc.) to further investigate the pathobiology of these murine KAs.One note of importance is that these mice were required by animal care guidelines to be sacrificed early because of tumor “burden”. Therefore exhaustive biologic assessment of the whole of the tumor set could not be completed. Accordingly, we cannot completely rule out eventual progression to SCC in some tumors; however, the large portion of KAs that had entered the regression phase did not show evidence for malignant transformation. Given that a large percentage of the KA tumor mass is keratin (similar substance to hair) and that they lack an ulcerated surface, it might be of value to discuss possibility of exemptions to traditional endpoints with Institutional Animal Care and Use Committees. This could allow investigators to further understand the biology of these tumors and result in better defined tumor regression events. These models could offer useful approaches to understanding why KAs develop, why they can appear aggregative yet eventually regress and open the door for new medical/surgical therapies. Our study of murine KAs has relevant implications for future investigations. First, the parallel development and biology of KAs of mice and humans suggests the lifecycle of these tumors are regulated by similar factors. Second, translational studies of KAs in mouse models may be useful to investigate mechanisms of tumor regression, novel therapies and potential for malignant transformation. Lastly, the local aggressiveness of murine KAs (which may be misdiagnosed as squamous cell malignancies) highlight the need in experimental models to validate tumor morphology with biology.We would like to thank the Histology Research Laboratory and Comparative Pathology Laboratory (Department of Pathology, University of Iowa Carver College of Medicine) for their technical expertise. Funding for this project included R01CA132962-01A2 (NCI) and Aiming For a Cure Foundation.KGC is the primary author of the manuscript and performed the histopathologic, immunohistochemical analyses and data collection. LR performed the animal studies in the Zimz1 and RAG-SB mouse models, collected the tissues and assisted with manuscript preparation. AD is the investigator overseeing the animal study work presented here and assisted with manuscript preparation. DM is the senior author who heavily contributed to the manuscript and the data collected.The authors declare no conflict of interest.

Med-MDPI/diseases/diseases-02-02-00120.txt

@@ -0,0 +1,21 @@
+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).A culture of exercise testing is firmly embedded in the management of pulmonary arterial hypertension (PAH) but its clinical relevance and utility have recently been under some debate. The six minute walk test (6MWT) has been used as a primary outcome measure to enable the licensing of many of the medications used for this condition. Recent reviews have questioned the validity of this test as a surrogate of clinical outcomes. At the same time, other questions are emerging where exercise testing may be the solution. With the rise in understanding of genetic markers of idiopathic PAH (IPAH), the screening of an otherwise healthy population for incipient pulmonary hypertension (PH) will be required. The proliferation in treatment choices and identification of populations with PH where PAH treatment is not indicated, such as left heart and lung disease, requires more definitive differentiation from patients with PAH. There is a continuing question about the existence and clinical relevance of exercise induced PAH as a cause of unexplained dyspnoea and fatigue and as a latent phase of resting PH. This review presents a summary and critical analysis of the current role of exercise testing in PAH and speculates on future trends.Exercise can be viewed as the process of transferring oxygen from atmosphere to capillaries and its utilisation there by exercising muscles. The movement of oxygen from the lungs to the capillaries is a convective process described mathematically by the Fick equation which states
+	  
+        VO2 = CO × (CaO2 ‒ CaO2)
+        (1)
+        
+	 where VO2 is oxygen uptake, CO is cardiac output and CaO2-CvO2 is the arteriovenous difference in oxygen content. This equation can be expanded as follows:
+	  
+        VO2 = SV × HR × Hb × (SaO2 ‒ SaO2)
+        (2)
+        
+	 where SV is stroke volume, HR heart rate, Hb blood haemoglobin concentration and SaO2-SvO2 the difference in oxygen saturation between arterial and mixed venous blood. Severe exercise intolerance due to impairment of oxygen transport is a hallmark of pulmonary arterial hypertension (PAH) and the reasons for that are evident from inspection of the Fick equation. In subjects with PAH, maximum SV is severely reduced [1], peak predicted HR is often not reached [2], relative anaemia is a common finding [3] and desaturation on exercise is present in the majority of cases due to a combination of precipitate fall in mixed venous oxygen saturation (SvO2) and ventilation-perfusion mismatch [4].There are other factors which can in general limit exercise such as ventilatory limitation and peripheral muscle dysfunction but their contribution to impairment of exercise capacity in PAH is less relevant. Ventilatory capacity can be reduced in PAH, where mild obstruction and restriction have been found at rest [5,6] and dynamic hyperinflation with reduced end exercise inspiratory capacity on exercise [7,8]. More striking in these patients is markedly increased ventilatory demand due to reduced ventilatory efficiency. The pathophysiological reasons for this can be illustrated by considering the ratio of ventilation (VE) to carbon dioxide production (VCO2) which is given by the following expression derived from the Bohr equation:
+	  
+        
+        (3)
+      
+	 where k is a constant, PaCO2 is the arterial carbon dioxide concentration and VD/VT is the ratio of dead space to tidal volume. In PAH, inefficient ventilation, i.e., the requirement for a high ventilation to remove the carbon dioxide produced by respiration, is due to a combination of both increased dead space and a lowered set point for arterial carbon dioxide concentration [9]. Despite this reduced ventilatory capacity and increased demand, ventilatory limitation is said not to occur in PAH unless there is concomitant lung disease. Peripheral muscle dysfunction has been shown in PAH in the form of reduced capillary density and oxidative enzymes in muscle biopsies of these patients [10]. This abnormality can lead to impairment of extraction of oxygen from muscle capillaries and should be reflected in abnormally high values of SvO2 at end-exercise [11]. However, data from studies in heart failure [12] and published in abstract form in PAH [13] suggest that this is not the case. Thus, although muscle dysfunction is present in PAH, the diffusion capacity for oxygen from the capillaries to the mitochondria still more than matches the reduced level of oxygen delivery to the capillaries.Since exercise limitation is present in the majority of patients with this condition, exercise testing has been widely used as a means of investigating its severity and assessing for change. The most commonly used tests have been incremental cardiopulmonary exercise tests (CPETs) and six minute walk tests (6MWTs) but these have both had their critics. When the former was used as the primary outcome measure in a trial of sitaxentan, it proved negative, whereas 6MWT, as the secondary outcome measure, was positive [14]. The failure of CPET to show a signal was attributed to its complexity and hence the quality of test performance [15]. Recently an attempt was made to quantify what proportion of the treatment effect of disease targeted therapy was captured by changes in 6MWT distance (6MWD) [16]. This proved surprisingly low at 22.1%, supporting earlier studies which suggested that changes in 6MWD were not of prognostic value [17].There is opportunity for development in the application of exercise testing techniques in PAH. There has been interest in both invasive and non-invasive measurements of exercise haemodynamics since this is one of the cardinal impairments of pathophysiology in this condition. Attempts have been made to enhance the value of CPET and 6MWT by modifications of protocol or measurement of additional variables. Finally studies are emerging of the use of activity monitoring in PAH under the premise that this test will more closely reflect clinical reality. The aim of this review is to summarise the current status of exercise testing in PAH and to highlight recent advances.The measurement of the pressure-flow behaviour of the pulmonary circulation during exercise by right heart catheterisation (RHC) continues to be of interest despite the invasive process involved [1,18,19]. It provides information which cannot be accurately acquired by other routes. Potential roles include differentiating between pulmonary venous and pulmonary arterial hypertension, investigation of exertional dyspnoea defying diagnosis by non-invasive tests and accurate assessment of treatment effect on pulmonary artery haemodynamics.Mean pulmonary artery pressure (mPAP), CO, pulmonary vascular resistance (PVR) and pulmonary artery occlusion pressure (PAOP) are interlinked by the equation
+		
+        
+        (4)
+      
+		Figure 1 contrasts the typical results seen for mean pulmonary artery pressure (mPAP) during exercise in healthy individuals and those with PAH. In health both mPAP and PAOP rise with exercise. The estimated range of normality for mPAP change on exercise is 0.5–3.0 mmHg.min.L−1 [20] whereas the normal range for PAOP change is less certain, one estimate being 0.3–1.93 mmHg.min.L−1 [19]. During supine exercise PVR falls slightly [21,22] and SvO2 falls from 75% at rest to 25% at maximal exercise [23]. By contrast in PAH there is a very steep rise in mPAP over a much more limited CO response. The data for PVR and PAOP in PAH are very limited. In the study by Janicki et al. [24] which included a mixture of subjects with PAH and pulmonary hypertension (PH) related to lung disease, PAOP rose from 8 mmHg at rest to 18 mmHg on exercise with PVR unchanged. As discussed earlier, there has been controversy over the SvO2 values achieved at maximal exercise in PAH. In a recent study of patients with precapillary pulmonary hypertension the mean SvO2 on maximal supine exercise was low at 22% [13].Mean data for healthy subjects taken from Kovacs et al. [22]. Mean data for subjects with pulmonary arterial hypertension (PAH) adapted from Provencher et al. [25]. Exercise performed in the supine position. mPAP: mean pulmonary artery pressure, PAOP: pulmonary artery occlusion pressure.There are obvious issues with the widespread utilisation of exercise haemodynamics. Firstly, it is an invasive test and therefore not well suited to serial measurements. There is no standardised exercise protocol followed in the studies which have investigated its use [26]. Difficulties arise in interpreting measurements at peak exercise due to the swings in pressure traces caused by increasing respiratory efforts, particularly marked with PAOP [27] but also noticeable in mPAP (see Figure 2). To overcome this problem which leads to exaggerated end-expiratory pressures, it has been suggested that pulmonary vascular pressures measured during exercise should be averaged over the respiratory cycle [18]. Lastly, exercise haemodynamics are most easily performed in the supine position but this does not lead to the same results as erect exercise. In particular, for any workload HR is lower and SV higher in the supine position [22] and the peak workrate achievable higher when erect [28].There is a role for exercise haemodynamics in established PH. It has provided insights into the physiological behaviour of the pulmonary circulation during exercise in these patients [29]. It can provide diagnostic information to differentiate between clinical classes of PH. Borlaug et al. [30] demonstrated that patients with pulmonary venous hypertension showed a marked increase in PAOP during exercise which could be used to discriminate between this condition and PAH. There is some, albeit limited, evidence for the prognostic value of exercise haemodynamics. Provencher et al. [25] showed that exercise haemodynamic variables could predict change in 6MWD on therapy whereas resting values did not. In a mixed population of subjects with PAH and chronic thromboembolic pulmonary hypertension, Blumberg et al. [31] showed that exercise cardiac index and the pressure/flow relationship were significant prognostic indicators. They may have greater sensitivity than resting values to detect treatment effect. Castelain et al. [29] demonstrated that pressure-flow slopes improved with epoprostenol whereas resting haemodynamic values did not.Right heart catheterisation traces of pulmonary artery pressure (PAP) and pulmonary artery occlusion pressure (PAOP) at rest and at peak exercise. Note both the significant elevation of PAP and the marked respiratory swing, most prominently affecting PAOP, which develops on exercise.There may be a role for exercise haemodynamics in evaluating subjects with dyspnoea or fatigue that is defying diagnosis particularly if combined with VO2 measurements. In subjects with normal resting measurements, elevated pulmonary artery pressure on exercise has been associated, not only with an impaired exercise capacity and CO response and higher values of PVR, but also with symptoms, reduced peak VO2, abnormal gas exchange and reduced quality of life [32,33,34]. Such results support exercise induced PAH (EIPAH) as a true pathophysiological entity which may be responsible for some cases of unexplained exercise intolerance. Since the concept of EIPAH was discouraged in the guidelines from Dana Point (2008) [35] and this advice was repeated in Nice (2013) [36], there has been neither consensus as to whether this is a true precursor of resting PAH nor a widely accepted haemodynamic definition by which to identify it. In a recent review, Naeije et al. [20] have proposed that either an mPAP of 30 mmHg at a CO of less than 10 L.min−1 (which approximately corresponds to a PVR greater than 3 Wood units) or a slope of mPAP-CO greater than 3 mmHg.min.L−1 could be used as diagnostic criteria for this condition.Exercise haemodynamics may also have a role in identifying EIPAH in subjects with substantial risk factors for PAH as early data suggest treatment may be indicated for this group. One fifth of patients with systemic sclerosis and EIPAH developed resting PAH after a mean follow up of 2.3 years [37]. Exercise haemodynamic measurements in scleroderma patients with borderline resting measurements [38,39] show a greater increase in mPAP on exercise. In this group there was progression in mPAP and PVR on serial measurements over a year and bosentan appeared to attenuate that change [40]. Finally Saggar et al. [41] showed an improvement in exercise haemodynamics in an open label trial of ambrisentan in patients with scleroderma and EIPAH.For greater utilisation of exercise haemodynamics to occur, the technique for measuring them should be non-invasive and there are a number of candidates for this.Transthoracic echocardiogram (TTE) surpasses RHC in the amount of information it supplies [42]. Not only can it give estimates of pressure and flow but it can also be used to evaluate right ventricular function. Its major problem, as with all indirect techniques, is the potential for error in patients with challenging anatomy (e.g., concomitant lung disease, obesity) or arising from inter-operator variability.There are a number of studies illustrating the feasibility of TTE during exercise in PAH and other conditions. These have shown the ability to estimate systolic pulmonary artery pressure (sPAP) [43,44,45,46,47,48], PAOP by inference from E/e’ ratio [49,50], CO from left ventricular outflow velocity-time integral [43,44], resistance [43,44], distensibility [43,44,51] and right ventricular function assessed by tricuspid annular plane systolic excursion (TAPSE) and tricuspid annular systolic velocity [52]. mPAP can be deduced from sPAP using one of the equations derived for this relationship [53,54]. Formal validation of non-invasive with invasive measurements during exercise has been limited [55,56,57].Early data show encouraging results for the use of exercise TTE in subjects at risk of PAH. Grunig et al. [58] looked at relatives of patients with IPAH or familial PAH with and without bone morphogenetic protein receptor 2 gene mutations and found a greater prevalence of a hypertensive tricuspid regurgitant velocity response in affected kindreds. Multiple studies have shown a high prevalence of TTE determined EIPAH in scleroderma patients [45,59,60,61,62,63]. Of these, Steen et al. [61] was the only one to proceed to RHC in those subjects with EIPAH and confirmed the non-invasive results in 81% of cases.Cardiac magnetic resonance (CMR) is a more complex imaging technique which also has potential to provide noninvasive haemodynamic data. It is more accurate than TTE in measuring CO [64] and similarly provides information on right ventricular function, resting values of which have been shown to be superior to PVR for predicting prognosis [65]. However, unlike TTE it cannot provide information on pressure and a significant minority of subjects fail to tolerate the test because of claustrophobia [66]. Additionally exercise measurements so far in PAH have been taken in the immediate post-exercise phase because of the difficulty in cycling whilst inside the bore of the scanner [67]. The validity of this is questionable as values of pressure and flow post-exercise fall rapidly [44,68]. This technique needs to be evaluated with imaging performed during exercise to assess its full value. The feasibility of such measurements has been illustrated in other conditions [64].Simpler physiological measurements have also been used to estimate exercise haemodynamic variables. The inert gas rebreathing technique utilises the high solubility in blood of gases such as acetylene or nitrous oxide to estimate pulmonary blood flow [69]. This technique gives no information on pulmonary artery pressure or other aspects of right ventricular function. It is simple to perform during exercise with a measurement possible every few minutes allowing for a washout period. Its major limitation is in patients with lung disease where ventilation heterogeneity leads to poor mixing of inspired gases and inaccurate results [70]. It is also compromised where there is significant intracardiac shunting.Nevertheless, the feasibility of the technique to estimate SV during exercise in PAH and detect the effects of disease targeted therapy on exercise SV, have both been demonstrated [67,71]. Lee et al. [71] also showed that, if SV is the variable of interest, then changes in supine resting SV provided as much information as erect exercise values, indicating the importance of preload as a stressor for assessing the pulmonary circulation. Validation data for use of the technique in PAH are limited to resting data [70,72].Impedance cardiography or bioimpedance uses the change in thoracic impedance as the fluid content of the chest varies during the cardiac cycle to estimate SV [73]. This technique is attractive as it has the potential to provide a continuous measurement of CO at rest or during activity. However, measurements are not possible in a quarter of patients and the quantitative validity of the data in the remaining proportion cannot be relied upon. Relative changes in measurements of cardiac index were able to identify subjects severely affected by PAH [74]. Bioreactance is a version of the technique which looks at phase rather than amplitude changes of alternating current and voltage applied across the thorax. Its signal is proportional to aortic blood flow and performed well in quantitative terms when validated against thermodilution and indirect Fick in a broad population of PH patients [75].Improvement in equipment design and widespread adoption of breath by breath measurement techniques have made CPET relatively easy to perform and widely available [76]. The minimum dataset collected during exercise includes VE, oxygen and carbon dioxide content of inspired and expired air, HR and oxygen saturation (SpO2). With these measurements the characteristic pathophysiological response of PAH patients can be seen, i.e., profound exercise limitation marked by impairment of oxygen transport and inefficient gas exchange with very high ventilatory demand [77,78,79,80,81,82,83,84,85]. In an incremental CPET, the oxygen transport abnormality is reflected in a low VO2 at anaerobic threshold, low VO2 - workrate slope, low peak oxygen (O2) pulse (defined as the ratio of peak VO2 to peak HR) and a steep HR-VO2 response, combined on occasions with a HR response which fails to reach predicted maximum. The gas exchange response is even more striking with high ventilatory equivalents (VE/VCO2 and VE/VO2) and low end-tidal carbon dioxide (PetCO2) combined with desaturation (see Figure 3). These features can be exaggerated by the use of walking rather than cycling exercise which causes more hypoxaemia [86] and are more prominent in the presence of a right to left shunt such as through a patent foramen ovale [87].The exercise response of disease are more subtle. Table 1 lists a number of studies which have examined this issue. Although PAH patients is very readily distinguished from normal subjects. However, differences in CPET results between PAH and other conditions complicated by PH such as left heart problems, hypoxic lung disease and chronic thromboembolic group mean measurements can differentiate between these conditions, using CPET results to make a definitive diagnosis for an individual is not often possible because of the overlap in the range of the abnormal variables. Thus CPET can suggest the presence of PH in an associated condition but is insufficient without other investigations to make a diagnosis of PAH. One study [88] has proposed an algorithm for diagnosing a pulmonary vascular problem which uses a decision tree based upon peak VO2, VO2 at anaerobic threshold (AT), breathing reserve and VE/VCO2 at AT. This achieved a sensitivity of 79%, specificity of 88% and accuracy of 85% of detecting a “pulmonary vascular limit” as the cause of exercise impairment. One major issue with this study was that the definition of “pulmonary vascular limit” used in the study was not a standard one and so these results need further confirmation.Several CPET variables have been demonstrated to have prognostic value for patients with PAH although some of the studies are limited by small numbers and lack of multivariate analyses [89,90]. Themes emerging from the heterogeneous results are summarised in Table 2. Of note both oxygen transport (peak VO2, O2 pulse, peak HR) and gas exchange variables (VE/VCO2 at AT) are represented in the table. A further study looked at prognostic information contained in change in variables following treatment with change in peak VO2 being predictive on multivariate analysis [91]. Only one study looked at “Time To Clinical Worsening” [92] as the prognostic outcome rather than death and this showed that peak VO2 was predictive.CPET variables in some but not all studies have been able to detect a change pre and post approved disease targeted therapy in patients treated for PAH. In an uncontrolled study in 16 subjects Wax et al. [93] showed an improvement in peak VO2 on intravenous prostacyclin therapy. In an uncontrolled study in 11 subjects [94] Wensel et al. was able to show an increase in peak VO2 and reduction in VE/VCO2 slope with nebulised iloprost. In a controlled study of 28 subjects [95] Oudiz et al was able to show changes at AT in VE/VCO2 ratio and PetCO2 with sildenafil. The largest treatment study utilising a CPET variable as a primary outcome measure [14] looked at the use of sitaxentan in 178 subjects. This failed to show an increase in the primary outcome measure (% predicted peak VO2) despite a positive exercise based secondary outcome measure (6MWD) which eventually lead to the drug’s approval. The reason for the failure of VO2 as an outcome measure has been attributed to the complexity of CPET and the multicentre design of STRIDE-1 [15]. It has been postulated that this lead to inaccurate results at the less experienced sites and failure of the outcome measure to show a signal. As support for this a single centre study in PAH subjects [96] achieved excellent reproducibility of CPET variables on repeated testing with coefficients of variation of 5.8%, 3.3%. 5.2% 6.5%, 1.0%, 2.8% and 3.3% respectively for peak values of VO2, HR and O2 pulse and AT values of VO2, PetO2, PetCO2 and VE/VCO2.Typical CPET responses of a patient with PAH. The solid lines in (A), (B) and (C) indicate the predicted peak values of the respective variables. The dashed line in (A) represents a VO2/WR slope of 10 mL.W./min−1 – a healthy response would parallel this line. Note the shallower VO2/WR slope and reduced peak VO2 in PAH. Reduced peak oxygen pulse is seen in (B). Steep heart rate response and VE/VCO2 slope are evident in (C) and (D) respectively with the predicted response corridors indicated by dashed lines. (E) displays a markedly elevated ventilatory equivalent of CO2 while (F) shows reduced end-tidal CO2, demonstrating key elements of the abnormal gas exchange response in PAH.Screening of at risk populations for the presence of resting PH is best performed by TTE. However, if it becomes important to detect EIPAH, then this may be an appropriate indication for CPET. Studies which examined CPET variable abnormalities in EIPAH are listed in Table 3. The ability of CPET to detect these differences adds support to the concept of EIPAH as a genuine pathophysiological entity.The safety of maximal incremental CPET in PAH subjects has been questioned. Multiple studies in adults [83,96,97] and children [89,98,99] have suggested that this is not a problem. In the largest study looking at CPET in high risk cardiovascular patients (n = 5060 CPET events) [97], adverse event rate was 0.16% with no fatalities. In a study dedicated to PAH [96], there were no events in 242 tests.Studies comparing cardiopulmonary exercise test (CPET) variable responses in PAH with left heart, hypoxic lung and chronic thromboembolic disease or in these conditions complicated by PH.higher dyspnoea scoreshigher VE/VCO2 at AT and peaklower peak O2 pulselower peak SpO2lower VO2-WR slope above ATlower peak SpO2lower SVsteeper HR-VO2 slopehigher peak RERhigh VO2-WR slopehigh VE/VCO2high P(a-et)CO2reduced peak SvO2reduced peak PaCO2reduced peak SpO2increased breathing reserveincreased peak RERreduced peak PetCO2increased VE/VCO2 slopehigher dyspnoea scoreshigher VE/VCO2 slopehigherP(a-ET)CO2 at AThigher peak VE, lower BR, higher peak RRhigher VE/VCO2 slope and VE/VCO2 at AT and lower PetCO2 at AThigher VD/VT, lower PaO2 and PaCO2 at peak exercisehigher VE/VCO2 slope and VE/VCO2 at AT and lower PetCO2 at AThigher VD/VT, Aa gradient, lower PaO2 at peak exerciseCPET: cardiopulmonary exercise test; PAH: pulmonary arterial hypertension; PH: pulmonary hypertension; LHD: left heart disease; COPD: chronic obstructive pulmonary disease; IPAH: idiopathic pulmonary arterial hypertension; RHC: right heart catheterisation; TTE: transthoracic echocardiogram; mPAP: mean pulmonary artery pressure; IPF: idiopathic pulmonary fibrosis; CTEPH: chronic thromboembolic pulmonary hypertension; CTED: chronic thromboembolic disease; VO2: oxygen uptake; VE: ventilation; VCO2: carbon dioxide production; O2 pulse: oxygen pulse; SpO2: oxygen saturation; WR: workrate; PetCO2: end-tidal carbon dioxide; SV: stroke volume; HR: heart rate; RER: respiratory exchange ratio; NYHA FC: New York Heart Association functional class; PAOP: pulmonary artery occlusion pressure; PeCO2: mixed-expired carbon dioxide; P(a-et)CO2: arterial to end-tidal carbon dioxide pressure difference; SvO2: mixed venous oxygen saturation; PaCO2: arterial partial pressure of carbon dioxide; Aa gradient: Alveolar-arterial gradient; PaO2: arterial partial pressure of oxygen.Studies of prognostic value of CPET variables in PAH subjected to multivariate analysis.CPET: cardiopulmonary exercise test; PAH: pulmonary arterial hypertension; VO2: oxygen uptake; BP: blood pressure; VE: ventilation; VCO2: carbon dioxide production; AT: anaerobic threshold; Δ O2 pulse: increase in oxygen pulse during exercise; HR: heart rate; ΔHR: increase in HR during exercise.Studies showing CPET variable abnormalities in exercise induced PAH (EIPAH).CPET: cardiopulmonary exercise test; EIPAH: exercise induced pulmonary arterial hypertension; PAH: pulmonary arterial hypertension; PH: pulmonary hypertension; sPAP: systolic pulmonary arterial pressure; TTE: transthoracic echocardiogram; mPAP: mean pulmonary arterial hypertension; RHC: right heart catheterisation; VO2: oxygen uptake; Aa gradient: alveolar-arterial pressure gradient; AT: anaerobic threshold; O2 pulse: oxygen pulse; WR: workrate; VE: ventilation; VCO2: carbon dioxide production; PetCO2: end-tidal partial pressure of carbon dioxide; VD/VT: ratio of dead space volume to tidal volume, P(a-et)CO2: arterial to end-tidal carbon dioxide pressure difference.In summary, studies of CPET in PAH have been encouraging but not unconditionally so. The test can discriminate readily between subjects with PAH and normals but overlap of CPET variable responses between PAH and other comorbidities such as heart and lung disease can reduce its usefulness as a discriminator between differential diagnoses. Several of the CPET variables have prognostic value but studies have had heterogeneous results and they are not as strongly predictive as simpler variables (6MWD). CPET variables can detect treatment effect but larger studies were perhaps compromised by the complexity of the test. Finally CPET variables can detect abnormalities present in EIPAH but this condition is of uncertain clinical significance.Several approaches for enhancing the utility of CPET in PAH have been proposed. One suggestion has been to use submaximal rather than maximal testing [71,116,117,118]. This simplifies the test, removing the subjective element of maximal effort, making it shorter and potentially more reliable to administer, and may not lose any of the responsiveness. Whilst most studies have concentrated solely on measurement of gas exchange variables in this context (PetCO2, VE/VCO2), Lee et al. [44] looked at isotime measurement of the complete array of CPET variables after three minutes of low level steady state exercise. They found that whereas both oxygen transport (VO2, O2 pulse) and gas exchange variables (VE/VCO2 at AT) distinguished between different severity of PAH, only the former showed change pre and post treatment.Another approach has been to introduce new CPET variables often based upon pattern of variable response rather than peak values [119]. Oxygen uptake kinetics are slower in these patients and represent one potential avenue which could be explored via time constants or post-exercise VO2 decline. Autonomic imbalance with increased sympathetic and reduced parasympathetic activity can be present leading to delay in heart rate recovery (HRR). The latter was shown to be reduced in PAH [2] and had prognostic significance on multivariate analysis when only CPET variables were considered [120]. Oxygen uptake efficiency slope (OUES) [121] which represents the slope of a plot of VO2 against log VE is thought to encapsulate in one variable a measure of oxygen transport and gas exchange. It can be used as an estimate of peak VO2 which requires only submaximal effort [121,122]. It has recently been shown to be of prognostic value in PAH [123].The application of field exercise tests in subjects with PAH has been overwhelmingly dominated by the 6MWT which has as outcome measurement 6MWD. As its name implies, this is a fixed time walking test whose major requirement is a quiet 30 m corridor. There is detailed guidance on how the test should be performed [124] and its reproducibility and validation are well documented. Deboeck et al. [125] demonstrated the physiological response to the test in PAH subjects to be a submaximal test performed using predominantly aerobic metabolism. In common with other conditions [126], the 6MWD in PAH correlates with both the peak VO2 achievable and the VO2 attained during the test [125,127]. The product of 6MWD and weight improves the correlation [15,125,128]. 6MWD correlates with resting haemodynamics including CO and total pulmonary resistance but not mPAP [127]. It also correlates with New York Heart Association Functional Class [127] and quality of life [129]. Similarly change in six minute walk distance (Δ6MWD) following PAH treatment correlated with changes in cardiac index, PVR [130] and quality of life [129].The short, fixed duration of administration makes it highly convenient for routine clinical use. Unfortunately it also has significant drawbacks. It can be surprisingly difficult to locate a 30 m corridor sufficiently quiet for regular use. Comorbidity with the pulmonary vascular axis which is particularly relevant in associated forms of PAH, such as scleroderma, compromise the reliability of the results [131,132]. The test has a ceiling effect as the linear relationship between peak VO2 and 6MWD is lost in less impaired subjects [71,133,134,135,136]. This makes it more responsive to change in more severely affected individuals and vice versa in fitter individuals. Additionally there is a learning effect [137] of approximately 2 to 4% between three consecutive daily walks, seen both at baseline and at two months follow up in healthy individuals. There was, however, no learning effect seen between two walks performed two months apart.The major strength of the 6MWT is the ability of a baseline measurement to predict prognosis in IPAH which has resulted in its inclusion in several PH registry models for predicting survival including the French PH registry [138], the risk calculator derived from REVEAL (the North American database) [139] and the Scottish Composite Score [140]. This was also demonstrable in a meta-analysis of the many treatment trials in PAH [141]. It retains significance in multivariate analyses even when other strong predictors are included such as patient demographics [127,140], baseline haemodynamics from right heart catheterisation or CMR [140,142,143], CPET variables [92,113] and presence of pericardial effusion [127]. Absolute values of 6MWD measured following PAH treatment retain prognostic power but do not improve on the baseline measurement [144]. The prognostic power is reduced and may be lost in cases of associated PAH such as connective tissue disease [37,92].Changes are seen in 6MWD with PAH treatment and it has been used as a surrogate outcome measure to obtain registration for most of the medications now used in this condition [145,146]. Although Δ6MWD correlates with change in haemodynamics following PAH treatment [130], it has proven very difficult to demonstrate any prognostic power of Δ6MWD with therapy despite being sought by several studies and meta-analyses [17,141,142,144,147]. The mechanism of this phenomenon has been explored recently by two further studies. In a meta-analysis of 22 PAH treatment studies, Savarese et al. [130] demonstrated that the change in haemodynamics seen with PAH treatment and correlating with Δ6MWD did not predict subsequent clinical events. In a pooled analysis of ten PAH treatment studies submitted to the US Food and Drug Administration (2404 patients), Gabler et al. [16] found that, despite a significant improvement in 6MWD, it accounted for only 22.1% of the treatment effect and they concluded that it was at best a modest surrogate endpoint for clinical events. To explain this anomaly it has been hypothesised that treatment improves both right ventricular function and haemodynamics but that only the former is strongly linked to clinical outcome [65]. Consequently Δ6MWD which correlates more strongly with change in haemodynamics [130] than change in right ventricular function [148] is poorly linked to clinical outcomes.Given the poor performance of Δ6MWD in predicting clinical outcome, it is unknown whether this represents an insurmountable drawback or whether change in implementation of the test can improve its performance. Use of minimal important differences may be of some value here as there will then be confidence that changes in 6MWD, although not prognostic, do have some bearing on the morbidity experienced by the patient. Estimates of this for the 6MWT in PAH subjects have suggested an increase in 6MWD of between 26 and 42 m to be clinically relevant [16,149,150]. Alternatively, absolute rather than predicted values of 6MWD are commonly used as targets in clinical practice, which may obscure prognostic information as the distance covered by a 2 m tall 30 year old male will be very different from that expected from a 1.5 m 70 year old female. However, analysis of the relative performance of absolute and % predicted 6MWD did not show any difference in the ability of the two values to predict prognosis [151].Measurements of HR and SpO2 routinely made during the 6MWT may confer additional value on the test. It has been shown that, as in left heart disease, patients with PAH have altered autonomic nervous system activity, the degree of which may reflect disease severity [152]. Such abnormalities may be visible in heart rate response during recovery from exercise and readily measured in variables such as HRR1, which is the fall in HR during the first minute of recovery. Minai et al. [153] have measured this following a 6MWT in idiopathic PAH and found it to complement the ability of 6MWD to predict clinical worsening. Similarly in an older study with a limited multivariate analysis on a small cohort (n = 34) [154], the size of fall in SpO2 during the 6MWT was found to be of prognostic value with a 27% increase in risk of death over the study period (1992–1997) for each percentage point fall in saturation. The relevance of this in the modern treatment era is uncertain.Waiting in the wings should the recent fall in fortune of the 6MWT prove irretrievable, there are a number of alternative exercise test candidates vying to displace it from its central position in the clinical management of PAH. Treadmill based walks have been proposed by several authors [155,156,157,158]. This is a convenient method of administering an exercise test not requiring a corridor with well-established protocols (such as Naughton-Balke) developed primarily for cardiac stress testing. The physiological stress imposed by such protocols is incremental in nature and avoids the ceiling effect issue seen with the 6MWT. Because of the fixed position of the subject, it can be combined with gas exchange measurements which moves it in complexity towards a formal CPET. SpO2 and other gas exchange measurements obtained during cycle and treadmill ergometry testing are not directly comparable because treadmill exercise leads to more desaturation and increased ventilatory inefficiency [86]. Changes in performance on treadmill testing were found to correlate with change in haemodynamics following PAH treatment [155] and treadmill exercise capacity was a predictor of mortality on multivariate analysis [156]. The REVEAL risk equation for PAH contains 6MWD. A study [158] which replaced this with treadmill test performance improved the predictive value of the equation. A treadmill version of the 6MWT has been developed where the speed of the treadmill is increased every 30 s following patient instructions [157] until a maximum is reached. If indicated by the subject, the speed can be decreased before the end of the test is reached. The outcome of the test is the distance walked on the treadmill in 6 min.Shuttle walk tests are popular field exercise tests which require only a 10 m corridor. The incremental shuttle walk test imposes a linear physiological stress avoiding a ceiling effect and has been used by some PH groups [159,160]. The walk distance achieved correlates more strongly with peak VO2 than 6MWD [160] but this is unsurprising given that it is an incremental test whereas the 6MWT is not. Endurance protocols are generally thought to be more sensitive ways of detecting a treatment effect than incremental tests [161]. There is only one study [162] comparing the relative ability of 6MWT, endurance shuttle walk test and constant load cycle ergometry CPET to detect treatment effect in PAH. The results suggest that the 6MWT is the most reproducible and responsive of the three.Step climbing tests have received some attention in PAH studies. Again this test has a very small footprint and, because of the fixed location of the test, it can be combined with more elaborate gas exchange measurements. Fox et al. [163] found that step test performance was impaired in PAH and was correlated with functional class and 6MWD. Bernstein et al. [164] assessed the use of change in PetCO2 measurements during a step test in patients with scleroderma with and without established PAH. They found a correlation with mPAP determined by RHC in a subset of the patients and the measurement performed well as a screening test for PH in this group.Activity monitoring is topical in many disease groups as daily activity is possibly a measurement more relevant to the burden of disease experienced by the patient than the performance of an exercise test. Both Mainguy et al. [165] and Pugh et al. [166] demonstrated a reduction in daily life physical activities and increased sedentary time in PAH. Preliminary data published by Ulrich et al. [167] have suggested poorer survival in those with reduced daytime activity and Frantz et al. [168] provided evidence that treatment can improve activity levels.There are roles for all the modes of exercise testing discussed above in the management of PAH and a summary of what these may be in the future is suggested in Table 4.Likely future Indications for exercise testing modalities in management of PAH.- little or no role; + some role; ++ major role.Invasive exercise haemodynamics remains the reference standard for investigating the possible diagnosis of EIPAH and its use in this context will hopefully answer the question of the clinical relevance of this condition. It is also useful as a test of last resort in the investigation of subjects with unexplained dyspnoea or fatigue. It will not have a major role in screening or follow-up of subjects and in that area it is likely that non-invasive methods of determining cardiac haemodynamics will increase their foothold.CPET is a cumbersome tool to use routinely. Its areas of growth are likely to include screening for subclinical disease and a focussing of the test towards follow up of fitter patients less well served by field tests. It is also likely that there will be increasing use of new variables such as OUES and HRR1 and application of cut-down submaximal tests more convenient for widespread clinical use.Given the major investment so far in 6MWTs and their acknowledged prognostic power in the more impaired patients, it seems unlikely that they will be replaced by alternative field tests. It is possible that more use will be made of additional variables such as HRR1. The major growth in this area may be in activity monitoring, particularly given the flexibility of this mode of measurement and its link with rehabilitation programmes.Exercise testing occupies a major role in the management of PAH. Critical review of its current utilisation shows scope for improvement of its implementation and expansion in the indications (screening, diagnosis, prognosis, monitoring of outcomes) for which it is used. This applies to a wide range of tests from invasive haemodynamics through to activity monitoring over prolonged periods. We predict continued and growing research interest in this modality of assessment of PAH.MKJ performed the literature review and wrote the article. ST produced the figures and reviewed the text.The authors declare no conflict of interest.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Aim: The present study investigates the role of NF-κB in Bmpr2-related pulmonary hypertension (PH) using a murine model of PH with inducible overexpression of a cytoplasmic tail Bmpr2 mutation. Methods and Results: Electrophoretic mobility shift assay for nuclear extracts in Bmpr2R899X mouse lung and immunohistochemistry for NF-κB p65 in human PAH lung demonstrate that NF-κB is activated in end-stage disease. Acute inflammation or expression of a constitutively active NF-κB elicits a strong suppression of the BMP pathway in mice inversely correlating to activation of NF-κB targets. However, Bmpr2 mutation does not result in NF-κB activation in early disease development as assessed by luciferase reporter mice. Moreover, Bmpr2 mutant mice in which NF-κB activation is genetically blocked develop PH indistinguishable from that without the block. Finally, delivery of a virus causing NF-κB activation strongly exacerbates development of PH in Bmpr2 mutant mice, associated with increased remodeling. Conclusion: NF-κB activation exacerbates, but is not required for Bmpr2-related PH. Pulmonary vascular-specific activation of NF-κB may be a “second hit” that drives penetrance in heritable PH.Pulmonary arterial hypertension (PAH), in animal models and probably in human patients, can derive from many primary insults. These include hypoxia (perhaps modeling human diseases such as COPD) [1], primary defects in vasoreactivity (perhaps modeling calcium-channel responsive human PAH patients) [2], metabolic problems (as in fawn-hooded rats) [3], proliferative problems (likely the defect in lymphangioleiomyomatosis) [4], and inflammation (as in schistosomiasis-related PAH, and probably sarcoidosis) [5]. From end-stage pathology and symptomology in patients, it is not clear which of these, if any, is the mechanism behind Group I PAH, the idiopathic and heritable forms.The majority of heritable PAH is associated with mutations in BMPR2 [6,7]. In the non heritable idiopathic form, even when BMPR2 is not mutated, it is suppressed, and the downstream molecular consequences are indistinguishable from mutated BMPR2 [8]. Mice with transgenic overexpression of Bmpr2 develop a form of PH which in many cases is associated with recruitment of inflammatory cells and increased expression of multiple cytokines [9,10,11,12]: in cell culture there is a clear negative feedback loop between the BMP pathway and cytokine expression [13]. Moreover, it has been hypothesized for decades that idiopathic PAH may be a primarily inflammatory disease [14], supported by numerous current lines of research [15,16,17,18].Perhaps the most central pathway in the regulation of inflammation is NF-κB [19]. The NF-κB complex is normally inhibited in the cytoplasm through binding to IκB. IκB is degraded as a result of inflammatory signaling, allowing NF-κB complex components including RelA and p50 to enter the nucleus and initiate transcription.The goal of the current project was to determine whether NF-κB activation is involved in the development of Bmpr2-related PH. We tested whether Bmpr2 mutation results in NF-κB activation in vivo using a triple-transgenic cross to an NF-κB luciferase reporter mouse line; we tested whether NF-κB activation was required for the development of Bmpr2-related PH using a triple-transgenic cross to a dominant negative IB line, and we tested whether NF-κB activation would increase disease penetrance in Bmpr2 mutant mice using a dominant RelA virus.For all of these studies we used mice with doxycycline inducible universal expression of a dominantly acting Bmpr2 mutation, R899X [20]. These mice are allowed to grow to adulthood before activating their mutation, to avoid developmental defects. The R899X mutation is a truncation mutation in the cytoplasmic tail domain of Bmpr2, which leaves canonical SMAD signaling intact [10], but which directly interacts with and modulates the activity of SRC, LIMK1, and TCTEX1 [21]. Cytoplasmic tail domain mutations are common in heritable PAH patients [22], and because they do not impact as many pathways as ligand binding or kinase domain mutations are used here as a sort of “least common denominator” for BMPR2 mutations.Snap frozen whole lung from Rosa26-only control mice or Rosa26-Bmpr2R899X mice shown to have elevated RVSP in prior studies [20] were prepared for nuclear extracts. EMSA for NF-κB binding activity were done as previously described [23]. A double-stranded oligonucleotide probe, containing consensus NF-κB motif (Stratagene, La Jolla, CA, USA) was used in these studies. For supershift studies, p50 antibody (Santa Cruz cat Sc-114) was added during incubation of nuclear extract with binding buffer on ice for 30 min, before incubation with labeled oligonucleotides.A stock solution of LPS derived from E. coli serotype 0111:B4 (Sigma-Aldrich) dissolved in PBS was stored in small aliquots at −20 °C. Immediately before each experiment, an aliquot of LPS was thawed, sonicated, and diluted in PBS yielding a final concentration of 1 mg/mL. Mice in the LPS-Group received 5 mg/kg LPS intraperitoneally (IP) (100 μL–125 μL). Control mice received the same amount of sterile saline IP. 6, 12 and 24 h after LPS administration a lethal dose of pentobarbital was injected. Immediately after a surgical level of anesthesia was achieved, the chest wall was opened, the left atria nicked, and the animal euthanized through injection of PBS (2 mL) into the right ventricle to remove all pulmonary intravascular blood. Lungs were divided and processed for molecular studies. These animal experiments were approved by the University of Colorado Intramural Animal Care and Use Committee.Tissues were homogenized in 500 μL of RIPA Buffer (PBS, 1% ipegal, 0.5% sodium deoxycholate, 0.1% SDS) with protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Tissues were centrifuged at 4 °C (15 min, 15,000 × g) and protein concentration was determined by Bradford microassay (Bio-Rad, Hercules, CA, USA) on the supernatant. Equal amounts of protein extracts were denatured at 95 °C in a denaturing sample buffer. Protein from each sample (15–70 μg) was separated by electrophoresis in a 10% or 8%–16% Tris-Glycine gel and transferred onto a PVDF membrane. The membrane was blocked for 45 min at room temperature with phosphate-buffered saline containing 5% non-fat dry milk and 0.05% Tween-20 and probed overnight at 4 °C with primary rabbit polyclonal antibody against Id1 (1:200 Dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), pSMAD1/5/8 and SMAD 1 (1:200 dilution, Cell Signaling Technology, Danvers, MA, USA) and β-actin (1:1,000, Abcam, Cambridge, MA, USA). The membrane was then incubated at 37 °C for 45 min with horseradish peroxidase-labeled donkey anti-mouse immunoglobulin secondary antibody (1:1,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Horseradish peroxidase was detected using the ECL+ Western blotting detection system (Amersham Biosciences, Piscataway, NJ, USA). Densitometry was performed using ImageJ (public domain software published by the NIH).RNA was made using an RNEasy mini kit (Qiagen, Valencia, CA, USA), and first strand cDNA was made using 1 μg total RNA using a QuantiTect® Reverse Transcription Kit (Qiagen, Valencia, CA, USA). Quantitative real-time PCR was performed using a total reaction volume of 25 μL, containing 5 μL of diluted cDNA, 12.5 μL iTaq SYBR Green Supermix with ROX (BioRad Laboratories, Hercules, CA, USA) and 0.78 μL of each oligonucleotide primer. Primer sequences were as follows: BMP4 (TAGCAAGAGTGCCGTCATTCC, ACCAGTGCTGTGGATCTGCTC), Grem2 (TTACAAGGACGGCAGCAGC, GCGTCTTGCACCAGTCACTC), Il1b (CGTGGACCTTCCAGGATGAG, AATGGGAACGTCACACACCAG), HPRT (TGCTCGAGATGTCATGAAGGAG, TTTAATGTAATCCAGCAGGTCAGC). HPRT expression was used for normalization.HLL mice are a previously described NF-B reporter strain that express luciferase under the control of an NF-B dependent promoter [24]. These were bred to Rosa26-Bmpr2R899X mice to make Rosa26/HLL or Rosa26-Bmpr2R899X/HLL double and triple transgenic mice. Adult mice were fed doxycycline in chow, then NF-B -dependent luciferase activity measured as previously described [25] on day 1, and week 1, 2, and 3 in live mice. Briefly, mice are anesthetized, have chest and abdomen shaved, are injected with Luciferin, and imaged under an intensified charge-coupled device. This is a survival procedure: the same mice are used at each time point. After week 3, mice received 5 mg/kg LPS IP, and were measured at 6 h, 1 day, 2 days, and 3 days. Mice had returned to baseline by the end of 3 days. At the end of week 4, mice were sacrificed, and organs collected for measurement of luciferase activity across tissues as previously described [25]. All animal procedures were approved by the Vanderbilt IACUC.Two-dimensional echocardiography was performed using Vivo 770© High-Resolution Image System (VisualSonics© Toronto, ON, Canada). Echocardiograms including B-mode, M-mode and spectral Doppler images were obtained the day prior to sacrifice under isoflurane anesthetic. Velocity time integral and heart rate were measured in the ascending aorta, diameter measured in the same location. Stroke volume [SV(calc)] was derived using the formula SV = {[π (Aortic diameter)2/4] * [Aortic velocity time integral]}. Cardiac output was [CO (calc)] derived using formula CO = SV(calc) * heart rate (HR) [26,27].Hemodynamic phenotyping was performed as previously described [10,20]. Briefly, mice are anesthetized with tribromoethanol, systemic pressure checked by tail cuff, and then undergo closed-chested intrajugular right cardiac catheterization.Pulmonary vascular resistance (PVR) was calculated based on an approximation of the standard formulation of 80 * (mean PA pressure − mean pa wedge)/cardiac output, where mean PA pressure is calculated using RV pressures rather than PA pressures and setting wedge pressure to zero (both of which are unobtainable in mice). Mean PA pressure is calculated as (2/3) diastolic pressure + (1/3) systolic pressure, but we set diastolic pressures to zero, which would tend to underestimate PVR, to avoid artifacts associated with changing pressures due to breathing in a closed-chested system. The final calculation is thus PVR = (80/3) * (RVSP/CO).All animal procedures were approved by the Vanderbilt IACUC.To prevent NF-κB activation, Rosa26-only or Rosa26-Bmpr2R899X mice were crossed to previously reported TetO7-dnIkB mice to create double or triple transgenics [10,20]. TetO7-dnIκB mice express a mutant avian IB that cannot be degraded [28]. Rosa26-rtTA2 mice express the reverse tetracycline transactivator universally; when used alone these were controls. Rosa26-Bmpr2R899X mice are the Rosa26-rtTA2 mice crossed to TetO7-Bmpr2R899X mice, which then express the dominant negative tail domain mutation R899X in all tissue types, when transgene expression is induced with doxycycline [20]. Adult mice were given doxycycline ad libitum at 0.2 g/kg in chow to activate transgenes, and after six weeks underwent hemodynamic phenotyping as above.Adult Rosa26-only or Rosa26-Bmpr2R899X mice had transgene induced by 0.2 g/kg doxycycline in chow. After two weeks 1 L of 1012pfu/mL RelA virus was administered intra-trachial in 50 L of sterile PBS. Control animals received 1 L of LUC 1-5z11 in 50 L of sterile PBS. RelA and control adenovirus were created as previously described [25]. After an additional four weeks, animals underwent hemodynamic phenotyping as above.IKTA mice (IKKβ Trans-Activated) selectively expressing a constitutively active form of human IKKβ in airway epithelium have been reported previously [29]. We used 8 week-old male IKTA mice and WT littermate controls on the original FVB background. To activate transgene expression, mice were provided ad lib with 0.5 mg/mL doxycycline (dox) in drinking water for 2 weeks. Then mice were sacrificed, lungs were perfused with PBS until free of blood by visual inspection and snap frozen in liquid nitrogen.After euthanasia, the left atria were removed and the pulmonary circulation was perfused with PBS using syringe-generated flow. The left lung was inflated with 0.8% low melt agarose at constant inflation pressure and embedded in paraffin. Samples were transversely sectioned at 6 μm thickness, and immunostained with antibody against smooth muscle alpha actin (αSMA, Sigma-Aldrich, St. Louis, MO, USA) with a fluorescent (GFP) secondary, and counterstained with DAPI. Random fields were selected with DAPI, avoiding fields with large airways or bronchi, and then vessel size and muscularization measured for each vessel identified by αSMA staining. Ten fields are used per lung, and lungs from at least three mice per group are surveyed.Statistical tests were performed using the JMP program (SAS, Cary, NC, USA). One-way or two way ANOVA were used to determine effects of interacting variables, with post-hoc Fischer’s LSD used to determine difference between individual groups.NF-κB activation has been extensively studied in the monocrotaline rat model of inflammatory PH [30], and has been recently extensively studied in human end-stage PAH [31], but we were interested in whether we saw it in our Rosa26-Bmpr2R899X model. To determine whether there was evidence of NF-κB activation in our murine model of Bmpr2, we performed EMSA on flash frozen whole lung from Rosa26-Bmpr2R899X or control mice. The mice used had eight weeks of transgene activation from a previous study [20], and the Bmpr2R899X mutants had developed elevated right ventricular systolic pressures (RVSP).We found that Rosa26-Bmpr2R899X mice had significantly increased nuclear NF-κB compared to Rosa26-only controls (Figure 1A, left 6 lanes). Because only nuclear extracts were used, and because only active NF-κB is found in the nucleus, the darker bands in the Bmpr2R899X lanes indicate increased activity. Specificity is demonstrated by elimination of bands through addition of p50 antibodies (Figure 1A, right 4 lanes). The p50 antibodies used interfere with DNA binding, resulting in disappearance of the band, rather than supershift. Immunohistochemistry for cells with nuclear RelA (indicating activation) shows isolated cells with strong nuclear staining in human idiopathic PAH (Figure 1B) similar to seen in Bmpr2-related PAH in our recent publication (42). A similar finding is presented in a recent study of NF-κB activation in human end-stage PAH [31].The rationale for these experiments was to determine whether acute inflammation, and activation of NF-κB, resulted in modulation of the BMP pathway in live animals. The purpose of these experiments was to establish a signaling link, not as necessarily directly bearing on the etiology of PAH.To determine whether there was evidence for a role for the BMP pathway in regulating inflammation in vivo, we treated wild-type mice with IP LPS, and sacrificed four mice each at 6, 12, and 24 h, compared to controls. We found that at the 6 h time point, at the peak of inflammation [32], there was almost complete abrogation of BMP signaling, as assessed by Smad1/5/8 phosphorylation (Figure 2A). Protein expression of canonical Smad1 target Id1 is suppressed at both 6 and 12 h (Figure 2B). Quantitative RT-PCR for expression of BMP pathway components found no change in Bmpr2 expression (not shown), but a greater than tenfold downregulation of Bmp4 at 6 h, strongly inversely correlated to canonical NF-κB target Il1β, coupled with a long-term decrease in secreted BMP inhibitor Grem2 (PRDC) (Figure 2C). One explanation for this decrease in Grem2, which reaches a minimum at the timepoint associated with return of pSmad activation, is that it is counter-regulatory: NF-κB activation results in suppression of BMP signaling, as well as suppression of BMP inhibitor Grem2, with the result that this suppression is, in normal mice, temporary.NF-κB activation is present in pulmonary arterial hypertension. (A, left) Electrophoretic Mobility Shift Assay (EMSA) using lung nuclear protein extracts from Rosa26-only (control) or Rosa26-Bmpr2R899X mice with elevated right ventricular systolic pressures (RVSP). Two NF-κB bands composed of RelA/p50 heterodimers and p50 homodimers are identified. Each lane is the result from a separate mouse. White space indicates non-adjacent lanes, but identically processed and from the same blot. (A, right) The rightmost four lanes demonstrate specificity of the EMSA bands using p50 antibodies to eliminate the p50 bands. (B) Lung from control (left), idiopathic PAH (IPAH), and heritable PAH (HPAH) (42) patients shows that the latter conditions are associated with increased NF-κB and increased nuclear localization (red) in some cells.To determine specificity of effect, we measured protein levels of Smad1 target Id1 in whole lung from mice with doxycycline-inducible expression of IKTA, a constitutively active form of human IKKβ, resulting in constitutive NF-κB activation. We found on average 3× suppression of Id1 in IKTA mouse lung (Figure 2D), suggesting that it is NF-κB, not other consequences of LPS, that suppresses BMP pathway activity.BMP pathway activity is transiently suppressed during LPS-induced lung injury. (A) Smad1/5/8 phosphorylation is almost completely abolished at six hours after 5 mg/kg IP LPS. (B) Canonical Smad1 target Id1 is greatly reduced at 6 and 12 h after 5 mg/kg IP LPS. (C) Expression of BMP4 in whole mouse lung is strongly suppressed at six hours, partially recovering at 12 and 24 h (p = 0.0003 by ANOVA, p = 0.0037 by Wilcoxon); expression of secreted BMP pathway inhibitor Grem2 remains suppressed throughout (p = 0.0005 by ANOVA, p = 0.0090 by Wilcoxon). Canonical NF-κB target Il1 expression correlates inversely (−0.8) with BMP4 expression. N = 4 mice per time point. (D) Canonical Smad1 target Id1 is reduced roughly threefold in lungs from mice engineered to have constitutive activation of NF-κB, as determined by densitometry (plotted below western blot, with Id1 protein normalized to beta actin), p = 0.012 by ANOVA/p = 0.06 by signed rank (nonparametric test). The rationale for these experiments was to determine whether induction of disease-causing Bmpr2 mutation resulted in modulation of NF-κB in vivo. Based on the late stage NF-κB activation seen in Figure 1, and on the apparently reciprocal relationship between inflammation and BMP expression suggested by Figure 2, we hypothesized that expression of the Bmpr2R899X transgene would result in NF-κB activation at early time points.HLL mice express luciferase under the control of an NF-κB dependent promoter [24]. To determine whether activation of the Bmpr2R899X transgene was pro-inflammatory, we created triple-transgenic Rosa26-rtTA2M2 X TetO7-Bmpr2R899X X HLL mice, or Rosa26-rtTA2M2 X HLL double transgenic mice as controls. Adult mice were fed doxycycline for three weeks, with NF-κB activity assessed the day after induction, and weekly for three weeks. After three weeks, mice received IP LPS, and NF-κB activity assessed at 6 h, 1 day, 2 days, and 3 days. At seven days post-LPS, mice were sacrificed and organs removed to determine organ-specific NF-κB activity. The overall time course was intended to capture mice prior to the development of elevated RVSP, which might increase NF-κB activity for mechanical reasons.We found that in measuring luciferase activity over the abdomen, Bmpr2R899X mice had roughly half the NF-κB activity of controls, and this was stable over time (Figure 3A). However, Bmpr2R899X mice were equally capable of responding to an acute inflammatory event (IP LPS injection). In measuring luciferase over the chest, Bmpr2R899X mice had NF-κB activity more similar to controls (Figure 3B). Of the 10 organs assessed, NF-κB activity was reduced on average twofold in Bmpr2R899X mice in six (Figure 3C), with lung being one of the exceptions. In no tissue was NF-κB activity increased. This is not a result of differential gene targeting; Bmpr2R899X transgene expression is consistent across organs [20].In order to determine whether NF-κB activation is required for murine Bmpr2-related PH, we compared four groups of mice. All mice had Rosa26-rtTA2-M2, which produces universal expression of the rtTA2-M2 variant of the reverse tetracycline transactivator [20]. Two of the groups also had expression of the well-characterized NF-κB inhibitor transgene, dnIkB; two also had expression of our previously published dominant negative Bmpr2 transgene, R899X. Adult mice were maintained on doxycycline for six weeks to activate transgenes, and then were subjected to measurement of systemic pressure by tail cuff, closed-chested intrajugular heart catheterization, blood glucose measurement, and various morphologic measurements.Rosa26-Bmpr2R899X mice had significantly higher RVSP compared to controls, but this was not altered by expression of dnIkB (Figure 4A). The low penetrance in the Bmpr2R899X mice is typical in our experience for Rosa26-Bmpr2R899X not stressed by altitude, diet, age, or other factors. Non-fasting blood glucose was also higher in Bmpr2R899X mice compared to controls (178 vs. 153 mg/dL, p < 0.0001 by t-test, p = 0.0184 by Wilcoxon) as previously reported [33], but this also was not affected by dnIkB. Systemic pressures were equivalent across all groups. Mice expressing the dnIkB transgene had significantly lower proportional left heart weights (by about 7%, p = 0.02 by t-test, p = 0.0049 by Wilcoxon), a phenomenon previously seen with NFkB inhibition [34], implying strength of expression was sufficient to produce phenotype. Expression of the dnIkB had no effect on Smad phosphorylation (Figure 4B) or on muscularization of vessels (Figure 4C), although there was a trend to increased partially muscularized small vessels.Expression of Bmpr2R899X transgene does not result in NF-κB activation in live mice (A) NF-κB activity in abdomen of live mice, assayed by transgenic luciferase-reporter construct, with or without additional Bmpr2R899X transgene. Taken as a whole the Bmpr2R899X curve is lower than the control curve at p = 0.0006 by ANOVA, p = 0.0037 v Wilcoxon rank sum prior to intraperitoneally lipopolysaccharide (IP LPS) injection but the difference disappears when LPS is injected. These are raw luciferase/unit area. N = 4–6 per group. (B) NF-κB activity in chest of live mice, assayed by transgenic luciferase-reporter construct, with or without additional Bmpr2R899X transgene. No individual time point is significantly different, but taken as a whole the Bmpr2R899X curve is lower than the control curve at p = 0.05. These are raw luciferase/unit area numbers, n = 5 per group. The first four time points (weeks 0–4) were statistically indistinguishable, and have been grouped as “base” (C) NF-κB activity in individual organs of Bmpr2R899X mice (n = 5–6 per organ) normalized to activity in the same organ in control mice. The majority of organs from Bmpr2R899X mice have significantly lower NF-κB activity than those from controls.NF-κB activation is not required for murine Bmpr2-related pulmonary arterial hypertension (PAH). (A) While Rosa26-Bmpr2R899X mice had significantly higher RVSP compared to controls (p = 0.0005 by Fisher’s LSD after two-way ANOVA, p = 0.0017 by Wilcoxon), this was not altered by expression of dnIκB. Each circle corresponds to measurements from one mouse; grey bars are means. (B) Smad phosphorylation is not altered by dnIκB or by expression of Bmpr2R899X; densitometry is normalized to beta-actin levels. The Bmpr2R899X has been previously shown to leave Smad signaling intact [10], and so this result was expected. Numbers under each blot are densitometry normalized to beta-actin; the plot at bottom is phosphorylated normalized to total protein. (C) Expression of dnIκB produces a trend towards increased muscularization in vessels in the lungs, but it is not statistically significant by two-way ANOVA.While the previous results indicate NF-κB activation is not required for murine Bmpr2-related PH, NF-κB activation could still contribute. To test this, Rosa26-only or Rosa26-Bmpr2R899X mice had transgene induced for two weeks, then had intratracheal instillation of either empty adenovirus or adenovirus encoding the active NF-κB molecule, RelA [25]. Intratracheal instillation of the RelA virus results in primarily epithelial infection [35,36], and so inflammatory effect on the vasculature was likely a secondary effect. After an additional 4 weeks, after either virus was cleared (quantitative PCR showed no sign of virus in lung or liver, not shown), we performed hemodynamic phenotyping as above.We found that RelA virus increased average pressure by 4 mm Hg in Rosa26-only control mice and 8 mm Hg in Rosa26-Bmpr2R899X mice (Figure 5A). Cardiac output in control mice with control and RelA adenovirus were the same, averaging 8.8 mL/min and 8.4 mL/min respectively; cardiac output in Rosa26-Bmpr2R899X mice dropped from 8.2 to 6.9 mL/min, reflecting a significant increase in pulmonary vascular resistance (Figure 5B). RelA virus increased PVR by 31% in control mice, and to 220% of control values (an average of 155 dyn*s/cm5) in Rosa26-Bmpr2R899X mice. Every mutant mouse receiving RelA virus had a PVR of over 100, making measurable disease penetrance 100% by this metric. RelA virus had no impact on other measured variables (weight, blood glucose, etc., not shown), and by the time of sacrifice had no impact on Bmpr2 activity as assessed by Smad phosphorylation (Figure 5C), although based on Figure 2, we expect that it would have had the effect of at least transient suppression of canonical Bmp signaling when first injected.RelA virus exacerbates pulmonary arterial hypertension in Bmpr2R899X mice. (A) By two-way ANOVA, Bmpr2R899X genotype increased RVSP at p = 0.0042 and Rel-A virus increased RVSP at p = 0.03. Using non-parametric statistics (Wilcoxon) difference between groups is p = 0.0021, with rel-A causing a significant increase in RVSP in Bmpr2R899X mice. Each circle corresponds to an individual mouse: grey bars are means. (B) By two-way ANOVA, Bmpr2R899X genotype increased pulmonary vascular resistance (PVR) at p = 0.0078 and Rel-A virus increased RVSP at p = 0.0126. Using non-parametric statistics (Wilcoxon) difference between groups is p = 0.0020. (C) Neither Rel-A virus nor Bmpr2R899X expression had a significant effect on Smad1 phosphorylation in whole mouse lung. By the time of sacrifice, though, Rel-A virus would have been cleared for at least a week. Numbers under each blot are densitometry normalized to beta-actin; the plot at bottom is phosphorylated normalized to total protein. (D) The mechanism of continuing increased RVSP resulting from Rel-A virus is likely through increased pulmonary vascular remodeling. There is a near doubling of partially and fully muscularized 25–50 m vessels in mutant mice receiving Rel-A virus. (E) Lung sections stained for smooth muscle actin (green) show a dramatic increase in muscularized small vessels (arrowheads) in Bmpr2R899X expression mice receiving Rel-A virus.We quantified numbers of fully and partially muscularized vessels of different sizes in 10 random fields from each of 4 mice from each group. We found that Bmpr2 mutant mice that had received RelA virus had near doubling in muscularized small vessels (Figure 5D,E), not found in control mice. This increase in NF-κB driven muscularization is the most likely explanation for the persistent increase in RVSP in Bmpr2R899X mice, long after clearance of the virus.While inflammation is probably causative in some secondary forms of PAH such as schistosomiasis and sarcoidosis [5], whether or not activation of NF-κB (or any inflammatory pathway) is causative in idiopathic PAH is unknown. NF-κB is activated in end-stage disease in human idiopathic and heritable PAH patients [31]. However, animal models of PH have firmly established that the inflammatory response, including NF-κB activation, are part of the response to high pressure, and mediate phenomena such as increased muscularization of vessels and thickening of adventitia [30,37]. Further, lungs from patients in the Prostacyclin era have substantially worse inflammation than those that were never treated [38], suggesting that perhaps inflammation is an epiphenomenon, or even protective, in human disease. Neither classical animal models nor human data thus clarify the issue of whether NF-κB activation is part of initial pathogenesis.To study this, we turned to BMPR2 mutant mice. BMPR2 mutation is the cause of roughly 80% of heritable PAH, and it is suppressed in all forms of idiopathic PAH [39,40,41], with the molecular signature of disease nearly indistinguishable between patients with and without BMPR2 mutation [8]. It is one of the only known direct causes of PH in humans (the other being use of serotonergic drugs). The relationship between NF-κB activation and development of elevated pressure with BMPR2 mutation is thus potentially important for understanding the etiology and possible interventions of most classes of PAH.The results of this study are summarized in Figure 6, combined with some data from the literature. The effect of NF-κB activation is most straightforward. It is activated in late PH, both in humans and in mice (Figure 1). NF-κB activation worsens disease, probably through increases in muscularization (Figure 5) but also through alteration in the behavior of inflammatory cells [42]. NF-κB activation also suppresses BMP pathway function, either with acute inflammation or with long-term genetic activation (Figure 2), which probably at least partially explains why the BMP pathway is suppressed in all forms of PH in humans. This might explain the common findings of active virus in IPAH [43]. While there has been no consistency in the type of virus found, by this hypothesis, the type of virus is really less important than its presence: Just about any virus will cause NF-B activation and thus suppression of the BMP pathway.Conversely, we found that Bmpr2 tail domain mutation did not lead directly to NF-κB activation (Figure 3), and in fact was associated with a moderate decrease in some organs. The BMP pathway has been published to regulate NF-κB through X-linked inhibitor of apoptosis [44] mediated by the type I receptor. However, the Bmpr2 mutation used in this study does not affect type I receptor-mediated signaling. Studies using a different Bmpr2 mutation, that does affect type I receptor-mediated signaling, did lead to increased NF-κB activation, but only in circulating cells [42].This study suggests that BMPR2 activation of NF-κB is indirect, occurring only after pulmonary vascular pressures are already increased (Figure 1, Figure 3 and Figure 4), while NF-κB suppression of BMP signaling is more direct, occurring acutely (Figure 2) and worsening muscularization and pulmonary pressures (Figure 5).Further, inhibition of NF-B using a dominant negative IB line in vivo had no effect, either positive or negative, on RVSP or penetrance in Bmpr2R899X mice (Figure 4), indicating that NF-B-dependent inflammation is not required for Bmpr2-dependent PH.The technical limitations of this study is, experiments are all done in whole mice or whole tissues, and so we plan to perform more targeted cell specific induction or suppression NF-B activity. Based on histology in Figure 1, it seems likely that the majority of the signal arises from circulating cells. Next, the mouse models all use inducible expression of dominantly negative Bmpr2 or IkB—they thus do not represent normal regulation. For instance, it is possible that for the endogenous genes, suppression of NF-B would result in induction of the BMP pathway. Because our transgenes are not under normal regulation, this would not be possible at the level of transcription in this model. Next, BMPR2 has been shown to regulate NF-B through a mechanism involving TAB1/TAK1 and the type 1 BMP receptor [45]; we explicitly chose to use a BMPR2 mutation which left this signaling intact, since both in mice and in humans mutations affecting this pathway are not required for the development of PH. These technical limitations do not alter our primary conclusions, but do limit their scope.Perhaps more important are the conceptual limitations: questions that this study does not answer, and was not designed to answer. This study does not show that inflammatory processes or cells are not required for the initiation or progression of Bmpr2-related PH. Human pulmonary arterial hypertension is heavily associated with increased local and circulating cytokines, and, especially in the prostacyclin era, massive recruitment of inflammatory cells to the lungs [14,46,47,48,49]. Based on animal models, it seems very likely that this inflammation is a necessary part of remodeling [50], which, although it may not be important in initiation of disease, is probably important in maintenance and progression of disease, not studied in the current project. Even for initiation of disease in Bmpr2 mutants, it seems very likely that inflammatory processes are required. The current study shows that these processes must not be NF-B dependent, but they could easily be dependent on other inflammatory pathways. For instance, both Stat3 [51] and Stat5 [52] have been linked to PAH, as has the MAPK pathway [53,54], all of which could be operating independently of NF-B.In summary, while NF-κB is activated in end-stage PH, mutation of the cytoplasmic tail domain in Bmpr2 does not directly result in NF-κB activation, and causes suppression of NF-κB in many tissues. The present data support the conclusion that NF-κB activation exacerbates, but is not required for Bmpr2-related PH.The work was funded by RO1 Grants HL82694 and HL095797, the North Carolina Chapter of National Lung Cancer Partnership, and Vanderbilt Allergy, Pulmonary and Critical Care Division internal funds.JW: had full access to all of the data in this study and takes responsibility for the integrity of the data and accuracy of the data analysis. JW: Contributed to the study design, data acquisition, analysis, and interpretation, writing and editing the manuscript and served as a principal author. MT and HM: Contributed to the study design, data acquisition, analysis, and interpretation, writing and editing the manuscript. KL, WH and OM: contributed to study design, acquisition, analysis, and interpretation of the data, writing manuscript and approved the final manuscript. Tom B and RZ: contributed to conducting experiments and data anaylsis and approved the final manuscript. AH and Timothy B: contributed to study design; acquisition, analysis, and interpretation of the data; writing and editing the manuscript; and approved the final manuscript.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Since the discovery of Helicobacter pylori (H. pylori), many efforts have been made to establish animal models for the investigation of the pathological features and molecular mechanisms of gastric carcinogenesis. Among the animal models, Mongolian gerbils and mice are particularly useful for the analysis of H. pylori-associated inflammatory reactions and gastric cancer development. Inhibitors of oxidative stress, cyclooxygenase-2 (COX-2) and nuclear factor-κB, exert preventive effects on chronic gastritis and the development of adenocarcinomas in H. pylori-infected gerbils. Genetically-modified mouse models, including transgenic and knockout mice, have also revealed the importance of p53, COX-2/prostaglandin, Wnt/β-catenin, proinflammatory cytokines, gastrin and type III mucin in the molecular mechanisms of gastric carcinogenesis. Microarray technology is available for comprehensive gene analysis in the gastric mucosa of mouse models, and epigenetics, such as DNA methylation, could be an alternative approach to correlate the observations in animal models with the etiology in humans.Gastric cancer is the fourth most common cancer and second leading cause of cancer-related death in the world [1]. Since Helicobacter pylori (H. pylori) was discovered about 30 years ago [2,3], a lot of epidemiological and experimental studies have revealed a significant relationship between H. pylori infection and chronic/atrophic gastritis, peptic ulcer, intestinal metaplasia, gastric lymphoma or cancer development [4,5,6,7,8,9,10,11,12,13,14]. In 2001, Uemura et al. confirmed that stomach cancers develop only in H. pylori-infected patients, but none of the uninfected group [15]. Based on the epidemiological findings, H. pylori was defined as a “definite carcinogen” by the World Health Organization/International Agency for Research on Cancer (WHO/IARC) in 1994 [16]. Since then, many experimental studies have been performed to investigate the mechanisms of gastric carcinogenesis using animal models, as well as clinical samples. Among animal models, Mongolian gerbils and mice are particularly useful for the analysis of H. pylori-associated gastric disorders [17,18]. In this article, we will review the research to date for the analysis of the molecular mechanisms of H. pylori-associated gastric carcinogenesis using rodent models.Several rodent models have been established to investigate the pathological features and molecular mechanisms of stomach carcinogenesis. These include rats, mice and Mongolian gerbils, and chemical carcinogens were used to induce gastric cancer. Some chemicals used in the early studies, such as benzo[a]pyrene [19], 3-methylcholanthrene [20] and 2-acethylaminofluorene [21], showed a low incidence of gastric lesions in rats. However, it has been reported that oral administration of 4-nitroquinoline 1-oxide (4-NQO) and 4-hydroxyaminoquinoline 1-oxide (4-HAQO) could induce adenocarcinomas of glandular stomach, as well as various other tissues in rats and mice [22,23]. In addition, Sugimura and Fujimura established a rat model using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in drinking water to induce gastric adenocarcinomas with relatively high incidence [24]. Similarly, N-methyl-N-nitrosourea (MNU) was shown to induce adenocarcinomas in the glandular stomach of mice, including BALB/c [25], C3H [26] and other strains [27]. In the MNU-treated mice, both differentiated and undifferentiated adenocarcinomas typically develop, showing more significant cellular atypia compared with the MNNG-treated rats. In 1998, Tatematsu et al. have demonstrated that stomach cancers, including well- and poorly-differentiated adenocarcinoma and signet-ring cell carcinoma, also develop in Mongolian gerbils (Meriones unguiculatus), both by MNU or MNNG treatment [28]. The establishment of mouse models has provided new approaches to clarify the molecular mechanisms of gastric carcinogenesis by using genetically-modified animals.Since the discovery of H. pylori, various types of experimental animals have been attempted to develop H. pylori infection models, but none of the early models could sufficiently reproduce the human situation [29,30]. In 1990, Helicobacter felis (H. felis), a novel H. pylori-related bacterium isolated from cat stomach, was found to be able to colonize the glandular stomach and induce acute and chronic gastritis in germ-free mice [31]. After that, H. pylori isolated from human clinical samples were also shown to cause chronic active gastritis in mouse models [32,33,34]. The Sydney strain of H. pylori (SS1) was established by screening of clinical isolates and showed high colonizing ability in C57BL/6 and BALB/c mice [35]. In addition, a Mongolian gerbil model was successfully established and shown to induce H. pylori-associated gastric disorders, including chronic active gastritis, peptic ulcers and intestinal metaplasia, which are closely similar to human [36].H. pylori infection increases the incidence of MNU- and MNNG-induced adenocarcinomas in gerbils [37,38,39]. Although early reports suggested that H. pylori infection alone can induce gastric adenocarcinomas in gerbils [40,41,42], subsequent studies have demonstrated by detailed histopathological assessment that gastric carcinomas are rarely observed in animals treated only with H. pylori infection [28,37,38,39,43], suggesting that H. pylori is a strong promoter of gastric carcinogenesis, rather than an initiator. The H. pylori-infected and carcinogen-treated gerbil model has been widely used for the investigation of the underlying mechanisms of gastric cancer development [17,18].While a number of new insights have been provided from the Helicobacter-infected rodent models, there are limitations that should be considered. Most rodent models take months to a year to develop the gastric lesion expected, and almost all gastric cancers in these models are not metastatic. Cytotoxin-associated gene A (CagA), one of the bacterial virulence factors located in cag pathogenicity islands (cagPAI) of the bacterial genome, is known to be associated with the risk of human stomach cancers [44]. H. felis lacks cagPAI and VacA, a vacuolating toxin [45]. Although both two major strains of H. pylori, ATCC43504 (also known as NCTC11637) and SS1, possess CagA, the SS1 may not express functional CagA protein [46,47]. Other considerations include the dependency on the gender, diet and genetic background of mice [48]. Taken together, it is considered that CagA-containing strains of H. pylori play an especially important role in the investigation of gastric carcinogenesis.Three general factors are considered to impact H. pylori-related gastric carcinogenesis: bacterial virulence factors, including CagA, the genetic susceptibility of the host and the environment [49]. As the environmental factor, diet has particularly attracted attention as a major determinant, and epidemiological and experimental studies have demonstrated that consumption of certain natural products can lower gastrointestinal cancer risk in humans and animal models through activation of antioxidative activity and inhibition of inflammatory pathways, such as COX-2, NF-κB and other proinflammatory cytokines. Among the host genetic factors, gene polymorphisms of proinflammatory cytokines are shown to be associated with the susceptibility to gastric carcinogenesis. Gene manipulation technology enabled us to examine the roles of the specific gene in the mechanisms of H. pylori-induced chronic gastritis and cancer development. It has been suggested that inflammation-associated oxidative stress exerts significant effects on gastric carcinogenesis through upregulation of DNA damage [50]. Inhibition of H. pylori-induced gastritis and oxidative stress is considered as one of the promising approaches to prevent gastric cancer, because the major determinant factor of stomach carcinogenesis is the degree of H. pylori-induced inflammation [51]. To address this hypothesis, Cao et al. examined the inhibitory effect of 4-vinyl-2,6-dimethoxyphenol (canolol), an antioxidant obtained from crude canola oil, on chronic gastritis and gastric cancer development in H. pylori-infected gerbils [52]. As a result, H. pylori-induced gastritis, the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and serum 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels were significantly decreased in canolol-treated gerbils compared with the non-treated groups. In addition, the incidence of gastric adenocarcinomas was also reduced by canolol treatment, indicating that oxygen radical scavengers could prevent H. pylori-associated gastritis and carcinogenesis in gerbils. Interestingly, the canolol treatment did not affect the viable H. pylori count, suggesting that the degree of H. pylori-induced inflammation is a more important determining factor than the existence of the bacteria. Other naturally-derived compounds, such as sulforaphane from broccoli sprouts [53,54], and nordihydroguaiaretic acid found in the creosote bush [55], a common shrub of North America, have been also shown to be effective for the prevention of H. pylori-induced gastric disorders in rodent models through the antioxidative activity.COX-2 has been shown to be involved in the processes of inflammation and carcinogenesis [56], and previous studies indicate that COX-2 expression is associated with H. pylori-induced gastritis and stomach carcinogenesis in humans [57,58]. H. pylori infection and excessive salt intake synergistically enhance COX-2 and iNOS expression in the gastric mucosa of gerbils [59]. Thus, the inhibition of COX-2 may be a promising target for reducing the risk of gastric cancer. Actually, COX-2 selective inhibitors, such as etodolac and celecoxib, have been shown to exert preventive effects on the stomach cancer of H. pylori-infected gerbils [60,61].Recent epidemiological studies have demonstrated that eradication of H. pylori has preventive effects on gastric cancer development [62,63]. However, since not all tumors are prevented by H. pylori eradication [64], the search for new approaches and alternative therapies for the prevention of stomach carcinogenesis after eradication continues to be very important. Because inflammatory reactions against the bacterium disappear after eradication, it is necessary to target factors directly regulating the proliferation and progression of tumor or precancerous cells for the prevention of gastric carcinogenesis after eradication. It has been demonstrated that COX-2 inhibitors could regress the early stage tumors in the intestine of mice and rats [65,66]. Therefore, there is another possibility that COX-2 inhibitors could be applicable to the regression of the remaining precancerous lesion and prevention of gastric cancer occurrence after H. pylori eradication. Nuclear factor-κB (NF-κB) plays a central role in host inflammatory responses and carcinogenesis [67]. H. pylori infection activates the NF-κB signaling pathway, and NF-κB-mediated cytokine expression is essentially involved with H. pylori-induced gastritis in gerbils and mice [68,69,70,71]. Ogura et al. reported that the degree of gastritis induced by a mutant strain of H. pylori lacking capacity for NF-κB activation is lower than that with wild-type infection in gerbils [72]. Therefore, it is considered that inhibition of NF-κB could be a target for the prevention of H. pylori-associated gastric cancer [73]. Caffeic acid phenethyl ester (CAPE), a naturally-derived NF-κB inhibitor, was analyzed on H. pylori-induced chronic gastritis using the gerbil model [74]. CAPE treatment significantly attenuated infiltration of neutrophils and mononuclear cells and the expression of the NF-κB p50 subunit and phospho-IκB-α in the antrum of H. pylori-infected gerbils. The proliferative activity of epithelial cells, both in the antrum and corpus, were markedly reduced by CAPE treatment. In addition, mRNA expression of inflammatory factors, such as tumor necrosis factor-α (Tnf-α), interferon-γ, interleukin (Il)-2, Il-6, KC (Il-8 homologue) and iNos, was significantly decreased in the pyloric mucosa. These results indicate that CAPE has inhibitory effects on H. pylori-induced gastritis in gerbils through the suppression of NF-κB activation and may have the potential for the prevention and therapy of H. pylori-associated gastric disorders [74].Statins, potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are widely used drugs for the treatment of hypercholesterolemia, with beneficial effects on cardiovascular disease [75,76]. Several studies have suggested that statins have chemopreventive effects on various types of cancers [77,78,79]. To clarify the effects of statins on gastric carcinogenesis, pitavastatin was examined in H. pylori-infected gerbil and mouse models [80]. The incidences of H. pylori-associated gastric adenocarcinomas and the degrees of chronic gastritis were not decreased by pitavastatin with upregulation of I1-1β and Tnf-α mRNA expression in the pyloric mucosa. Interestingly, serum total cholesterol (T-Chol), triglyceride (TG) and low-density lipoprotein (LDL) levels were significantly increased by pitavastatin treatment in the H. pylori-infected groups. In the short-term study, H. pylori-infected gerbils and mice also showed upregulation of TG levels by pitavastatin, whereas T-Chol was markedly reduced, and LDL exhibited a tendency for decrease in non-infected animals. These findings indicate that pitavastatin has no suppressive effects on chronic gastritis and stomach carcinogenesis in H. pylori-infected rodent models, and the H. pylori infection and following severe chronic gastritis interfere with the cholesterol-lowering effects of pitavastatin [80].Genetically-modified rodent models have been used for the investigation of the mechanisms of gastric carcinogenesis. The target genes include the oncogenes and tumor-suppressor genes directly associated with the transformation of gastric epithelial cells, the signaling pathways and cytokines involved in the proinflammatory responses induced by Helicobacter infection; and the environmental regulatory factors for colonization and proliferation of H. pylori in the glandular stomach.3.2.1. p53 Tumor Suppressor GeneThe p53 tumor suppressor gene is known to be frequently mutated in various types of human cancer [81]. However, MNNG-induced rat stomach adenocarcinomas had a mutation of the p53 gene in only one of 10 cases [82]. In addition, no mutations in exons 5–8 were found in a total of 30 gastric tumors in MNU-treated mice [83]. In 1992, the p53 knockout mouse has been established by Donehower et al. as a powerful tool for functional analysis of carcinogenesis [84,85,86]. Yamamoto et al. have investigated the susceptibility of p53 nullizygote (−/−), heterozygote (+/−) and wild-type (+/+) mice to MNU-induced pepsinogen altered pyloric glands (PAPGs) and stomach carcinogenesis [87]. PAPGs are putative precancerous lesions immunohistochemically stained weakly or negative for pepsinogen 1 (Pg 1) in mice and rats [88,89,90]. At five weeks, there were more PAPGs both in slightly irregular glands and the normal-looking mucosa of MNU-treated p53 (−/−) mice than the control mice with consistent expression of Pg 1 in the pyloric gland. The frequency of PAPGs in MNU-treated p53 (−/−) mice was significantly elevated compared with the values for MNU-treated p53 (+/+) and (+/−) mice and the control groups. At 15 weeks, adenomas were observed in two of 21 p53 (+/−) and six of 10 p53 (−/−) mice, and adenocarcinoma was detected in one of 10 p53 (−/−) animals. There was a significant tendency toward malignancy in the tumors developed in p53 (−/−) mice compared with (+/−) and wild-type mice. At 40 weeks, no significant difference in the incidences of gastric adenoma and adenocarcinoma between p53 (+/+) and (+/−) mice was observed [87,91]. PCR-single strand conformation polymorphism (SSCP) analysis revealed that none of the 68 gastric tumors derived from the 15- and 40-week experiments has any mutations in the p53 gene, although the simultaneous development of lymphoma and sarcoma in MNU-treated groups showed mutations in the wild-type allele of p53 [87]. These results indicate that p53 (−/−) mice are more susceptible to MNU-induced gastric carcinogenesis than p53 (+/−) and wild-type mice. Thus, it is suggested that p53 may act as a gatekeeper in the gastric carcinogenesis of rodents, rather than as a direct target of chemical carcinogen.COX-2 is a rate-limiting enzyme for prostanoid biosynthesis, and prostaglandin E2 (PGE2) is known to be most important for cancer development. In 2004, Oshima et al. established K19-C2mE mice simultaneously expressing COX-2 and microsomal prostaglandin E synthase (mPGES)-1 under a keratin 19 (K19) promoter in the gastric epithelial cells, and the transgenic mice developed proliferative lesions in the glandular stomach with significant infiltration of macrophages [92]. They revealed the importance of the COX-2/mPGES-1 pathway in Helicobacter-associated gastric carcinogenesis, since H. felis infection also upregulated epithelial expression of COX-2 and PGE2. TNF-α-dependent inflammation is required for the development of proliferative lesions in the COX-2/mPGES-1 mice [93]. COX-2 and mPGES-1 expression progressed metaplastic lesions observed in K19-Wnt1 transgenic mice to dysplastic gastric tumors [94]. In addition, additional expression of noggin, a bone morphogenetic protein (BMP) antagonist, in K19-C2mE mice caused gastric hamartoma that resembles human juvenile polyposis syndrome [95]. CD44-positive stem-like cells are candidates for the origin of gastric tumor in K19-Wnt1/C2mE mice and triggered by PGE2-induced inflammation and Wnt signaling [96]. The germ-free colony of K19-Wnt1/C2mE mice develops less gastric tumors, and additional H. felis infection recovers the tumorigenesis, suggesting that bacterial infection and inflammation play a critical role in gastric carcinogenesis [97]. Takasu et al. has demonstrated that H. pylori (SS1) infection and MNU treatment could induce gastric adenocarcinomas, not only in the antrum, but also in the corpus of K19-C2mE mice, providing a better model for increasing proximal gastric cancers [98].The activation of Wnt/β-catenin signaling mainly caused by mutations in exon 3 of the β-catenin gene is found in 30%–50% of gastric cancers, suggesting an important role of Wnt signaling in stomach carcinogenesis [99]. In MNNG-induced rat adenocarcinomas, four of twenty-two tumors showed nuclear localization of β-catenin with the characteristic mutation of exon 3 [100]. This study suggested that β-catenin mutations could be associated with the progression of chemical-induced rat adenocarcinoma at the late stage. In the gerbil model, only one of forty-five adenocarcinomas induced by H. pylori infection and MNU treatment showed nuclear accumulation of β-catenin with gene mutation [101]. Nuclear localization of β-catenin in stomach cancers is more frequently observed in mouse models. It has been reported that H. pylori infection enhances the activation of β-catenin in the gastric carcinogenesis of the pyloric region, especially in K19-C2mE transgenic mice [98]. Du et al. suggested that Sox17 expression prevents malignant progression of gastric tumors in K19-Wnt1/C2mE mice through the regulation of Wnt activity [102].H. pylori infection-mediated cytokine expression is essentially involved in the development of chronic gastritis and stomach tumors. TNF-α and IL-1β are considered to be particularly important, because epidemiological studies have suggested that their polymorphisms are associated with the increased risk of gastric carcinogenesis [103,104,105]. Oshima et al. recently reported that additional knockout of TNF-α or TNF-α receptor results in the significant suppression of gastric tumor development in the K19-Wnt1/C2mE mice [106]. H. pylori-associated gastric carcinogenesis was also attenuated in IL-1β knockout mice with decreased infiltration of neutrophils and macrophages [107]. MyD88 is one of the adaptor molecules in host inflammatory responses, and a recent study demonstrated that MyD88-deficient mice show the early onset of Helicobacter-induced gastric dysplasia with increased expression of TNF-α, IL-1β, INF-γ and IL-6 [108]. Thus, these studies indicate that proinflammatory cytokines and related pathways could be effective targets of preventive and therapeutic strategies for stomach cancer.The association between hypergastrinemia and gastric carcinogenesis remains unclear. Most patients infected with H. pylori exhibit relatively low serum gastrin levels along with the progression of atrophic gastritis. Actually, it has been reported that gastrin-deficient mice show increased gastric inflammation and tumor development mainly in the antrum [109,110]. On the other hand, Wang and their colleagues have demonstrated that chronic hypergastrinemia in the insulin-gastrin (INS-GAS) transgenic mice could induce gastric cancer in the corpus, and H. felis and H. pylori infection synergistically enhances the progression of tumors [111,112], suggesting that gastrin exerts distinct functions in the antrum and corpus. The INS-GAS mice have been widely used for various fields of investigation of gastric carcinogenesis [113].The gastric mucosa are covered with two types of mucin, surface and gland mucins. Gland mucin is secreted from mucous neck cells and pyloric gland cells and contains O-linked oligosaccharides (O-glycans) with terminal α1,4-linked N-acetylglucosamine residues (αGlcNAc) [114]. It has been revealed that H. pylori could colonize and proliferate not in the gland mucous layer, but in the surface mucous layer, because the O-glycans have antimicrobial effects on H. pylori [115,116]. Karasawa et al. established A4gnt(−/−) mice, completely lacking αGlcNAc expression in the gastric gland mucin, and showed the spontaneous development of gastric adenocarcinomas without H. pylori infection [117]. The expression of pro-inflammatory cytokines and growth factors is increased in the gastric mucosa of A4gnt(−/−) mice, suggesting that the absence of αGlcNAc triggered inflammatory responses followed by gastric tumorigenesis. In human cases, the loss of αGlcNAc in well-differentiated adenocarcinoma with MUC6 expression is shown to be significantly associated with tumor malignancy and poor prognosis [118].Microarray technology has been applied to investigate global gene expression patterns in both human samples and animal models of gastric disorder [119,120,121,122,123]. While there are many reports investigating the gene expression profiles of H. pylori-treated gastric cell lines, cell culture studies do not always reflect the in vivo microenvironment, including host immune responses and stromal-epithelial interactions in cancers. Since there is little information available for the gerbil genome, attention has focused on mouse models. Itadani et al. demonstrated that gastric tumors in K19-Wnt1/C2mE transgenic mice are closely similar to intestinal-type stomach cancer regarding the gene expression profiles [124]. Among thirty-five candidate genes upregulated in H. pylori-infected and high-salt diet-treated mice, Cd177 expression is found to be a novel prognostic factor of patients with advanced gastric adenocarcinoma [125]. Thus, it is considered that the mouse model is suitable for the investigation of the gene expression profile associated with stomach carcinogenesis.DNA methylation is one of the epigenetic mechanisms for gene regulation and deeply associated with cancer development of various tissues. Aberrant DNA methylation is induced by aging and chronic inflammation, including ulcerative colitis, chronic hepatitis and H. pylori-induced gastritis [126]. Niwa et al. revealed that H. pylori-associated inflammation, rather than infection itself, is responsible for inducing the aberrant DNA methylation in gastric epithelial cells using the gerbil model [127]. The authors also demonstrated that specific types of inflammation caused by Helicobacter infection, not the increased cell proliferation, has a strong potential to induce aberrant DNA methylation in the gastric mucosa [46]. A DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), could prevent gastric carcinogenesis in H. pylori-infected gerbils, suggesting that the suppression of aberrant DNA methylation is a target for the prevention of stomach cancers [128]. In addition, novel risk markers of gastric cancer were identified by the microarray analysis for hypermethylated CpG islands [129]. A recent study revealed that CagA of H. pylori causes aberrant epigenetic silencing of let-7 followed by upregulation of Ras oncoprotein using in vitro experiments and an in vivo mouse model [130].H. pylori infection is one of the most important risk factors for gastric carcinogenesis in human stomach. Since the discovery of H. pylori, the Mongolian gerbil and mouse have become useful model animals for the investigation of stomach carcinogenesis, the search for chemopreventive agents and molecular mechanisms. As already revealed by the gerbil model, H. pylori itself is a potent promoter in stomach carcinogenesis through chronic gastritis. The genetically-modified mouse models play a significant role to clarify what kind of molecules or genes act as a bridge between the H. pylori-induced inflammatory process and gastric cancer development. The global gene expression analysis and epigenetic approaches will become much more important for solving the complicated interaction. This study was supported by Grant-in-Aid for the Third-term Comprehensive 10-year Strategy for Cancer Control and the Health Labour Sciences Research Grant from the Ministry of Health, Labour and Welfare, Japan, and Grant-in-Aid for Young Scientists (B) (22700935) and Scientific Research (C) (26430132) from the Japan Society for the Promotion of Science (JSPS), Japan.T. Toyoda, M. Yamamoto, S. Takasu, K. Ogawa, M. Tatematsu and T. Tsukamoto all participated in designing, writing and editing of the review.The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Helicobacter pylori colonizes the gastric mucosa of more than half of the human population worldwide. Soon after its discovery, the causative relationships between H. pylori infection and chronic atrophic gastritis, peptic ulcer and gastric mucosa-associated lymphoid tissue lymphoma were evidenced. Then, a significantly increased risk of developing gastric cancer was found to be associated with H. pylori infection. The efficacy of the treatment for H. pylori, based on a proton pump inhibitor plus antibiotics, has dropped below 80%, mainly due to antibiotic resistance. Vaccination would overcome antibiotic resistance and would lead to the eradication of this pathogen; however, in spite of almost twenty-five years of investigation on H. pylori vaccine candidates and good protective results obtained in animal models, no vaccine is currently licensed. This review focuses on the studies on the efficacy of those H. pylori vaccine candidates that underwent clinical trials. Efficacy trials have given unsatisfactory results, so far, with bacterial colonization remaining unaffected by vaccination. However, a vaccine able to counteract H. pylori-induced diseases, such as gastric cancer, even without providing sterilizing immunity, could be considered valuable.The presence of bacteria in mammalian stomach has been reported since the late 19th century; nevertheless, only at the beginning of the 1980s did Barry Marshall and Robin Warren describe a bacterium isolated and cultured from human gastric biopsies [1], initially classified as Campylobacter pylori and, then, as Helicobacter pylori [2]. Soon after the discovery of H. pylori, its causative relationship with gastritis and peptic ulcer in humans was proven [3]. In 2005, Marshall and Warren received the Nobel Prize in Physiology or Medicine for their revolutionary discovery.H. pylori is a curved or spiral-shaped, flagellated, microaerophilic, Gram-negative bacillus. It colonizes the gastric mucosa of more than 50% of human population, with much higher prevalence in developing than in developed countries [4,5], most probably as a consequence of different hygiene and living conditions.H. pylori is generally acquired during childhood, within the family [6,7]. The colonization is long-lasting, commonly for the entire life. The most likely route of transmission is oral-oral and/or fecal-oral [8]. Moreover, since H. pylori was detected both in water and milk [9,10,11], these could represent possible further routes of transmission, although to date, there is not a formal demonstration of this hypothesis. The H. pylori colonization remains asymptomatic in the majority of the subjects, but a subset of the H. pylori-infected population develops chronic gastritis, peptic ulcer or gastric mucosa-associated lymphoid tissue (MALT) lymphoma [3,12,13]. Moreover, H. pylori colonization was found associated with an increased risk of gastric carcinogenesis [14,15]: thus, the WHO has included H. pylori among the Category 1 carcinogens [16]. H. pylori is the only bacterium that has been clearly associated with the development of cancer to date [17]. In particular, H. pylori CagA (the product of cytotoxin-associated Gene A, cagA) has been shown to trigger many cancer-related signaling pathways in vitro and in vivo, as will be described more in detail below. Several studies have tried to define the possible association of H. pylori with other carcinomas: significant association was found with squamous cell laryngeal cancer and squamous cell cancer of the upper aerodigestive tract (excluding the esophagus), while for other carcinomas, to date, the results are controversial or the size of the studies too small to reach definitive conclusions [18]. H. pylori may also induce or worsen gastric autoimmunity through the activation of cytolytic CD4+ Th1 cells specific for H. pylori peptides cross-reactive with human H+, K+-ATPase: these cells infiltrate the gastric mucosa and may contribute to developing gastric atrophy [19,20].The diagnosis of H. pylori infection in symptomatic subjects is generally followed by eradication therapy. The eradication causes the regression of H. pylori-induced peptic ulcer and MALT lymphoma [21] and may represent a tool for the reduction of gastric cancer incidence in populations at risk [22]. Current therapies against H. pylori are based on the use of one proton pump inhibitor plus two or more antibiotics for one–two weeks, with various approaches that also vary according to the geographic area [23,24,25]. Antibiotic-based therapy presents some drawbacks. If eradication is done at late stages of colonization, when pre-malignant lesions are already present, it could be too late for reverting the mechanisms of carcinogenesis already triggered [26]. The efficacy of the treatments has dropped even below 80% in some cases [27], mainly due to the increase of antibiotic resistance; thus, modifications in the combination and in the sequence of drug administration are continuously under investigation [23,28]. Moreover, after treatment, recurrence and/or reinfection can occur [29], especially in developing countries [30]. Vaccination would represent a valid and cost-effective [31,32] alternative approach to overcome the problems with the antibiotic-based therapy. Several animal models of infection with H. pylori have been described [33]; among them, the mouse model was the most exploited. These animal models allowed the production of a large body of data demonstrating the feasibility and the efficacy of vaccination against H. pylori [34,35]. Since H. pylori colonization occurs at the mucosal level, particular emphasis has been given to oral immunization, although other mucosal routes of immunization [36], as well as the parenteral route [35] or prime-boost regimens, have been considered [37,38,39]. Mucosal immunization requires the use of strong mucosal adjuvants, as proteins are poor immunogens when administered mucosally. Some of the strongest mucosal adjuvants presently known are bacterial toxins, such as cholera toxin (CT) and the E. coli heat-labile enterotoxin (LT), which, however, induce severe diarrhea in humans, seriously limiting their use; thus, non-toxic mutants of these molecules were developed to be used as mucosal adjuvants [40]. The feasibility of mucosal immunization was first demonstrated in mice immunized orally with bacterial lysates or inactivated whole-cell bacteria together with CT, LT or their non-toxic mutants [35], obtaining a high rate of protection against H. pylori. Presently, the use in humans of whole-cell based vaccines could be hampered by the presence of bacterial components that may induce unwanted responses, as, for instance, antigens that have homology with human ones. In particular, H. pylori expresses Lewis antigens identical to those occurring on the surface of gastric epithelial cells [41] and peptides cross-reactive with human H+, K+-ATPase [19,20]. Thus, the current research on an H. pylori vaccine is focused on recombinant protein antigens.The efficacy of prophylactic and therapeutic immunization against H. pylori has been demonstrated for a variety of native or recombinant antigens, such as urease, heat shock proteins, VacA, CagA, HP-NAP, catalase [35], HpaA (Helicobacter pylori adhesin A) [42] and SOD (superoxide dismutase) [43]. Furthermore, DNA vaccines and different delivery systems, such as live Salmonella or poliovirus vectors, have been used in animals [44]. In most cases partial protection was observed, i.e., gastric colonization was reduced, but not abolished, suggesting that higher efficacy could be achieved by combining more than one antigen. The most relevant non-clinical studies carried out with H. pylori antigens that subsequently were included in vaccines that underwent clinical trials are described in the next section of this review, dedicated to these antigens.Recent studies attempted to identify further protective antigens in mice, such as Hp0410 (neuraminyllactose-binding hemagglutinin HpaA homologue) [45], Tpx (thiol peroxidase) [46], outer membrane proteins [47] and alkyl hydroperoxide reductase [48].Other studies attempted to use previously known protective antigens in novel forms (e.g., fusion proteins, portions encompassing defined epitopes, constructs epitope-based vaccines), demonstrating either therapeutic or prophylactic efficacy in mice [49,50,51,52,53,54,55,56]. All of these studies confirmed that protection against H. pylori can be achieved by vaccination in animal models; however, complete protection is rarely achieved, suggesting that the optimization of the antigen combination, adjuvant and route and regimen of immunization is still required. Moreover, efficacy in animals is not necessarily predictive of efficacy in humans; unfortunately, this seems to have happened so far with H. pylori, as highlighted in further sections of this review. The studies in animals led to identifying some H. pylori factors relevant to the colonization and/or the disease, thus representing suitable vaccine targets. Some of these factors exerted protective efficacy when used as a vaccine in animal models. In the following paragraphs, the antigens that were included in vaccines that underwent clinical trials are described; the non-clinical studies that led to propose them as potential vaccine candidates in humans and thus to test them in clinical trials are also reported.H. pylori colonizes a particularly harsh niche, due to the acidic gastric juices. H. pylori urease is required for bacterial colonization [57]; it catalyzes the conversion of urea to carbon dioxide and ammonia: carbon dioxide diffuses into the blood and is exhaled through the lungs, while ammonia forms ammonium hydroxide, which neutralizes the local acidity in favor of H. pylori survival. H. pylori urease consists of two moieties, UreA (27 kDa) and UreB (62 kDa), and constitutes up to 10% of the total bacterial protein content. The production of ammonia is able to produce histological damage in the gastric mucosa [58]. Moreover, urease may contribute to mucosal damage through local activation of inflammatory cells [59,60], The first evidence of the protective efficacy exerted by urease immunization was provided in a mouse model of H. felis infection [61]: in this study, recombinant UreA or UreB vaccine was administered orally, and it was shown that both subunits were efficacious, though with different kinetics, in clearing the experimental colonization. Native, purified urease oral vaccine, including E. coli heat-labile enterotoxin (LT) as an adjuvant, was then assessed in a mouse model of H. pylori infection, and its protective efficacy was confirmed [62,63]. Other mucosal routes of vaccination (intranasal or rectal, besides oral) with recombinant urease were investigated, using LT as an adjuvant, achieving protection by all routes [64], as evaluated by the significant reduction of urease activity and bacterial load in vaccinated mice as compared with sham-immunized controls. In the same study, mucosal vaccination, and, in particular, the rectal route, was also found to induce specific mucosal IgA [64]. Systemic immunization with recombinant urease was then demonstrated to be protective in mice: urease was administered subcutaneously along with different systemic adjuvants, or orally with CT or LT, resulting in good protection with both routes [65]. Interestingly, this study also indicated that urease immunization achieved better protection if adjuvants that induced strong Th1/Th2 responses were used, in comparison with those inducing the Th2 response only.The protective efficacy of urease was also assessed using systems of antigen delivery at the mucosal level by live, engineered Salmonella. The live attenuated Salmonella typhimurium phoPc strain was engineered to express H. pylori urease and administered intranasally in mice; the specific Th1/Th2 response was elicited and significant protection against gastric colonization obtained [66]. A similar study was carried out using the UreA- and UreB-expressing attenuated S. typhimurium SL3261 strain, administered orally to mice: immunization elicited a specific humoral and mucosal antibody response and conferred complete protection [67]. The encouraging results obtained with urease in mice led to assessing its protective efficacy in non-human primates (reviewed in [68]). The immunogenicity of urease administered orally with LT was proven in squirrel monkeys (Saimiri species) [69], a model that, however, was found not suitable for a challenge study. Further studies were conducted in rhesus monkeys (Macaca mulatta), primates that naturally acquire H. pylori infection. In a first study, female, nine-month-old rhesus monkeys were selected on the basis of seronegativity to H. pylori sonicate and H. pylori urease, then orally immunized with five administrations of 8 mg of recombinant urease along with 25 µg of LT [70]. Vaccination elicited specific IgG and IgA in both serum and saliva. Ten months later, the number of individuals that had acquired H. pylori infection was significantly lower for the vaccinated group than for the control group: moreover, antral gastritis was lower in vaccinated than in control animals. A second study was conducted in H. pylori-infected rhesus monkeys, to assess the possible efficacy of therapeutic urease vaccination [71]. Animals received six oral administrations of 40 mg of recombinant urease plus 25 µg of LT: specific serum IgG and IgA, as well as salivary IgA, were induced by the vaccination, but no therapeutic efficacy was observed. This study was continued with the eradication of H. pylori by standard antimicrobial + omeprazole treatment, and then, monkeys received a further administration of vaccine: the subsequent experimental challenge with H. pylori showed that gastric colonization was significantly lower in vaccinated than in control animals, accompanied by a trend toward the reduction of gastric inflammation. This confirmed the feasibility of H. pylori prophylactic vaccination in primates. Another study in rhesus monkeys evaluated the efficacy of prime-boost vaccination strategy [72]. Animals were subjected to antimicrobial + omeprazole therapy, then received either four parenteral (intramuscular) vaccine administrations or one oral administration followed by three intramuscular boosts. The parenteral vaccine consisted of 100 µg of recombinant urease with either 100 µg of aluminum hydroxide (alum) or 600 µg of the synthetic glycolipid adjuvant N-[(2R,3R,4R,5S,6R)-3-[[(2S)-2-amino-3-methylbutanoyl]amino]-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]-N-octadecyldodecanamide (BAY-R 1005); the oral vaccine consisted of 4 mg of urease plus 100 µg of LT. After experimental H. pylori challenge, only animals vaccinated with the prime-boost regimen showed gastric colonization significantly lower than that of control animals that received placebo. A further study included H. pylori-negative rhesus monkeys that received either oral (four administrations of 4 mg of urease plus LT) or intramuscular vaccination with recombinant urease (four administrations of 100 µg of urease plus 600 µg of BAY R 1005) [73]. Serum IgG and salivary IgA were elicited by the vaccination; nevertheless, after experimental H. pylori challenge, no efficacy of vaccination against gastric colonization was observed. The lack of efficacy by parenteral vaccination observed in this study is consistent with a previous one [72]; as for the lack of efficacy of the oral vaccination, it must be noted that these results are not directly comparable with those of a previous study that found protective efficacy [72], as the dose of urease (4 vs. 40 mg) and the number of administrations (four vs. six) were quite different, and in addition, monkeys in the previous study were H. pylori-primed.The results obtained with urease vaccination in non-human primates, although often resulting in only partial protection, provided indications on the antigen dose, adjuvant and regimen to be used in the subsequent trials conducted in humans.HP-NAP consists of dodecamers of 17-kDa protein subunits [74,75]. It induces chemotaxis and both the direct and indirect activation of neutrophils and monocytes [76,77], with the production of oxygen radicals, and promotes neutrophil adhesion [78]. HP-NAP is now considered a crucial factor in driving the Th1 inflammation in H. pylori infection, due to its inflammatory and immunomodulatory properties [79]. The protective efficacy of HP-NAP was assessed in the mouse model [76]. Mice were orally immunized with recombinant HP-NAP along with LTK63, the non-toxic mutant of LT, as an adjuvant. Subsequent experimental challenge with H. pylori revealed protection against gastric colonization in the majority of vaccinated mice. No further reports on the protective efficacy of HP-NAP alone are available. HP-NAP was used in non-clinical studies in combination with other H. pylori antigens, as described in the last subsection of this section, “Multi-Component Vaccines”.In early studies, it was found that H. pylori culture supernatants were able to induce vacuolization on several epithelial cell lines [80]. Then, the cytotoxin responsible for this activity was purified and characterized [81]. VacA toxin is now among the best characterized H. pylori molecules. It is composed by hexa- or heptamers of 88-kDa subunits [82,83]. Each subunit consists of two moieties, p33 and p55, both required for exerting the toxicity: p55 is necessary for binding to target cells, while p33 is essential for the internalization of the toxin [84]. The VacA sequence is well conserved among different isolates, but with some allelic variations: in the signal (s) region (alleles s1a, s1b, s1c and s2), in the mid- (m) region (alleles m1 and m2), localized in the p55 domain, and in the intermediate (i) region (alleles i1 and i2), localized in the p33 domain [85,86]. The m1 allele associates most frequently with s1 allele: this s1m1 form of VacA is related to the most severe pathological outcomes, including gastric cancer [87].Oral immunization with native purified VacA given along with LT as an adjuvant was shown to be protective in mice [62]. A further study confirmed the prophylactic protective efficacy of immunization with both native and recombinant VacA, administered intragastrically along with LTK63 [63]: in this study, it was also demonstrated that the whole VacA molecule was required to achieve protection, as the two VacA moieties individually expressed, purified and used as an immunogen were unable to elicit significant protection against experimental H. pylori challenge. In a study of therapeutic vaccination, animals were infected with H. pylori and then received intragastric immunization with recombinant VacA plus LTK63: a high rate of non-infected mice was observed in vaccinated mice, demonstrating the feasibility and the efficacy of therapeutic vaccination [88]. VacA was then used in non-clinical studies in combination with other H. pylori antigens, as described in the last subsection of this section, “Multi-Component Vaccines”.Early studies on H. pylori virulence determined the classification of the strains as type I or type II, based on the presence or the absence, respectively, of the cytotoxin-associated gene pathogenicity island (cag PAI) in their genome. Type I constitutes the majority of H. pylori clinical isolates, associated with the more toxic s1 allele of VacA, and with the most severe outcome of the infection [89,90,91]. Type II strains are cag negative, most frequently associated with the less toxic s2m2 type of VacA, and induce mainly asymptomatic gastritis [92].Cag PAI encompasses several genes, including cagA, which encodes the CagA protein [93,94], and the components of a type IV secretion system (T4SS) [94,95,96,97]. T4SS, through its component, CagL, binds the α5β1 integrin on the host cell surface and translocates CagA, which is then phosphorylated by the host’s enzymes [98,99]. CagA is also able to bind the host β1 integrin or phosphatidylserine to mediate its own internalization [100,101]. The translocation and phosphorylation of CagA are followed by the induction of IL-8 production by epithelial cells, the activation of NF-κB, the remodeling of the cytoskeleton, the formation of cellular pedestals and the induction of abnormal cell proliferation [93,94].The CagA properties led to defining it as an oncoprotein [100,102], explaining its association with the most severe pathological outcome and giving suggestions on the early mechanisms of H. pylori-induced carcinogenesis. Besides the studies that linked CagA to the activation of oncogenic pathways, its effects on tumor suppressor pathways have been also reported [103,104,105], confirming the strong relationships between the infection with type I H. pylori strains and the risk of developing gastric cancer.Recombinant CagA administered intragastrically to mice along with LTK63 was found to be protective against gastric colonization upon subsequent H. pylori experimental intragastric challenge [63,88]. It was also shown that a recombinant protein encompassing a short fragment of CagA that contains immunodominant epitopes in humans was ineffective in eliciting protection in the mouse model, suggesting that a full-length molecule should be used to maintain protective efficacy [63]. CagA was further used in non-clinical studies in combination with VacA and HP-NAP, as described in the last subsection of this section, “Multi-Component Vaccines”. The combination of CagA, VacA and HP-NAP was assessed for its efficacy as a therapeutic vaccine [106] in the model of H. pylori experimental infection of beagle dog that was previously set up [107]. The dog model presents some advantages as compared with the murine one, in particular those related to the body size, which allows taking gastric biopsies repeatedly from the same animal, without sacrificing it, thus providing a more reliable time course of the evolution of both H. pylori colonization and gastric inflammation. Previous, unpublished results indicated that the combination of these antigens was protective as a prophylactic vaccine administered parenterally [106]. For the therapeutic study, dogs were infected by intragastric administration of H. pylori and then vaccinated intramuscularly with the CagA + VacA + HP-NAP recombinant proteins along with aluminum hydroxide as an adjuvant, giving three immunizations, either one week or one month apart. No side-effects due to immunization were observed. In vaccinated dogs, the reduction of both H. pylori colonization and gastric inflammation was achieved as compared with the controls. Both regimens appeared similar in terms of H. pylori eradication, while the monthly regimen appeared slightly superior in reducing gastritis. A specific IgG response was elicited against each of the vaccine components.Although a very large number of studies of H. pylori vaccination have been conducted in animals, achieving protective results, very few clinical studies have been carried out so far in humans, and no vaccines are currently licensed. Several of the trials conducted focused on the use of the most abundant H. pylori antigen, i.e., urease. In addition, with one exception, all trials were carried out using the mucosal route of immunization or delivery. The number of clinical trials with these mucosal vaccines has been limited also due to the still incomplete optimization of mucosal adjuvants for human use [108] and of their formulation. A model of experimental H. pylori infection in human volunteers has been set up; it is useful not only for studying the vaccine efficacy in humans, but also for better understanding H. pylori pathogenesis [109].The efficacy of inactivated whole-cell vaccines against H. pylori has been shown in a large number of studies carried out in animals immunized either mucosally or parenterally [35]. Whole-cell vaccines offer the advantage of eliciting immune responses against a wide variety of antigens; however, they may include potentially dangerous components of the bacterium, such as those sharing homologies with the self-antigens and potentially able to induce autoimmune responses, e.g., H. pylori lipopolysaccharide (LPS), which mimics human Lewis antigens [110], or, as already mentioned, H. pylori peptides cross-reactive with human H+, K+-ATPase [19,20].A formalin-inactivated whole-cell H. pylori vaccine was evaluated in a phase I trial in both H. pylori-negative and H. pylori-positive subjects [111]. The vaccine, containing various amounts of bacterial cells, was given orally three times, on Days 0, 14 and 28, together with 25 µg of the LTR192G mutant of the LT toxin. The first part of the trial was an open-label, dose-response study in which H. pylori-infected or -uninfected individuals received 2.5 × 106 to 2.5 × 1010 inactivated bacterial cells together with the LT mutant. Vaccination elicited H. pylori-specific antibody responses only in subjects receiving the highest dose of the vaccine. A marginal increase of IgA and IgG titers was observed only in H. pylori-positive patients. The number of specific antibody-secreting cells induced by the vaccine remained negligible; however, some detectable responses were observed at the duodenal level in H. pylori-negative subjects [112]. While some antibody response was induced only in H. pylori-positive patients, proliferative responses of peripheral blood mononuclear cells and the production of IFN-γ were observed only in uninfected volunteers who had received the highest dose of the vaccine, following in vitro re-stimulation with a bacterial sonicate (and not with a purified antigen). The second part of the trial was a randomized, double-blind study in which H. pylori-positive individuals received either 2.5 × 1010 bacteria plus 25 µg of LTR192G or placebo plus 25 µg of LTR192G. Vaccinated subjects had significantly higher IgA antibody titers in the stools than subjects receiving the placebo. The co-administration of the vaccine with the adjuvant influenced only marginally the serum antigen-specific IgA antibody response. Vaccination of H. pylori-infected patients did not achieve bacterial eradication, since in both parts of the trial, the [13C]urea breath test remained positive up to 7.5 months after vaccination [111]. It is not known, however, whether vaccination affected the degree of H. pylori gastric colonization, since no microbiological data were reported in this study. Finally, diarrhea occurred in five out of 18 subjects vaccinated with the highest dose of bacteria plus the LTR192G mutant and in one out of three who received the LTR192G mutant. Conceivably, diarrhea was due to LTR192G, which retains partial toxic activity in vitro and in vivo [113].In the first study conducted with recombinant urease as a vaccine, H. pylori-infected volunteers were orally immunized with high doses of recombinant urease (20, 60 or 180 mg once weekly for four weeks) together with 5 µg of LT [114]. Diarrhea was observed in 16 out 24 of the vaccines, regardless of the dose of urease, thus conceivably due to LT toxicity. Volunteers receiving the highest doses of urease (60 or 180 mg) showed high levels of anti-urease IgA serum antibodies. In spite of a significant decrease of gastric colonization being observed, neither eradication nor a decrease of the gastric inflammation was achieved. A subsequent trial attempted to reduce the side effects of the vaccine by decreasing the amount of LT administered [115]. In this study, 42 healthy H. pylori-negative subjects were immunized orally with 60 mg of recombinant urease either in soluble or acid-resistant, encapsulated form together with 2.5, 0.5 or 0.1 µg of LT. The vaccine was given on Days 1, 8, 28 and 57. The subjects who received the highest dose of LT showed a slightly better urease-specific antibody response and an increase of CD4+, CD45RO+ and CD69+ cells; however, diarrhea was evident in 50% of the subjects of this group. These data confirm, as already known from in vitro assays and animal studies, that LT toxicity, immunogenicity and adjuvanticity are dose-dependent and that a fine tuning is required in order to induce protective immune responses against the vaccine without causing unacceptable side effects, such as diarrhea.Another attempt, made to circumvent the safety issues inherent to the use of the oral administration of wild-type LT, has been made by replacing the oral with the rectal route of vaccination, which had been previously successfully used in mice [64]. Furthermore, in this study, recombinant urease was used at a dose of 60 mg, administered as a rectal enema to 18 healthy, H. pylori-negative adults, together with either 5 or 25 µg of LT, given three times on Days 0, 14 and 28 [116]. A strong systemic antibody response to LT was detectable in the majority of the vaccines, mainly in the group of subjects receiving the lowest dose of LT (5 µg). Only a small minority of subjects developed systemic anti-urease IgG or IgA antibody responses. However, no anti-urease or anti-LT IgA antibodies were detectable in stool or in salivary samples. Finally, the urease-driven proliferative response and IFN-γ production were negligible.All of these studies clearly show that oral immunization with recombinant antigens, urease in particular, requires the use of strong and safe mucosal adjuvants to be given at doses sufficiently high to exert their adjuvant effects, but still unable to induce unwanted effects, such as diarrhea. Both the oral and the rectal routes require the development of appropriate formulations and regimens able to induce adequate protective immune responses to H. pylori.Several clinical trials have been conducted so far using recombinant Salmonella strains as a live vaccine vector for H. pylori urease. Unfortunately, in marked contrast with the results previously obtained in mice, all of these attempts resulted in poor immunogenicity and efficacy. Nevertheless, the most recent trials provided some useful information relevant to the possible cell-mediated mechanisms of protections.In the first study with Salmonella-vectored urease, one or two oral administrations of 1010 CFU of a Salmonella enterica serovar Typhi strain, attenuated by the deletion of the phoP/phoQ virulence regulon and expressing H. pylori urease, induced mucosal and systemic immune responses to Salmonella antigens, while no detectable responses to urease were observed, even after an oral booster dose of recombinant urease plus wild-type LT [117]. Slightly better results were reported in a subsequent study, in which six volunteers were immunized orally (5 to 8 × 107 CFU) with S. enterica serovar Typhimurium harboring the same phoP/phoQ deletion [118]. Only one of the six volunteers had detectable urease-specific IgA antibody secreting cells; two others had slight amounts of urease-specific antibodies produced in vitro by cultured peripheral blood mononuclear cells; two subjects had some specific serum IgA antibodies detectable by ELISA, but not by western blotting.Another study was carried out in volunteers immunized orally with S. enterica serovar Typhi Ty21a expressing H. pylori urease. None of the nine vaccinated volunteers showed any detectable antibody response against urease, but five subjects developed cellular response [119]. Similar results were obtained in a further study, in which five out of nine vaccinated subjects that were pre-immunized with the carrier strain developed cellular, but not antibody, response against urease; however, in this study, vaccinated subjects that were not pre-immunized with the carrier strain did not develop any kind of specific immune response [120]. In one of the latest studies with Salmonella-vectored vaccine, which included the experimental challenge of volunteers, a small number of subjects cleared the infection, but protection was not related to vaccination, as it was similarly distributed between vaccines and controls [121]. In these subjects, a specific T-cell response was observed that could be helpful in understanding the mechanisms of protective response.It appears that Salmonella-vectored H. pylori urease vaccines still require optimization, in particular in terms of the increase of urease expression [120]; also, co-expression of an adjuvant or immunostimulatory, as shown in mice [122], could increase the efficacy of this vaccine. The only multi-component vaccine against H. pylori that underwent clinical trial so far consisted of recombinant VacA, CagA and HP-NAP. Differently from the H. pylori vaccines described above, this vaccine was administered parenterally rather than mucosally. The safety and the immunogenicity of this multicomponent vaccine were evaluated in human volunteers. H. pylori-uninfected individuals were immunized intramuscularly three times, following three different immunization regimes, with a vaccine consisting of either 10 or 25 µg each of CagA, VacA and HP-NAP, plus aluminum hydroxide as an adjuvant [123]. This vaccine was extremely safe and highly immunogenic, inducing antibody and cellular responses to the three antigens. Months after the last immunization, most of the subjects had still detectable antibody responses to each of the three antigens. Interestingly, parenteral vaccination with VacA, CagA and HP-NAP induced very strong and sustained antigen-driven cellular proliferative responses and IFN-γ production, as well as strong and long-lasting immunological memory [123].No further results on clinical trials of H. pylori vaccines, and, in particular, of efficacy trials, have been disclosed in the recent literature. Presently, there is not any licensed anti-H. pylori vaccine. There are several reasons, besides the disappointing results of the efficacy trials conducted with urease-based vaccines, that, currently, the development of a H. pylori vaccine seems to be discontinued (reviewed in [124,125]).A major issue is the still incomplete knowledge of the mechanisms behind protective immunity against H. pylori. The host immune response against H. pylori will be not treated in the present review; several updated reviews are available that extensively describe the abundance of the immunomodulatory effects of H. pylori on the host immune system [124,125,126,127,128,129,130,131,132]. The majority of non-clinical studies resulted in a significant decrease of bacterial colonization rather than complete, sterilizing protection. This extent of efficacy against experimental infection in animals could be insufficient when translated to human infection. Moreover, ideally, an H. pylori vaccine would be both prophylactic and therapeutic, given the high rate of the currently infected population. Therefore, further research is needed to understand the protective mechanisms and to identify vaccine formulations able to prevent and cure the infection. The current lack of immunological correlates of protection for H. pylori constitutes per se a severe drawback for the further development of a vaccine, as the vaccine efficacy could only be proven by appropriate phase III clinical trials. This would be considered by companies as too expensive and risky. A further element that may limit the development of an H. pylori vaccine is represented by reports suggesting some beneficial roles of H. pylori colonization for the host, as, for instance, the reduction of the risk of developing allergic and chronic inflammatory disorders [133,134]. The beneficial effects would be exerted in the first part of the host’s life, while detrimental effects start to appear over 50 years of age [133]. If, on the one hand, these observations represent an interesting scientific field that deserves further investigation, on the other hand, most of them still appear controversial [135]. Similarly, some epidemiological studies suggested the reduction of the risk of developing esophageal adenocarcinoma for H. pylori-infected subjects, but the results of other studies were in contrast with this hypothesis [18]. It is now evident that the highest risk of developing the most severe disease upon H. pylori infection is related to the combination of both particular pathogen characteristics (such as, for instance, the cag PAI presence) and host genetic background (such as specific polymorphisms of inflammation-related genes) [136,137]; although, it is clear that, when such a combination of H. pylori virulence factors with particular host susceptibility occurs, H. pylori infection can eventually lead to the development of gastric cancer, the feeling that it could also provide the host with some advantages could adversely affect compliance with a vaccination campaign. Although vaccination against H. pylori was successful in animal models with various antigens and administration routes, the disclosed results of efficacy trials in humans have been disappointing so far, resulting in similar bacterial colonization for both vaccinated and placebo groups. Rather than abandoning hope of having a vaccine against H. pylori, in the frame of all of the above considerations, an objective different from that of obtaining sterilizing immunity could be now proposed for H. pylori vaccination, which currently seems unlikely to be achieved. Since some H. pylori factors have been already well characterized and its particular dangerousness for the host has been proven, a vaccine specifically targeting those factors could be proposed: such a vaccine should be aimed at affecting H. pylori-induced disease rather than bacterial colonization. In other words, a vaccine against H. pylori able to prevent gastric cancer, even without providing sterilizing immunity, would be a valuable vaccine. This could be the case for CagA, whose relationships with H. pylori-induced carcinogenesis are now well documented [104].The ongoing studies on H. pylori pathogenesis may also help in identifying novel vaccine targets. One promising aspect is the study of T-regulatory (Treg) cells. Treg cells increase in the gastric mucosa of H. pylori-infected subjects. This suggests the involvement of these cells in suppressing mucosal immune responses, therefore contributing to the infection persistence and to the modulation of H. pylori-induced gastritis [138,139,140,141,142]. A study in a neonatal mouse model suggested that, while in adults, the suppression of immune response to H. pylori infection may lead to gastric disease, in neonates, the same mechanism induces tolerance and may prevent the development of gastric disease and preneoplastic lesions [143]. This relatively recent finding of the involvement of Treg cells in the development of H. pylori-related gastric diseases may help to focus the studies also on the H. pylori factors that drive the Treg activation, as potential vaccine targets. The research on novel possible vaccine candidates against H. pylori is still active, as well as studies on more efficacious adjuvants, regimens and routes of administration. Moreover, the identification of novel vaccine candidates may benefit from “reverse vaccinology” and “-omics” approaches [144,145], which can exploit the availability of the complete genome sequence of H. pylori. The authors declare potential conflicts of interest, being Novartis Vaccines employees. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Hepatocellular carcinoma (HCC) is one of the most lethal malignancies due to underlying co-morbid cirrhosis and chemo-resistance. Vaccination and improved treatment for hepatitis are the most effective means to reduce the burden of liver cancer worldwide. Expression of biomarkers such as AFP (alpha-fetoprotein), DDK1 (Dickkopf WNT Signaling Pathway Inhibitor 1) and microRNAs in blood are being tested for early screening of liver cancer. Since 2008, sorafenib has been used as the standard molecular targeting agent for HCC. However, overall outcomes for sorafenib alone or in combination with other tyrosine kinase inhibitors are unsatisfactory. Whether simultaneously or sequentially, addiction switches and compensatory pathway activation in HCC, induced by sorafenib treatment, may induce acquired resistance. Forkhead box M1 (FOXM1) and metadherin (MTDH) have been shown to be master regulators of different aspects of tumorigenesis, including angiogenesis, invasion, metastasis and drug resistance. Elevated expression of both FOXM1 and MTDH is known to be a consequence of both activating mutations in oncogenes such as PI3K, Ras, myc and loss of function mutations in tumor suppressor genes such as p53 and PTEN in various types of cancers including HCC. The role of FOXM1 and MTDH as potential prognostic markers as well as therapeutic targets in HCC will be discussed. In addition, microRNAs (miRNAs), endogenous small non-coding RNAs involved in the regulation of gene expression, are involved in HCC and interact with both FOXM1 and MTDH in several ways. Thus, altered expression of miRNAs in HCCs will also be discussed as potential tools for diagnosis, prognosis and therapy in HCC.Hepatocellular carcinomas (HCC) are genetically heterogeneous tumors. TP53 mutations have been found in HCC, especially in HCC patients with HCV or HBV positive [1]. In addition to prior HBV/HCV infection, metabolic abnormalities such as diabetes and obesity are also involved in HCC carcinogenesis. In contrast to other epithelial cancers such as breast, ovarian and colon, no mutations of common oncogenes and tumor suppressor genes except TP53 have been identified in HCC cells. However, many growth factor receptors such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and fibroblast growth factor receptor (FGFR) as well as key oncogenic signaling pathways related to proliferation and angiogenesis such as Ras/Raf/MEK/ERK (MAPK), phoshoinositol-3 kinase (PI3k)/Akt/mTOR, hepatocyte growth factor (HGF)/c-mesenchymal epithelial transition factor (c-Met), insulin growth factor receptor(IGF), transforming growth factor-β (TGF-β), Wnt/β-catenin, Hedgehog and Notch have been shown to be involved in hepatocarcinogenesis [2,3]. Therefore, the tyrosine kinase inhibitor sorafenib has been used to block both tumor cell proliferation and angiogenesis by targeting Raf-1/B-Raf kinases and VEGFR-2/-3 and PDGFR-β tyrosine kinases. Sorafenib administration has led to significant improvement in overall survival (OS) in patients with advanced HCC but disease stabilization does not last due to acquired resistance [4,5,6]. Understanding the mechanism of sorafenib resistance would improve sorafenib therapy in patients with HCC. One mechanism of sorafenib resistance in cultured HCC cells may be associated with a switch from the MAPK pathway to the parallel PI3K/Akt pathway when vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), Ret, c-kit and the downstream MAPK pathway are inhibited by sorafenib inhibition of tyrosine kinases. Activation of Akt and upregulation of phosphorylation of its downstream targets, including S6K and 4EBP1, have been shown in sorafenib treated HCC cells [7]. In HCC cells in which resistance is the result of long-term exposure to sorafenib, increased expression of phosphorylated Akt and the p85 regulatory subunit of PI3K was observed. Sorafenib resistance could be reversed by knockdown of Akt or by administration of the Akt inhibitor MK-2206 [8]. In addition to the above described mechanisms, multidrug resistance protein 2 (MDRP2) and nuclear factor κB (NF-κB) also appear to be involved in acquired resistance to sorafenib in HCC [9,10,11].Activation of the oncogenic signaling pathway PI3K/Akt can induce expression of both FOXM1 and MTDH, two critical master regulator genes involved in metastasis and drug resistance in numerous solid tumors including HCC [8,12]. FOXM1 expression can also be induced by other mechanisms including (i) increased transcription through the action of transcription factors E2F, c-Myc, and hypoxia-inducible factor-1 (HIF-1) [13,14,15]; (ii) loss transcription repression due to mutations in the tumor suppressor p53 (seen in approximately half of HCC patients) [16,17,18] and (iii) decreased expression of microRNAs (miRNAs) such as miR-149 and miR-134 which down-regulate FOXM1 post-transcriptionally [19,20]. Bioinformatic analyses of genome-wide transcriptional sequencing data in HCC have identified alterations of the FOXM1 transcription network including downstream FOXM1 effectors AURKB, BIRC5, CCNB1, CCNB2 and NEK2 [21,22].Conditionally deleted FOXM1 mouse hepatocytes are highly resistant to developing HCC in response to a Diethylnitrosamine (DEN)/Phenobarbital (PB) liver tumor-induction protocol suggesting a pivotal role for FOXM1 in HCC development [23,24]. HBV-associated hepatocarcinogenesis involves the activation of FOXM1 expression by hepatitis B virus X (HBx) through the ERK/CREB pathway [25]. Induction of MMP-7, RhoC, and ROCK1 expression by FOXM1 can promote hepatoma cell invasion and metastasis [25]. FOXM1 expression was highly correlated with the recurrence and poor survival of patients with HBV-HCC after surgical resection. Another downstream effector of FOXM1, acid phosphatase 5 (ACP5), is also involved in progression and metastasis of HCC mediated by FOXM1. Knockdown of ACP5 significantly reduced FOXM1-mediated invasion and lung metastases [26]. Co-expression of ACP5 and FOXM1 was associated with poor prognosis. Overexpression of miR-135a favors invasive and metastatic behavior in vitro. MiR-135a is transcribed by FOXM1 and metastasis suppressor 1 (MTSS1) was identified as a direct target of miR-135a [27]. Further analysis revealed the relevance of miR-135a with respect to the prognosis and survival of HCC patients with Portal vein tumor thrombus (PVTT). PVTT has been correlated with poor prognosis of hepatocellular carcinoma and 50%–80% of HCC is accompanied by portal vein invasion. MiR-135a is highly over-expressed in PVTT [27].Thiazole compounds, Siomycin A and thiostrepton, and other proteasome inhibitors such as bortezomib, have been shown to repress transcription of FOXM1 at both the mRNA and protein levels in cancer cells [28]. These agents could be attractive therapeutic drugs to overcome drug resistance when combined with standard regimens for cancers with FOXM1 overexpression. FOXM1 is also an inhibitory target of tumor suppressor gene p19 (ARF) [23,29,30]. A p19(ARF) position 26-44 peptide fragment containing nine D-Arg residues to enhance cellular uptake was sufficient to significantly reduce FOXM1 transcriptional activity. HCC in both wild-type mice or in Arf(−/−)Rosa26-FOXM1 Tg mice was induced by diethylnitrosamine/phenobarbital. These HCC-bearing mice were then injected daily with the cell-penetrating ARF(26-44) peptide inhibitor to pharmacologically reduce FOXM1 activity. After 4 weeks of this treatment, HCC regions displayed reduced tumor cell proliferation and angiogenesis within the HCC region but not in the adjacent normal liver tissue. Significant downregulation of FOXM1 to normal cell levels has been reached in cancer cells by depleting NPM1, an interacting partner of FOXM1 [31]. Peptides or small molecules specifically targeting the interaction between FOXM1 and NPM1 could potentially lead to a FOXM1 targeted therapy. Various DNA repair genes including BRIP1 (BRCA1-associated BACH1 helicase), Rad51, XRCC1 (X-ray repair cross-complementing protein 1) and BRCA2 (breast cancer-associated gene 2) have been transcriptionally regulated by FOXM1 [32,33,34,35]. The function of FOXM1 as transcription regulator of DNA repair genes indicates that FOXM1 might be targeted to increase the sensitivity of tumor cells to DNA damage induced by chemotherapy and radiotherapy (Figure 1A).Mechanisms regulating the overexpression of FOXM1 (Forkhead box M1), MTDH (metadherin) and downstream effectors and strategies used to target FOXM1 in HCC (Hepatocellular carcinoma). (A) Thiazole compounds siomycin and thiostrepton, proteasome inhibitors (highlighted in green boxes), ARF peptides and NPM1 siRNA (highlighted in blue boxes) can reduce the expression of FOXM1. Chemotherapy can increase FOXM1 stability via CHK2. HBV/HCV can increase FOXM1 through ERK/CREB.Up-regulated FOXM1 subsequently controls cell cycle transition, DNA repair and metastasis via positive (pink boxes) or negative (blue boxes) regulating downstream effectors. (B) Multiple mechanisms including chromosome amplification at 8q22, Ras, PI3K/Akt, myc activation, increased CEBP1, reduced miR-375, and HBV infection can cause up-regulation of MTDH. Increased MTDH can regulate various downstream effectors to control drug resistance and metastasis via RNA or protein interactions. Increased expression of anti-MTDH auto-antibody, TSPAN8 and reduced expression of IGFBP7 in HCC patient blood might be used as poor prognosis biomarker. (C) Multiple feedback loops are involved in the expression regulation of MTDH, FOXM1 and c-myc in HCC.MTDH has also been shown to play a critical role in DNA damage response (DDR) in a transgenic mouse model with hepatocyte-specific expression of MTDH (Alb/MTDH) [36,37]. Compared to control hepatocytes, reduced reactive oxygen species (ROS) levels and delayed activation of ATM, ATR, CHK1 and CHK2 have been observed in Alb/MTDH hepatocytes following isolation and culture. Endogenous MTDH is mainly distributed in the nucleus or nucleolus in hepatocytes of wild type mice, however, MTDH is mainly localized at cytoplasm in hepatocytes of the transgenic (Alb/MTDH) mouse model. Our previous report in which we used RNA-binding protein immunoprecipitation followed by microarray analysis (RIP-chip) showed that MTDH can associate with various mRNA targets including multiple members of the Fanconi Anemia pathway (FANCA, FANCC, FANCD2, FANCI) and functionally related Rad18 [38]. MTDH might function as a RNA binding protein to regulate the levels of important DNA repair factors at the post-transcriptional level. Previous work has shown that MTDH can promote translation of the multiple drug resistance gene MDR1 and the angiogenesis gene factor XII (FXII) [36,39,40]. Therefore, targeting MTDH might impair the ability of cancer cells to rapidly repair their DNA and increase therapeutic efficacy.Overexpression of MTDH is detected in >90% of HCC patients by immunohistochemistry in tissue microarrays (TMAs) and the level of MTDH expression is negatively correlated with overall survival and cumulative recurrence rates [41]. MTDH was identified by characterizing the copy number aberration (CNA) landscape in HCC from the recurrent focal amplification peaks [42] as one of the oncogenic drivers playing a significant role in HCC growth and survival. Multivariate analyses of 288 HCC patients revealed that high MTDH expression was an independent predictor of shorter disease-free survival (DFS) after curative hepatectomy [43]. MTDH overexpression in HCC was associated with elevated copy numbers due to gains of large regions of chromosome 8q22. Transcription of MTDH is induced by direct binding of c-myc to the MTDH promoter upon activation of Ha-ras/PI3K/Akt signaling [44]. Overexpression of MTDH in HCC cells might evade apoptosis and increase cell growth through the regulation of PLZF repression on the activation of c-myc, which can activate transcription of MTDH through a positive feedback loop [45]. MTDH is a confirmed target of miR375 for post-transcriptional regulation. MiR-375 has been shown to be downregulated in 60 human HCC compared to normal adjacent tissues [46]. Downregulation of N-cadherin and snail, upregulation of E-cadherin, and translocation of β-catenin by depletion expression of MTDH in HCC cell lines indicates that MTDH may promote HCC metastasis through the induction of the epithelial-mesenchymal transition (EMT) process [47,48]. Moreover, MTDH overexpression was correlated with clinicopathologic characters including cell proliferation, invasion, metastasis, chemoresistance, angiogenesis and poor clinical outcome [40,49,50]. A gradual increase in MTDH expression in normal liver tissue to hepatitis B and HBV-related HCC tissue levels was observed [51]. Significant correlation between MTDH expression with the American Joint Committee on Cancer (AJCC, seventh edition) stage, T classification, N classification, vascular invasion, and histological differentiation was seen in the HBV-related HCC patients. Cytoplasmic polyadenylation element-binding protein 1 (CEBP1) binds to the 3'-UTR of MTDH mRNA and promotes its translation. Monoubiquitination of MTDH protein in cancer cells leads to increased stabilization and cytoplasmic accumulation. In summary, MTDH overexpression in human HCC is caused by multiple mechanisms, such as genomic amplification at 8q22 as well as myc mediated transcription activation and reduced post-transcriptional regulation by microRNAs, monoubiquitination and association with CEBP1 (Figure 1B). HCC with steatotic features was induced by the carcinogen N-nitrosodiethylamine in a transgenic mouse with hepatocyte-specific expression of MTDH (Alb/MTDH), but not in wild-type (WT) mice. Novel aspects of MTDH function, including induction of steatosis, inhibition of senescence, and activation of the coagulation pathway to augment aggressive hepatocarcinogenesis have been observed in hepatocyte-specific MTDH transgenic mice [36]. Inhibition of hepatocarcinogenesis induced by N-nitrosodiethylamine was also observed in MTDH knockout mice [52].MTDH is primarily located in the membrane and the cytoplasm of HCC cells compared to nuclear distribution in normal hepatocytes. Autoantibodies against tumor-associated antigens present in the blood of cancer patients can be developed as biomarkers. As a consequence of overexpression of MTDH in cancer cell membranes, elevated levels of anti-MTDH antibody in the serum of cancer patients was detected [53]. Anti-MTDH antibody at titers of ≥1:50 from the sera of liver cancer patients was detected in 48 out of 96 (50%) HCC patients by ELISA using the lung-homing domain (aa 381–443) of human MTDH as the antigen [53,54]. In contrast, no anti-MTDH antibody was detected in the serum of 230 normal controls. Anti-MTDH antibody was detected in eight of 31 (26%) stage I and II HCC patients and in 40 of 65 (62%) stage III and IV HCC patients. Activation of the Akt and NF-κB pathways are the two major signal transduction pathways correlated with MTDH and drug resistance in HCC [55]. Although the detailed mechanism of PI3K/Akt pathway activation by MTDH is unclear, involvement of MTDH has been confirmed by several laboratories. MTDH may play a role in the chronic inflammatory changes preceding HCC development by activating the NF-κB pathway. MTDH was seen to interact with the p65 subunit of NF-κB as well as with CREB-binding protein (CBP) [56]. Though MTDH itself does not have a DNA-binding domain, MTDH may function as a transcriptional coactivator in the NF-κB complex bound to IL-8 promoter as a bridging factor among NF-κB, CBP, and basal transcription machinery to activate downstream genes of NF-κB pathway. Further, MTDH is involved in Wnt/β-catenin signaling through inducing ERK42/44 activation and is associated with protective autophagy by inducing AMPK activation.Genes downstream of MTDH were identified via an Affymetrix oligonucleotide microarray by comparing differential gene expression in control and exogenous MTDH expressing HepG2 cells. One cluster of genes in the Wnt signaling pathway, including lymphoid-enhancing factor 1/T cell factor 1 (LEF1/TCF1), CTBP2 and APC, were regulated by MTDH. A second cluster of genes downstream of MTDH was associated with chemoresistance including drug-metabolizing enzymes, such as dihydropyrimidine dehydrogenase (DPYD), cytochrome P4502B6 (CYP2B6), dihydrodiol dehydrogenase (AKR1C2), and the ATP-binding cassette transporter ABCC11 (also known as MRP8). Transcription factor CP2 (TFCP2, also known as LSF) was also significantly upregulated by MTDH in HCC cells. Other important genes downstream of MTDH include claudin 4 (CLDN4), tetraspanin 8 (TSPAN8), transgelin (TAGLN, a suppressor of MMP-9), IGF-binding protein 7 (IGFBP7) and pyruvate kinase (PK).Late SV40 factor (LSF) was up-regulated by MTDH in HCC [41]. LSF is a ubiquitous transcription factor, first identified as a transcriptional activator of the major late Simian Virus 40 promoter. LSF belongs to the LSF/CP2 family related to grainyhead family of proteins and is involved in many biological functions, including regulation of cellular proliferation, viral promoters and drug resistance. Similar to MTDH, LSF overexpression is detected in more than 90% of human HCC patients, compared to normal hepatocytes. A network of proteins including osteopontin (OPN), Matrix metalloproteinase-9 (MMP-9) and c-Met were identified as downstream effectors of LSF in LSF-induced hepatocarcinogenesis [41,57,58]. A small molecule inhibitor of LSF, Factor Quinolinone inhibitor 1 (FQI1), was identified by a high throughput screening [59]. FQI1 can profoundly inhibit viability in human HCC cell lines in vitro and markedly inhibit growth of human HCC xenografts as well as angiogenesis in vivo. MET/HGF targeted anticancer therapies with tivantinib, cabozantinib, onartuzumab, crizotinib, rilotumumab, and ficlatuzumab have shown encouraging results in clinical trials. Patients with high MTDH and LSF expression may benefit from targeted MET /HGF inhibitor treatment.Tspan8 (also called D6.1A/CO-029 cancer antigen) is associated with metastasis and pro-angiogenesis. Tspan8 is another gene upregulated by MTDH in HCC cells [36]. Tspan8 expression is induced 30-fold in liver after DEN treatment of MTDH transgenic mice compared to wild type mice. Tspan8 contributes to a selective recruitment of proteins and mRNA into extra-cellular microvesicles called exosomes. This includes CD49d, which is implicated in exosome-endothelial cell (EC) binding and EC internalization. EC uptake of Tspan8-CD49d complex-containing exosomes was accompanied by enhanced EC proliferation, migration, sprouting, and maturation of EC progenitors. Tumor-derived exosomes containing the Tspan8 can efficiently induce angiogenesis.Insulin-like growth factor-binding protein-7 (IGFBP7) is a downstream gene repressed by MTDH in HCC cells [60]. IGFBP7 is a secreted member of the IGFBP family. The IGFBP family (IGFBP1-7), two growth factors (IGF-I and IGF-II) and their receptors (IGF-IR and IGF-IIR) are components of IGF signal transduction pathway. Using an immunohistochemical approach in tissue microarray, high IGFBP7 expression was detected in normal human liver while a significantly decreased expression was seen in 104 HCC patients. Thus, IGFBP7 downregulation might be a prognostic marker for aggressive HCC. Although stable overexpression of IGFBP7 in aggressive human HCC cells resulted in modest inhibition compared to controls, profound inhibition of tumorigenesis by IGFBP7-overexpression was observed in a subcutaneous xenograft assay using athymic nude mice. Marked inhibition of angiogenesis and induction of senescence may cause the difference in the IGFBP7 in vitro effect. Replication-incompetent adenovirus-delivered IGFBP7 (Ad.IGFBP7) potently induces apoptosis in multiple HCC cell lines in vitro and profoundly inhibits primary tumor growth and metastasis in vivo [60].MicroRNAs (miRNAs) are small (21–23 nucleotide) RNA species whose role in both normal cellular functions and human diseases, particularly cancers, is well established [61]. A direct role for miRNAs in HCC drug resistance has been shown. Over-expression of miR-122, the liver-specific miRNA, in HCC cells, including Hep3B, HepG2 and Huh7, increased sensitivity to adriamycin and vincristine [62]. More generally, use of several drug resistant Huh7 HCC sublines identified an array of significant miRNA expression changes when compared to control Huh7 cells [63]. Among the miRNAs displaying significant differential expression were miR-146a/b and miR-181d. Both of these miRNA families have been shown in other cancers to influence chemoresistance [63].In addition to therapeutic resistance, a number of studies have implicated miRNAs in various aspects of HCC development and pathology. One of the most direct demonstrations of the role of miRNAs in HCC is related to the tight linkage of several miRNAs to the hyperactivation of the Myc oncogene that is characteristic of both HCC and the rare childhood hepatoblastoma (HB) [64]. MiRNAs miR-100, miR-125b, miR-26, miR-23a/b, miR-371 and let-7a are all validated Myc targets and all are important effectors of Myc-mediated oncogenesis in HCC. Hou et al. [65] examined the miRNAomes of both normal human liver tissues (n = 3) and hepatocellular carcinomas (n = 3) using next-generation deep sequencing. They found that the majority of miRNA expression in both samples could be accounted for by very few miRNAs. Among the most abundantly expressed miRNAs was the liver-specific miR-122 [66] and miR-192. Giordano and Columbano [67] note that miR-21 is the most over-expressed miRNA in HCC as compared with normal tissues.While the vast majority of miRNA studies in cancers have focused on the role of particular miRNAs, in particular cancers or cancer processes, the advent of The Cancer Genome Atlas project has made it possible to examine multiple biologic and genetic characteristics in numerous cancers simultaneously [68,69,70]. These, in turn, have allowed researchers to re-examine previous, more focused studies in a much broader context.We recently compared expression levels of 1,046 miRNAs in seven cancers using deep sequencing profiles of hundreds of tumors produced by The Cancer Genome Atlas (TCGA) project [71]. Expression levels were compared via Spearman Rank Order Correlation [72] and included uterine corpus adenocarcinoma (n = 415 patients), ovarian serous adenocarcinoma (n = 970 patients), breast adenocarcinoma (n = 770 patients), prostate adenocarcinoma (n = 286 patients), pancreatic adenocarcinoma (n = 62 patients), colorectal adenocarcinoma (n = 478 patients), and lung adenocarcinoma (n = 482 patients). The patterns we observed show that overall miRNA expression is very highly correlated among the cancers (mean ρ = 0.94 ± 0.03). We also observed that all of the most highly expressed miRNAs among these cancers are members of evolutionarily ancient miRNA families, the vast majority of which appeared in animal genomes prior to the emergence of land animals 380 million years ago.TCGA has produced similar miRNA expression data on 197 patients diagnosed with hepatocellular carcinoma (HCC). We have incorporated these data into our previous analyses and compared expression levels with those of uterine corpus adenocarcinoma, breast adenocarcinoma, prostate adenocarcinoma, pancreatic adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma. Rank order scatter plots and correlations are shown in Figure 2. Again, the Spearman Rank Order Correlations are very high (mean ρ = 0.95 ± 0.01) and all are statistically significant (p < 0.0001).Scatter plots of the expression rank of 1,046 miRNAs in hepatocellular carcinoma versus six adenocarcinomas with the Spearman Rank Correlation for each. All correlations are significant (p < 0.001).Moreover, the previously seen pattern of domination by ancient miRNA families is obtained in HCC as well. Budhu et al. [73] identified 19 miRNAs related to metastasis in HCC. Consistent with our prior observation, only one of these 19 miRNAs, miR-148, is a member of a miRNA family whose emergence in animal genomes occurred more recently than 380 million years ago.In TCGA HCC data, miR-21 is the most highly expressed miRNA and it ranks either first or second in each of the other tumors as well. Indeed, miR-21 has been called the most dysregulated miRNA in cancer and noted the many biological processes, both normal and pathologic, in which miR-21 plays a major role [74]. In the HCC TCGA data, both miR-122 and miR-192 were very highly expressed. In comparison with data from the six other cancers, miR-192 was also highly expressed whereas, not surprisingly, miR-122 was barely detected. Interestingly, among the most highly expressed miRNAs in other tumors are the five members of the miR-200 family (miR-200a,b,c; miR-141 and miR-429). In the HCC TCGA data, members of the miR-200 family are barely expressed. These miRNAs are involved in the crucial epithelial to mesenchymal transition and are routinely over-expressed in adenocarcinomas [75]. HCC is not an adenocarcinoma and its etiology is tied to exposure to certain viruses such as hepatitis B (HBV) and hepatitis C (HCV) as well as other liver-specific pathologies such as cirrhosis. Takahashi et al. [76] note that miR-200a is down-regulated in chronic liver tissue injury, including HCV infection. Thus, the very different expression pattern of the miR-200 family members in HCC compared to other cancers is related to the difference in the route of carcinogenesis in HCC. Other miRNAs displaying HCC-specific expression in the TCGA rankings are miR-194-1,2, miR-217, and miR-885. It has been shown in mice that miR-217 levels are dramatically increased in liver cells in response to chronic ethanol exposure and that such increases result in excess fat accumulation [77]. Chronic alcohol exposure in humans leads to cirrhosis, a well-established HCC precursor. MiR-194 is a marker of hepatic epithelial cells but over-expression in liver cancer cells in mice inhibits metastases [78]. Finally, miR-885 has been reported to activate the tumor suppressor p53 and to inhibit proliferation and survival [79]. Thus, it is not surprising to see that this miRNA exhibits quite low expression in adenocarcinoma TCGA ranks. That it is substantially more highly expressed in HCC by comparison is puzzling since the rate of p53 mutation in HCC is very low except in areas of the world where aflatoxin-contaminated foods are routinely consumed [80]. However, Gui et al. [81] identified significantly high levels of circulating miR-885 to be characteristic of both cirrhosis and HCC. Thus, miR-885 may well be important to a liver-specific pathologic process independent of p53. In summary, the mechanisms of the drug resistance in HCC are complicated and remain unclear. Genetic heterogeneity may be the primary resistance mechanism of HCC to chemotherapy. Multiple mechanisms can induce the overexpression of FOXM1 and MTDH, two pivotal master regulators involved in drug resistance and metastasis. As shown in Figure 1C, there are several positive feedback loops between the activation of FOXM1 and MTDH. First, MTDH can activate the PI3K/AKT pathway, suppressing FOXO3a mediated transcription repression to FOXM1 and activating c-myc [82,83]. MTDH may also activate FOXM1 by interacting with NPM1 [31,38]. Second, MTDH can release the transcription repression to c-myc by interacting with the promyelocytic leukemia zinc finger protein (PLZF) [45]. C-myc can directly increase MTDH transcription and inhibits the suppression by miR-26a [84]. Third, FOXM1 can directly regulate c-myc transcription and c-myc can release the suppression of FOXM1 by miR-134 and miR-149 [19,84]. Chemotherapy can also simultaneously activate the addiction switches and compensatory pathways, such as FOXM1 protein stability induction by CHK2, leading to acquired resistance and metastasis [85,86]. Further investigation of the feedback loops of FOXM1, MTDH, c-myc and microRNAs will better our understanding of the mechanisms of drug resistance in HCC and inform strategies for mitigating it. This work was partially supported by NIH Grant R01CA99908 to Kimberly K. Leslie, the Department of Obstetrics and Gynecology Research Development Fund, and Institutional Research Grant no. IRG-77-004-31 from the American Cancer Society to Shujie Yang, administered through the Holden Comprehensive Cancer Center at the University of Iowa.X.M., and S.Y. wrote part 1 to 7, E.J.D and B.M.S wrote part 8, X.M.,E.J.D and K.K.L edited the paper. All authors read and approved the final manuscript. The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Hepatocellular carcinoma (HCC) is an aggressive disease with poor prognosis due to its high rate of recurrence after the initial curative treatment. Therefore, development of effective therapeutic strategies that can prevent recurrence and secondary tumor formation is required to improve the clinical outcomes of HCC patients. Malfunctioning of the retinoid X receptor-s (RXRs) of HCC patient by activation of the Ras- mitogen-activated protein kinase (MAPK) signaling pathway is strongly associated with hepatocarcinogenesis. Acyclic retinoid (ACR), a synthetic retinoid, prevents HCC recurrence by inhibiting Ras-MAPK activation and the subsequent RXRα phosphorylation, thereby improving patient prognosis. Here, we have reviewed the detailed effects of ACR on the prevention of HCC development, with particular references to the results of our previous basic and clinical research.Liver cancer is the sixth most common neoplasm and the third leading cause of cancer-related deaths in the world. Hepatocellular carcinoma (HCC) accounts for more than 90% of primary liver cancers and is therefore a major health problem worldwide. HCC primarily develops from chronic liver inflammation and subsequent cirrhosis. In Western countries, HCC occurs in a cirrhotic background in up to 90% of the cases [1]. In addition, cirrhosis has been well-established as a major risk factor for HCC, independent of any underlying liver disease [2].Currently, the surveillance for patients at high risk of HCC has increased the possibility of early diagnosis [1]; however, the overall patient survival rate continues to remain poor (7%, 5-year survival) [3]. Potentially curative therapies, including surgical resection and percutaneous ablation, can provide long-term control in patients with early stage HCC [4,5,6]. However, the 5-year recurrence rates of HCC following these curative treatments exceed 70%, due to intrahepatic metastases (true recurrence) or the development of de novo tumors [2]. In addition, systemic therapies such as those using standard chemotherapeutic agents hvae not had significant effects on HCC in previous randomized trials [7]. Thus, the lack of effective treatments is also one of the main reasons for the poor prognosis of HCC patients, especially with advanced-stage HCC. Therefore, the development of preventative strategies is essential to improve the clinical outcomes for HCC patients.Several effective strategies for the prevention of development of primary HCC have been presented in clinical trials [8,9,10,11,12,13,14,15,16,17]. It is widely known that chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) causes liver cirrhosis, with both infections together accounting for 75%–80% of the global HCC cases [8]. Primary prevention in the form of HBV vaccination has led to a significant decrease in the number of HBV-related HCC cases [9,10], and antiviral treatment for chronic HBV and HCV infections has reduced the risk of HBV- and HCV-related HCC [11,12,13,14]. Thus, antiviral treatment is a promising approach for the prevention of HCC.In addition to the studies on antiviral therapies, other important trials using specific agents have been conducted to explore the methods of preventing HCC development. One clinical trial demonstrated that long-term oral supplementation with branched-chain amino acids (BCAA) reduced the frequency of HCC in obese cirrhotic patients [14]. In our randomized controlled study, we found that oral intake of acyclic retinoid (ACR) significantly prevented the development of second primary liver cancer after the initial treatment [15], thereby improving the patient survival [16]. Moreover, a long-term follow-up of the study subjects revealed that 1-year administration of ACR was effective in suppressing secondary liver cancer for up to 3 years [17]. Thus, chemoprevention is one of the key strategies for preventing liver carcinogenesis.Cancer chemoprevention is defined as an approach in which a natural or synthetic chemical compound regulates premalignant cells via physiological pathways [18]. ACR is a synthetic retinoid developed for the purpose of chemoprevention of HCC [19]. Several experimental studies have reported pleiotropic effects of ACR on either the prevention of HCC development or on the growth suppression of cancer cells [20,21,22]. In this review, we have summarized the important roles of ACR in preventing the development of HCC based on previous basic and clinical studies and the relevant recent literature. We have also discussed the possibility of “combination chemoprevention” using ACR as the key drug, which may be a potential preventive treatment against HCC.Retinoids, which are derivatives of vitamin A, are physiological signaling molecules involved in the regulation of cell growth, tissue differentiation, and development of an organism [23,24]. Retinoic acids (RAs) are active metabolites of natural retinoids; they exert their biological functions by regulating the transcription of target genes through two distinct nuclear receptors—RA receptors (RARs) and retinoid X receptors (RXRs). Two isomers of RA, all-trans-RA and 9-cis-RA, have similar binding affinities for RARs, whereas only 9-cis-RA binds to the other RXR nuclear receptors. Both types of nuclear receptors consist of three subtypes (α, β, and γ), characterized by a modular domain structure [23,25]. Different RXRs demonstrate different expression patterns: RXRα is predominantly expressed in the liver, kidney, epidermis, and intestine [26,27,28], RXRβ is ubiquitously distributed and can be detected in almost every tissue [26,28,29,30], while RXRγ is primarily limited to the muscle, certain parts of the brain, and to the pituitary gland [26,28,31,32].Similar to other members of the nuclear receptor superfamily, the nuclear retinoid receptors regulate target gene transcription in a ligand-dependent manner. After binding with the ligand, RXRs form homodimers or heterodimers with other RARs and then interact with their respective DNA-response elements (RXRE or RARE) located in the promoter region of the target genes to modulate gene expression [23,25,33]. In addition, RXRs are cofactors required for transcriptional activation by several members of the steroid/thyroid hormone nuclear receptor superfamily such as thyroid hormone receptors (TRs), vitamin D receptors (VDRs), and peroxisome proliferator-activated receptors (PPARs) [24,34]. Moreover, interactions with RXRs enhances the DNA-binding efficiency of the partner molecule [33]. Thus, RXRs function as master regulators of nuclear receptors.Several of the nuclear receptors that form heterodimers with RXRs are implicated as important regulators of genes involved in the liver metabolism [35]. RXRα is the most abundant subtype of RXR in the adult liver [28]. Therefore, it is believed that RXRα plays a prominent role in the regulation of hepatic metabolism. Indeed, RXRα is an obligate heterodimeric partner for nuclear receptors involved in lipid physiology, such as PPARs, liver X receptors (LXRs), and farnesoid X receptors (FXRs) [36,37]. In addition, studies using liver-specific RXRα-deficient mice have revealed that the absence of RXRα reduces the activation of its dimeric partners and results in the impairment of fatty acid and cholesterol metabolism in the liver [35,38,39]. Thus, RXRα and its dimeric receptors are involved in the mediation of normal hepatic lipid metabolism.Retinoids and their receptors play important roles in the regulation of normal cell proliferation, differentiation, and apoptosis [40]; therefore, impaired expression or function of these molecules is strongly associated with the development of various human malignancies such as HCC. Indeed, surgically resected HCC tissues contain low levels of vitamin A [41,42]. In a rodent model, 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB)-induced liver tumors had lower levels of retinol than the surrounding noncancerous liver tissues [19]. Notably, a marked reduction in the levels of both retinol and retinyl ester were observed even at the precancerous, hyperplastic liver nodule stage [42]. In addition, the expression levels of RXRα, which is the most abundant retinoid in normal liver tissues, were also reduced, not only in HCC specimens but also in some precancerous lesions obtained from a 3'-MeDAB-induced rat liver carcinogenesis model [43]. In contrast, increased retinoid signaling of lecithin:retinol acyltransferase-deficient mice suppressed diethylnitrosamine (DEN)-induced hepatocarcinogenesis [44]. Thus, these findings indicate that the expression levels of retinoids or their nuclear receptors, especially RXRα, are closely related to the development of HCC.Dysfunction of nuclear retinoid receptors is also associated with hepatocarcinogenesis. Our studies showed that the malfunction of RXRα due to post-translational modification by phosphorylation is associated with HCC development [45,46]. It has also been reported that phospho-modification of the nuclear receptors enhances or reduces their transcriptional activity in a context-dependent manner [47]. For instance, phosphorylation of RXRα that occurs in its N-terminal A region induces the expression of several RA-responsive genes and results in RA-induced endodermal differentiation [48]. In contrast, a mitogen-activated protein kinase (MAPK)-mediated phosphorylation of RXRα within its ligand-binding domain impairs the transcriptional activity of RAR-RXR [49] and VDR-RXR heterodimers [50]. Notably, correlation of impaired receptor functions due to phospho-modification and therapy-resistant phenotypes has been reported in several human malignancies [47].In HCC, Ras-extracellular signal-regulated kinase 1/2 (ERK 1/2) is highly activated, and the phosphorylation of RXRα occurs at both serine 260 and threonine 82, which are recognized as the consensus sites for phosphorylation via the Ras/MAPK/ERK signaling pathways [46]. We found that phosphorylated RXRα is highly accumulated in HCC tissues as well as in HCC cell lines, which prevents its normal degradation through the ubiquitin-proteasome pathway [51]. The accumulated phosphorylated RXRα (non-functional RXRα) abrogates the function of the remaining normal RXRα in a dominant-negative manner, thereby inhibiting the formation of heterodimers with the partner molecules such as RARβ [52]. RARβ has been suggested to be a tumor suppressor gene [53,54,55]. Therefore, impaired RARβ function due to the accumulation of non-functional RXRα may promote the development of HCC. In addition, we have reported in our previous study that phosphorylated RXRα is refractory to its potent ligand, 9cRA, and evades 9cRA-induced apoptosis [56]. These observations suggest that not only the depletion of retinoids but also the dysfunction of retinoid receptors, especially phospho-modification of RXRα, plays a critical role in the development of HCC (Figure 1).ACR (equivalent to NIK-333 and Peretinoin; Kowa Pharmaceutical Co., Tokyo, Japan) is a synthetic retinoid developed for chemoprevention of HCC [19]. The chemopreventive effects of ACR have been reported in our previous studies [19,57,58,59,60]. In human HCC cell lines, ACR induces apoptosis and inhibits cell proliferation by inducing cell differentiation or by regulating cell-cycle progression [56,61,62,63,64]. We have found that ACR functions as an agonist for both RAR and RXR and activates RARE and RXRE in hepatoma cells [61,65]. Indeed, increased expression levels of RA-target genes such as RARβ and p21 were observed in ACR-treated HCC cell lines [56,62,63,64,66,67,68,69,70,71]. Thus, ACR inhibits the development of HCC by acting as an effective ligand for nuclear retinoid receptors.The role of phosphorylated retinoid X receptor (RXR)α in hepatocellular carcinoma (HCC) development. In normal hepatocytes, while all-trans-RA binds only to retinoic acids receptors (RARs), 9-cis-RA binds to both RARs and RXRs as a ligand (a). After activation by their specific ligands, the nuclear receptors bind to their specific response elements and regulate cell proliferation, differentiation, and apoptosis through induction of their target gene expressions (b). Nuclear receptors (RARs and RXRs) cease functioning owing to their degradation through the ubiquitin-proteasome pathway (c). In HCC cells, RXRα is phosphorylated by the Ras/ mitogen-activated protein kinase (MAPK)/Erk pathways, which are constitutively activated in HCC (d), and accumulates in the nucleus, preventing degradation through the ubiquitin-proteasome pathway (e). The phosphorylated RXRα inhibits normal RXRα function in a dominant-negative manner (f) and shows refractoriness to 9-cis-RA (g), thus impairing normal RXRα function (h). The impaired receptor function results in the down-regulation of its target gene expression and leads to hepatocarcinogenesis.In addition to its role as a ligand for nuclear retinoid receptors, ACR restores the impaired receptor functions of RXRα by inhibiting RXRα phosphorylation. In HCC, the Ras/MAPK/ERK signaling pathways are highly activated and involved in inducing constitutive phosphorylation within the consensus sites of RXRα [46]. We have reported that ACR inhibits the activated Ras-Erk 1/2 pathways independent of RXRα and consequently prevents phospho-modification of RXRα, thereby restoring the function of RXRα in HCC cells [66]. Moreover, our recent study has revealed that ACR inhibits not only Ras-Erk 1/2 pathways but also several types of growth factors and their corresponding receptor tyrosine kinases (RTKs) in several malignancies, including HCC [58,59,62,72,73,74]. RTKs transmit signals that regulate cell proliferation, differentiation, and survival. The MAPK cascade comprised of Ras-Erk kinases is an essential effector cascade required for most RTK functions [75]. Therefore, the inhibitory effect of ACR on RTKs suppresses the development of HCC via inhibition of the downstream Ras-Erk 1/2 pathways and the subsequent RXRα phosphorylation, in addition to its direct effect on the Ras-Erk 1/2 pathways. Thus, ACR functions not only as a ligand for RXRα but also as a suppressor of the RTK-Ras-MAPK signaling pathways, thereby restoring the function of RXRα and activating the transcriptional activity of its response element.Recently, a new target of ACR was elucidated in an in vitro study by using Huh-7.5 cells infected with HCV-RNA [76]. This study revealed that ACR inhibits HCV-RNA replication and infectious virus release by modulating several aspects of lipid metabolism, such as triglyceride abundance and the expression of mature sterol regulatory element-binding protein 1c (SREBP1c). Considering that HCV infection is a major cause of HCC development, the inhibitory effect of ACR on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-positive patients (Figure 2).The role of acyclic retinoid in the prevention of HCC. Acyclic retinoid (ACR) itself functions as a ligand for RXRα and regulates expression of its downstream genes such as p21, RARβ, and Cyclin D1, thus preventing HCC development through induction of cell proliferation, differentiation, and apoptosis in HCC cells (a). ACR inhibits the activated Ras/MAPK/Erk pathway independent of RXRα (b). ACR also inhibits several types of growth factors and their corresponding receptor tyrosine kinases (RTKs) (c). Thus, ACR restores the impaired RXRα function. Recently, new possible targets of ACR have been reported in either in vivo or in vitro studies [76,77,78] (d). Thus, the pleiotropic responses of ACR target molecules, including phosphorylated RXRα, may play a role in preventing hepatocarcinogenesis.A major concern in HCC patients is the high rate of recurrence and de novo tumors following the initial curative treatments [2], which results in poor prognosis of the malignancy [79]. We have discussed the inhibitory effects of ACR in the development of HCC in our basic studies [19,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74]. In order to further investigate the chemopreventive effects of ACR on the recurrent and secondary HCC clinically, a double-blind and placebo-controlled clinical study was performed on patients who had received anticancer treatment for initial HCC [15,16,17]. Oral administration of ACR (n = 44 patients; dose = 600 mg/day) for 12 months significantly reduced the incidence of post-therapeutic recurrence or new HCC development compared to administration of placebo (n = 45 patients) (median follow-up time = 38 months; P = 0.04) [15]. After a median follow-up time of 62 months, ACR administration improved both the recurrence-free survival (P = 0.002) and overall survival (P = 0.04) rates [16]. Moreover, the preventive effects of ACR lasted for up to 3 years following the completion of ACR administration [17]. Thus, a short-term administration of ACR for only 12 months yielded a long-term effect on the prevention of secondary HCC, without causing any severe adverse effects.A safety-evaluation study was conducted in a Phase I pharmacokinetics clinical trial to determine the dose-limiting toxicities and pharmacokinetics of ACR [80]. In this trial, no adverse effects or dose-limiting toxicities were observed in either of the groups administered with doses of 300 or 600 mg/day, although a dose-limiting toxicity of Grade 3 hypertension was observed in the group receiving a dose of 900 mg/day [80].Based on the Phase I clinical study of ACR [80], a Phase II/III clinical multicenter, large-scale, randomized, placebo-controlled study (n = 401) was conducted to evaluate the effectiveness of ACR on the prevention of secondary HCC in HCV patients who had received curative treatment for initial HCC [81]. In this study, oral administration of ACR (n = 124 patients, dose = 600 mg/day) showed a reducing trend of incidence of secondary HCC as compared with placebo administration (n = 127 patients), although no significant differences were observed between the two groups at a median follow-up time of 2.5 years. However, at 3 years after treatment, the cumulative recurrence-free survival rate of the ACR-treated group (43.7%) was higher than that of the placebo group (29.3%). Notably, subgroup analysis of this data showed that ACR (n = 100 patients; dose = 600 mg/day) reduced the risk of HCC recurrence or death by approximately 40% as compared to placebo (n = 106 patients), especially in patients with Child-Pugh A and small tumors (size < 20 mm) (p = 0.0347). On the other hand, a 300 mg/day dose of ACR was insufficient for tumor control, showing no substantial difference as compared to placebo. Thus, these studies show that ACR administration to cirrhotic patients effectively inhibits the development of secondary HCC and thereby improves the clinical outcome of the patients. Especially, the inhibitory effects of ACR on secondary HCC were observed in patients with well-preserved liver function (Child-Pugh A) [81]. To this effect, currently, a confirmatory large-scale ACR study focused on Child-Pugh A patients is ongoing.The inhibitory effects of ACR on the development of secondary HCC have been reported by several clinical studies [15,16,17,81]; however, not much information is available on the mechanisms by which ACR inhibits the secondary HCC in humans in vivo. In response, Honda et al. [77] conducted a gene expression profile analysis under the same study conditions as in previous clinical studies and found that ACR treatment down-regulated the expression of platelet-derived growth factor C (PDGFC), other angiogenesis genes, and cancer stem cell marker genes, thereby successfully preventing HCC recurrence in humans in vivo [77]. This result not only supports the previous clinical data but also suggests the possibility of new targets for ACR for the prevention of HCC recurrence (Figure 2).“Clonal deletion” is a concept in which latent malignant or premalignant cells that are undetected by diagnostic images are removed from the organ in a hyper-carcinogenic state. A typical example of this can be seen in a cirrhosis-HCC sequence. The cirrhotic liver is recognized as a major risk factor for HCC due to a high incidence of tumors in the pathological background [2]. In particular, chronic hepatitis or cirrhosis induced by either HBV or HCV frequently results in the accumulation of DNA mutations across the liver by repeated necrosis and hepatocyte regeneration, and leads to a hyper-carcinogenic state. In cirrhotic patients with HCC, a high incidence of recurrence or de novo tumors is observed after the initial curative treatment [2], which may be explained by the characteristic clinical mode of liver carcinogenesis — “multicentric carcinogenesis” (or “field cancerization”) [82]. Thus, once the liver is exposed to continuous carcinogenic insults such as hepatitis virus infection, the whole liver is regarded as a precancerous field possessing multiple, independent, and premalignant or latent malignant clones.As reported by some previous studies [15,16,17], ACR effectively inhibits secondary HCC development after initial curative treatment and significantly improves the clinical outcome of HCC patients. These findings suggest that ACR may delete and/or inhibit the “clones of secondary HCC” from the hyper-carcinogenic liver. Indeed, ACR significantly reduced the serum levels of lectin-reactive alpha-fetoprotein factor 3 (AFP-L3) and protein induced by vitamin K absence/antagonist-II (PIVKA-II), both of which indicate the presence of latent HCC cells in the remnant liver [17,83]. This finding suggests that ACR eliminates malignant clones producing AFP-L3 or PIVKA-II before the clones expand to become clinically detectable tumors. Once such latent clones are eliminated from the remnant liver, it takes several years for the secondary HCC to be recognized clinically [17]. In fact, the inhibitory effects of ACR on the development of secondary HCC lasted for about 3 years after termination of its administration [17]. In addition, Zheng et al. [84] recently reported that ACR treatment decreased the emergence of precancerous cells and their progeny in a rat liver carcinogenesis model, consequently leading to suppression of HCC development. This experimental data sufficiently supports the previous clinical reports and the concept of “clonal deletion” [15,16,17]. Thus, we hereby suggest the concept of “clonal deletion” as a new concept in the chemoprevention of HCC using ACR, and that this chemopreventive approach may become a viable cancer therapy for eliminating malignant clones (Figure 3).The concept of “clonal deletion” and the therapeutic application of ACR on the basis of “clonal deletion.” Continuous exposure to genetic insults such as chronic- hepatitis B virus (HBV) and - hepatitis C virus (HCV) infections induce DNA mutation across the whole liver (a). The multiple premalignant and latent malignant clones occur on the background of this “precancerous field.” Primary HCCs arise from these latent clones in cirrhotic patients (b). After curative treatments, while secondary/de novo HCC development is observed at early time points during the natural history (multicentric carcinogenesis) (c), ACR deletes these latent premalignant clones from the remnant liver by restoring impaired RXRα function and inducing cell differentiation and apoptosis (d). This is the concept of “clonal deletion.” Since lectin-reactive alpha-fetoprotein factor 3 (AFP-L3) and protein induced by vitamin K absence/antagonist-II (PIVKA-II) indicate the presence of latent HCC cells in the remnant liver, reduced levels of these markers observed in ACR treatment sufficiently support this concept.The combined use of two or more agents not only yields the synergistic effects of each agent but also decreases the overall toxicity by enabling treatments with lower clinical dosages [85,86]. Therefore, it is expected that combinatorial treatment of HCC, ACR, and other clinical agents may exert synergistic effects against HCC development. In order to explore this possibility, we conducted studies of “combination chemoprevention” using ACR as the key agent [87,88,89,90]. The combination of ACR and interferon-β (IFN-β) synergistically inhibited cell growth and induced apoptosis in HCC cell lines by inducing the expression of type 1 IFN receptor and STAT1, located downstream of RXRα [68]. In addition, the combinatorial treatment of HCC cells with ACR and OSI-461, a potent derivative of sulindac sulfone, elicited strong synergistic expression levels of RARβ and p21, which are associated with the transcriptional activation of RARE, thereby inducing apoptosis of HCC cells [69]. In addition, the combined use of ACR and vitamin K2 (VK2) synergistically induced apoptosis and inhibited the growth of HCC cells by preventing RXRα phosphorylation through inhibition of the Ras/MAPK/Erk signaling pathway [67]. Thus, the combination of agents with different target sites and action mechanisms enables exertion of the pleiotropic and synergistic inhibitory effects on HCC development and growth of HCC cells. Moreover, if these combination treatments facilitate reduction of the dosage of agents for effective chemoprevention of HCC, the risk of adverse effects and toxicity of the agent would also be reduced. Sorafenib, an oral multi-tyrosine kinase inhibitor, is the first and the only drug that has demonstrated survival benefits in patients with advanced HCC [1]. While improved overall survival has been reported in sorafenib-treated HCC patients, the associated toxicities such as hand-foot skin reaction significantly affect the patients’ quality of life and, occasionally, cause early termination of the treatment [91]. We reported earlier that the combined use of ACR and trastuzumab (the humanized anti-HER2 monoclonal antibody) synergistically inhibited the activation of HER2 and its downstream signaling pathways, including RXRα phosphorylation, and subsequently inhibited the growth of HCC cells [70]. This finding suggests that ACR may be a practical candidate in combination therapy with other RTK antagonists. Considering that sorafenib partially targets the Ras/MAPK/Erk signaling pathway also, the combination of ACR and sorafenib are expected to reduce the probability of adverse events, providing beneficial inhibitory effects against HCC development. Thus, ACR may become a potential candidate for use in a combination therapy with sorafenib against advanced HCC.Poor clinical outcomes of HCC patients primarily result from the high incidence of secondary HCC following the initial curative treatment, proving to be a major concern in the treatment of this malignancy. Therefore, the establishment of a new effective strategy to prevent the recurrence of HCC has been recognized as an urgent task worldwide. Accordingly, ACR was originally developed as a chemoprevention agent to accomplish this purpose [19,80] and was administered to patients who had achieved complete cure of HCC in clinical trials [15,16,17,77,80,81]. The preventative effects of ACR on the development of secondary HCC suggest that ACR may play a role beyond that of a “chemopreventive drug”, as a “cancer therapy drug” that actively eliminates latent malignant lesions from the cirrhotic, hyper-carcinogenic liver. This novel therapeutic concept of “clonal deletion” using ACR may be a promising approach in the prevention of HCC.Our previous reports have shown that ACR exerts its chemopreventive effects by working as a ligand for retinoid receptors as well as by restoring impaired RXRα function through inhibition of Ras/MAPK/Erk activation [61,65,66]. Indeed, restored function of RXRα (unphosphorylated RXRα) regulates the expressions of its downstream genes such as p21, RARβ, and Cyclin D1 and inducts the apoptosis and cell-cycle arrest of HCC cells [64]. In contrast, cells transfected with phospho mimic-RXRα cDNA showed refractoriness to retinoid treatment and association with the acquisition of malignant phenotype [46,52]. These results suggest that phosphorylated RXRα plays an important role in hepatocarcinogenesis. Currently, no clinical agents that can directly target phosphorylated RXRα for the prevention of HCC are commercially available. However, considering its positive role in HCC development, the establishment of a new strategy directly targeting phosphorylated RXRα is highly intriguing.Although previous evidence suggests that phosphorylated RXRα is a target of ACR, the molecular mechanism by which ACR prevents HCC development is not fully understood [78]. Indeed, recent studies have revealed several targets for ACR that can contribute to the chemoprevention of HCC either in vivo or in vitro [76,77,78] (Figure 2). These reports suggest that the preventative effects of ACR on HCC development may result from the pleiotropic response of ACR target molecules, including phosphorylated RXRα. Further detailed studies are required to fully explore the molecular mechanisms of ACR.In conclusion, the accumulating evidence discussed in this review suggests that retinoid compounds, especially ACR, may be promising candidates for the chemoprevention of HCC. A Phase III, large-scale, randomized controlled trial on ACR is ongoing to this effect. In addition, ACR-based chemoprevention in combination with other agents may become a promising strategy for HCC chemoprevention.This work was supported by 1) Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan (No. 22790638 and 25460988), 2) Grant-in-Aid for the 3rd Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare of Japan, and 3) Takeda Science Foundation.H. Sakai, M. Shimizu, and H. Moriwaki all participated in designing, writing and editing of the review.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Pulmonary hypertension (PH) has been found to have significant morbidity and mortality. The treatment of PH has advanced considerably with increasingly more effective and safer options. With an increasing effort to diagnose patients early, non-invasive techniques are often used to screen those patients likely to have PH. Computerized tomography (CT) chest scans are increasingly utilized in the evaluation of patients with exertional dyspnea, including those with suspected PH. The main role of the CT scan is to evaluate for any associated underlying diseases. There have been attempts to address the utility of CT to predict the presence of PH. This article reviews previously published investigations to summarize the relationship between pulmonary artery dimensions and PH to determine both the strength of the correlation and its discriminatory ability for use in clinical practice.Although there have been significant advances in the treatment of pulmonary hypertension (PH), there remains significant morbidity and mortality [1,2,3]. With increasingly more effective and safer pharmacological therapy for pulmonary arterial hypertension (PAH), outcomes may be improved by earlier detection of PH [3]. Screening algorithms have been proposed to facilitate the timely and accurate diagnosis of PH, utilizing a combination of echocardiographic, physiologic (lung function), and radiologic non-invasive techniques [4,5], before proceeding to a definitive right heart catheterization (RHC) for confirmation.Computed tomography (CT) chest scans have largely supplanted chest x-rays in patients with PH, partly due to its ability to detect thromboembolism in some cases, but also to identify any diffuse parenchymal lung diseases that may not be evident in 15% of chest x-rays [6,7]. With advances in CT technology and its wide availability, there have been attempts to address the utility of CT to predict the presence of PH.The pulmonary artery (PA) is a more compliant vessel than the systemic arterial system, and is thus more sensitive to changes in pressure and volume. As a result, an increase in mean pulmonary arterial pressure (MPAP) should correlate with pulmonary artery diameter. A variety of PA dimensions have been explored to see if there is any association with both the presence and severity of PH, including the PA diameter, the cross-sectional area, the ratio of the diameter to the bronchus, the ratio of the diameter to the pulmonary vein, the ratio of diameter to the aortic diameter, and multiple regression methods assessing dimension of the main and branching pulmonary arteries [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]. Based on such observations, we have found radiologists formally reporting on the PA size and suggesting the presence or absence of PH, which often has led to changes in clinical behavior without a clear justification otherwise.In this review, we have compiled, for the first time, all published investigations exploring the relationship between PA dimensions and PH to determine both the strength of the correlation and whether it has adequate discriminatory ability for use in clinical practice. Extrapolating from our own series, we hypothesized that, although the PA size should correlate with PA pressures, its discriminatory ability is poor and over utilized clinically.An OVID Medline literature search was used to identify all English and human studies relating PA size as measured on a CT of the chest in patients with PH confirmed by RHC (or in some series as suggested on echocardiography) between 1991 and 2014. PA dimensions are expressed in millimeters (mm) with their standard deviation (±SD). In the brief report of our series, consecutive patients referred to the PH clinic were identified between 2006 and 2010. CT measures were blinded to clinical history and were done by three separate reviewers.The pulmonary circulation is a high flow, low pressure system. It has a lower resistance and is more compliant than the systemic circulation, owing to the abundance of vasculature in parallel and a lower transpulmonary pressure gradient. The factors that determine the size of a vessel depend on several physiologic variables and any underlying pathology. Fundamentally, it is the volume of blood within the PA that relates to the size of the PA as measured on the CT, with the indirect factors of pressure and vessel compliance contributing. From this, it is easy to understand how PA size will correlate with PA pressures, but with an often incorrect assumption of constant compliance and blood volume. Practically, we can measure the MPAP, cardiac output, PA occlusion pressure (as a surrogate for left ventricular end-diastolic pressure) from RHC and derive secondarily, the pulmonary vascular resistance [19,20]. Additionally, there are variations due to anthropomorphic factors that can be considered such as age, gender, height, and body surface area (BSA) [20,33]. Pathologic factors within the vessel itself may include atherosclerosis, endothelial proliferation, and occlusion from thrombi [20]. In idiopathic PAH (i.e., diagnostic group 1.1 pulmonary arterial hypertension), the peripheral pulmonary vessels are characterized by the degeneration of the elastic lamina with replacement by fibrous tissue, along with intimal proliferation and hypertrophy of the muscular layer in the media of the arteries [34,35,36]. This downstream occlusion with subsequent increase in resistance is what should lead to proximal increase in the main PA pressure and size. The conditions that cause mechanical changes to the mediastinal vasculature by compression, traction, or shifting, as well as pathologic changes in the heart or lungs, such as from congenital heart disease, prior surgeries, or radiation, can cause additional anatomic distortion. Despite limited investigations that address this [20,37,38,39], we suspect that technical issues in the imaging of the PA (e.g., body position, different acquisition protocols and reconstruction algorithms, depth of inspiration, the use of contrast, and reduced intra- and inter-rater reliability) may also affect the accurate measure of the PA size and further confound the relationship between the PA size and PA pressures. Although logical deduction would indicate that the PA size should correlate nicely with PH and PH severity, there are a multitude of factors that may potentially make the measurement unreliable or inaccurate. Lastly, the reader should appreciate that “PH” is simply a hemodynamic condition defined by a MPAP ≥ 25 mmHg at rest as documented by RHC; therefore, is more a pathophysiologic state than a specific disease or diagnosis.Many studies have tried to determine the normal range of the main PA size. Typically, the transverse axial diameter of the main PA at the level of its bifurcation is measured. This landmark is easy to define anatomically and is highly reproducible [20], and at this same level, the ascending aorta can also be measured to calculate the ratio of main PA to the aortic diameter (PA/Ao) (Figure 1), as a means of “normalizing” for differences in anthropomorphic factors. Several published studies provide normative data. Edwards et al. reported that the mean PA size, as measured on a non-contrast CT, was 27.2 mm (SD 0.6) in 100 study participants with no history of cardiopulmonary disease; however, no RHC was performed [15]. Karazincir et al. reported a mean diameter of the main PA of 26.6 mm (SD 2.9) in a cohort of 112 patients who had no evidence of pulmonary disease and documented normal PA pressures by echocardiogram (MPAP ≤ 25 mmHg) [40]. The Karazincir study was performed as clinically indicated rather than by research protocol; therefore, the mean PA diameter may be falsely elevated. Kuriyama et al. reported a somewhat smaller mean PA size of 24.2 mm (SD 2.2) [17]. The possible discrepancy may be due to the differences in CT techniques and race. The majority of Kuriyama et al.’s study participants were of Japanese origin, while Edward et al.’s participants were Caucasian. It is important to note that overlapping hemodynamic definitions of PH in the various studies may confound the results. Indeed, the Karizincir study uses a definition that actually includes PH, i.e., a MPAP of 25 mmHg. In addition, a normal MPAP is less than 20 mmHg as suggested by RHC data from 1,184 healthy individuals that reported a normal MPAP of 14 mmHg (SD 3.3) [41].Measurements of main pulmonary artery and ascending aorta at the level of bifurcation. The main pulmonary artery (PA) size is typically taken at the level of the bifurcation of the main pulmonary artery perpendicular to the vessel wall. The aortic dimension of the ascending aorta is taken at the same level to calculate the PA to the aortic diameter (PA/Ao) ratio. The diameter is determined using the internal diameter in the contrast-enhanced image.There are also reported gender differences, with men having a slightly higher average PA size of 27.0 mm (SD 2.8) versus 25.9 mm (SD 3.0) in women (P = 0.048). However, it appears the gender differences could largely be accounted for by differences in the BSA between men and women [20,42] for the main PA size, with the exception of the right PA. Similarly, gender differences in the size of the aorta could also be explained by adjusting for the BSA, but notably not the ascending aorta [42].The largest population cohort in which PA dimensions were assessed by CT comes from the Framingham Heart Study of 3171 participants (mean age 51 years, 51% men), of whom 706 were identified as asymptomatic without any cardiopulmonary risk factors. The main PA size in this reference subset was 24.7 mm (SD 2.7) and the PA/Ao was 0.80 (SD 0.09) [33]. Interestingly, there was a weak inverse correlation between age and the main PA size in men (r = −0.11, p = 0.04), although for the entire cohort, the correlation was direct (r = 0.10, p < 0.0001). Height also weakly correlated with the main PA size (men: r = 0.18, P < 0.0001; women r = 0.24, p < 0.0001), but was stronger again for the BSA (men: r = 0.41, p < 0.0001; women: r = 0.42; p < 0.0001). Using the subset of asymptomatic study participants without cardiopulmonary risk factors, they established a 90th percentile gender-specific cutoff value for main PA of 29 mm for men and 27 mm for women [33].Numerous studies have investigated the correlation between CT measurements of the PA and the presence and severity of PH (Table 1 and Table 2). Overall, the measurement of the main PA size by using CT shows a moderate to strong correlation with PH (r ~ 0.4–0.7).Studies of CT measurements in patients with PH.AUC: area under curve; BSA: body surface area; CAD: coronary artery disease; COPD: chronic obstructive pulmonary disease; CT: computed tomography; CTEPH: chronic thromboembolic pulmonary hypertension; ILD: interstitial lung disease; IPF: idiopathic pulmonary fibrosis; MPAP: mean pulmonary artery pressure; NPV: negative predictive value; PA: pulmonary artery; PAH: pulmonary arterial hypertension; PASP: pulmonary arterial systolic pressure; PA/Ao ratio: ratio of the diameter of the pulmonary artery to the diameter of the aorta; PH: pulmonary hypertension; PPV: positive predictive value; PVD: peripheral vascular disease; RHC: right heart catheterization; RV/RA: right ventricular/right atrial; WHO: World Health Organization.CT measurement studies in patients with parenchymal lung disease (Subset of WHO Group 3 with fibrotic lung disease).AUC: area under curve; BSA: body surface area; CT: computed tomography; FVC: forced vital capacity; ILD: interstitial lung disease; MPAP: mean pulmonary artery pressure; PA: pulmonary artery; PA/Ao ratio: ratio of the diameter of the pulmonary artery to the diameter of the aorta; PH: pulmonary hypertension; RHC: right heart catheterization.However, Moore et al. reported no correlation between main PA and MPAP in 24 patients with history of primary PH and chronic thromboembolic pulmonary hypertension (CTEPH) [19]. The study observed that increasing main PA diameter was associated with decreased cardiac output (r = −0.75, p < 0.001) and increased pulmonary vascular resistance (r = 0.61, p < 0.0005) in the patients with pulmonary vascular disease, explained by the obliteration of peripheral arteries [19].The presence of significant parenchymal lung disease, which can distort the great vessel anatomy, also appears to affect the correlation between PH and PA size. Tan et al. [23] evaluated 36 patients with confirmed PH by RHC (defined by MPAP of at least 20 mmHg), but found no correlation between the main PA size and the MPAP. The lack of correlation was attributed to the presence of parenchymal lung disease and architectural distortion, which was present in the majority (24/36). Similarly, Zisman et al. and Devaraj et al. [24,27] also reported no significant correlation between the main PA size and MPAP in patients with fibrotic lung disease. In contrast, Alhamad et al. did find a moderate correlation between main PA size and MPAP in a larger series of patients with interstitial lung disease (ILD) [12] (Table 2).In systemic sclerosis and other connective tissues diseases, PH can be another manifestation apart from an ILD. A weak correlation between the main PA diameter and MPAP in connective tissue disease can be found [30], but appears to be mostly unaffected by the presence of ILD. For example, when Condliffe et al. evaluated patients with limited systemic sclerosis, a weak correlation was found (r = 0.345, p = 0.002) between the main PA diameter and the MPAP, regardless of the extent of ILD [43]. In another cohort of 48 patients with scleroderma and ILD, the presence of mild to moderate fibrotic lung disease did not influence the correlation between the PA diameter and MPAP [32]. Notably, the correlation was reduced after adjusting for the BSA or the aortic diameter, and there was no correlation in the subset with more advanced restriction (FVC less than 70%) [32].Similarly, in a retrospective study of 65 patients with advance idiopathic pulmonary fibrosis, there was no significant correlation between the main PA diameter and MPAP [24], similar to observations by others [25]. These studies suggest that perhaps, in the context of fibrotic lung disease, the PA dimension is even less of a reliable parameter as an indicator for PH. The mechanism for this is unclear, though a traction effect on the pulmonary vessels has been suggested [24].So given that there is evidence of some correlation between the PA size and PH severity, investigators have reported on whether specific thresholds could be used to facilitate the diagnosis or exclusion of PH, and in particular, whether it might help with regards to the need for a confirmatory RHC. Depending on the study population, the cut-off selected for the PA diameter (25–33.3 mm), and the gold standard used, the reported sensitivity can range between 47%–100% and the specificity between 41%–100% [10,12,13,16,17,18,20,21,22,28]. As anticipated, there is considerable overlap in the cut-off value and some overlap with what has been reported in the population without PH (Table 1). Although using the PA/Ao ratio has the potential to improve the diagnostic accuracy, its discriminatory ability appears also highly variable with a sensitivity of 59%–73% and specificity of 76%–93% [18,20,21,28,29]. In these studies of PH patients compared to controls, identifying PH based on the PA size, or the correlation between the two, may be artificially affected by the separation in severity of PA pressures between the groups being selected. Additionally, PH in different clinical contexts may affect the discriminatory ability of the PA measures. For example, the diagnostic accuracy is reduced when looking at patients with ILD (AUC = 0.65), with a specificity of 41% and sensitivity of 86% for a PA diameter cut-off of 25 mm [12].There are a limited number of investigations on how much the measure of the PA size, when used in conjunction with other clinical tools such as the echocardiogram, will add to the diagnosis or exclusion of PH [14]. However, one interesting study used a regression model to adjust for other variables on CT in combination with other anthropomorphic variables. In this heterogeneous cohort of 101 hospitalized patients with cardiopulmonary diseases who had a RHC and chest CT performed a mean of three days apart, the main PA and the PA/Ao ratio were poorly predictive of PH. However, the accuracy of the model when adjusted for age, gender, BSA, thoracic diameter, ascending aortic diameter, and pulmonary wedge pressure, improved with an AUC of 0.93 (sensitivity 77%, specificity 89%) [9] confirming that multiple factors are at play in the relationship between the PA size and PH. Our own internal investigations to see if CT measurements of the PA size might help in the diagnostic algorithm for patients with suspected PH was similar to what others have thus far reported. We retrospectively reviewed 109 patients with suspected PH who underwent both a chest CT and RHC within 180 days apart. The diagnostic categories were; Group 1 43%, followed by Group 3 22%, Group 2 18%, Group 4 9%, and Group 5 5%. The main PA diameter normalized to the aortic diameter (PA/Ao) did significantly correlate with the MPAP as measured on a RHC, but weakly (r = 0.28, p = 0.0032). Furthermore, its discriminatory ability for PH was poor with an AUC of 0.61 [44], making its clinical utility in our population unlikely. It is possible that despite its overall variable diagnostic utility for PH, that the measurements of the PA may have other important clinical implications. However, such relationships appear to be specific to the disease being considered. For example, in a cohort of 3464 patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage II to IV chronic obstructive pulmonary disease (COPD), PA/Ao ratio greater than 1 was associated with subsequent or any COPD exacerbation, but after adjustment for age, presence of gastroesophageal reflux disease, FEV1, the St. George’s Respiratory Questionnaire, and prior exacerbations (odds ratio 3.44; 95% CI 2.78–4.25, p < 0.001) [45]. Although the PA enlargement in this setting could be due to several pathologic mechanisms (e.g., volume fluctuations, hypoxic stress), its ability to predict future exacerbations indicates the potential for PA measures to be clinically useful independent of its ability to diagnose or correlate with any PH.The association with exacerbations might also hold true for patients with ILD. Matsushita et al. observed that patients who had an acute exacerbation of their ILD had a greater increase in their PA diameter (by 3.15 mm, SD 0.54 vs. 2.89 mm, SD 0.6, p < 0.0001) from a baseline CT, and had a higher PA/Ao ratio (0.94, SD 0.19 vs. 0.85, SD 0.18, p < 0.0001) [46]. Alterations in hemodynamics and hypoxic vasoconstriction affecting the PA size may be more secondary rather than causal, and whether this might have a clinical role is yet to be determined [46].In contrast, Boerrigter et al. [47] observed that progressive PA dilatation by MRI during follow-up (942 days, range 242–2,359 days) did not reflect hemodynamic changes in MPAP or cardiac output by RHC in 51 patients with Group 1 PAH. The authors suggested that structural changes in elastin and collagen under the influence of an increased pressure might become a cause of PA dilatation independent of hemodynamics. The results suggest that serial PA dimensions are not useful in clinical practice to evaluate the course of the disease, therapeutic response, or change in MPAP.Finally, in 264 patients with inoperable CTEPH, the PA size was associated with unexpected death [48], even though the causes of death were heterogeneous [45]. Overall, these studies suggest that perhaps the PA dimensions might have other clinical relevance aside from its ability to predict PH, particularly in acute settings. Further study in its role in acute cardiopulmonary decompensations might be of further interest.In summary, although CT measurements of PA dimensions have shown a correlation with the presence of PH and PH severity, the strength of the correlation is highly variable and inadequate for its routine application in clinical practice. Significant variability exists within populations, particularly in the heterogeneous set of diseases that can cause PH. Additional heterogeneity comes from the complex set of physiologic and anatomic factors that disconnect the PA size from PA pressures. Future studies could focus on specific disease subsets and changes in the PA size as a marker of prognosis, disease activity, and treatment response, rather than as an isolated measure. CT measures of PA size are simple and can suggest a possible reason for dyspnea but it should be interpreted cautiously and not be used solely in either screening or guiding management in the patients with suspected PH.KU contributed to conception and design of the study, collection, analysis, and interpretation of the data, drafting and critical revision of the article, and collection/generation of the figures. JPW, TAL, and IDC contributed to the experiments, collection of data, and drafting of the article. CDB contributed to the conception and design of the study and critical revision of the article. ASL contributed to the conception and design of the study, analysis and interpretation of the data, and critical revision of the article. All authors gave final approval of the article.The authors declare no conflict of interest. 

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Pulmonary arterial hypertension (PAH) is a progressive lung disease diagnosed by an increase in pulmonary arterial blood pressure that is driven by a progressive vascular remodelling of small pulmonary arterioles. We have previously reported that tumor necrosis factor apoptosis-inducing ligand (TRAIL) protein expression is increased in pulmonary vascular lesions and pulmonary artery smooth muscle cells (PASMC) of patients with idiopathic PAH. The addition of recombinant TRAIL induces the proliferation and migration of PASMCs in vitro. TRAIL is required for hypoxia-induced pulmonary hypertension in mice, and blockade of TRAIL prevents and reduces disease development in other rodent models of PAH. Due to the availability of knockout and transgenic mice, murine models of disease are key to further advances in understanding the complex and heterogeneous pathogenesis of PAH. However, murine models vary in their disease severity, and are often criticized for lacking the proliferative pulmonary vascular lesions characteristic of PAH. The murine Sugen-hypoxic (SuHx) mouse model has recently been reported to have a more severe PAH phenotype consisting advanced pulmonary vascular remodelling. We therefore aimed to determine whether TRAIL was also required for the development of PAH in this model. C57BL/6 and TRAIL−/− mice were exposed to normoxia, Sugen5416 alone, hypoxia or both Sugen5416 and hypoxia (SuHx). We report here that SuHx treated C57BL/6 mice developed more severe PAH than hypoxia alone, and that TRAIL−/− mice were protected from disease development. These data further emphasise the importance of this pathway and support the use of the SuHx mouse model for investigating the importance of potential mediators in PAH pathogenesis.Pulmonary hypertension (PH) is a broad group of diseases defined by a mean pulmonary artery pressure (mPAP) of greater than or equal to 25 mmHg at right heart catheterisation [1]. Pulmonary arterial hypertension (PAH) is a sub-classification of PH and a progressive and degenerative disease driven by progressive arterial remodelling within the pulmonary circulation [2]. PAH is a rare disease, with an incidence of 1–3.3 per million per year (idiopathic PAH) and a prevalence of 15–52 per million [1] but has severe morbidity, a prognosis worse than many cancers and is ultimately fatal (without lung transplantation). Although PAH is a disease of the lungs, it is the impact on the right ventricle which ultimately defines a patients survival. The increased pulmonary vascular resistance results in a pressure afterload that drives a process of adaptive, then maladaptive right ventricular hypertrophy, dilation and stiffening. This leads to impaired stroke volume, inadequate organ perfusion and eventual right heart failure [3]. Current treatments for PAH are effective in alleviating vasoconstrictive symptoms, however they do not address the underlying pulmonary vascular or right ventricular remodelling and have only modest impact on patient survival. The increase in pulmonary pressure observed in PAH is largely due to underlying remodelling of small-to-medium sized pulmonary arterioles. The underlying mechanisms that lead to the pulmonary vascular remodelling are incredibly complex [4,5]. Although the exact trigger for pulmonary arteriole remodelling is currently unknown, pulmonary arterial endothelial cell (PA-EC) dysfunction and apoptosis is believed to be an early insult. PA-EC apoptosis causes the obliteration of distal pulmonary arterioles and exposes the underlying pulmonary arterial smooth muscle cells (PA-SMCs) to a whole host of mitogenic and vasoconstrictive factors, which then drive the vascular remodelling [4,5]. A population of dysfunctional, hyper-proliferative PA-ECs also proliferate to form plexiform lesions, a hallmark of severe PAH [6]. Growth factors, such as PDGF [7,8] and cytokines such as IL-1 and IL-6 [9,10,11,12,13] have also been implicated in the pathogenesis of PAH. More recently, we have described TRAIL as a critical downstream mediator of disease [14].TRAIL (TNF-related apoptosis inducing ligand, Apo2L) is a type two membrane protein that is predominantly expressed in the lung, spleen and prostate [15]. TRAIL binds to four membrane receptors: TRAIL R1 (death receptor 4, DR4) [16], TRAIL R2 (death receptor 5, DR5) [16,17], TRAIL R3 (decoy receptor 1, DcR1) [16,18] and TRAIL R4 (decoy receptor 2, DR2) [19,20]. There is also a fifth, soluble decoy receptor, osteoprotegerin (OPG) [21], a putative biomarker for IPAH [22]. TRAIL induces the proliferation and migration of both rat and human aortic SMCs, and human pulmonary artery smooth muscle cells predominately via TRAIL-R3 [14,23]. TRAIL protein expression has also been observed in concentric and plexiform pulmonary vascular lesions in patients with IPAH [24] and in animal models of disease [11,14]. In rodent models anti-TRAIL antibody prevents disease development [14]. Furthermore, in therapeutic studies, where antibody treatment was performed in models with established disease, an anti-TRAIL antibody successfully reduced pulmonary vascular remodelling and increased survival [14].Due to the availability of knockout and transgenic mice, murine models of disease are key to further advances in understanding the complex and heterogeneous pathogenesis of PAH. The most common murine model is the chronic hypoxic mouse model [25], however the mouse is a poor responder to hypoxia in comparison to other species, and therefore often criticized as a poor model of PAH [26]. More recently, an adaptation of the rat Sugen-hypoxic (SuHx) model, first developed by Taraseviciene-Stewart and colleagues [27], to mice has been shown to display a more severe disease phenotype, with higher right heart pressure and more severe pulmonary vascular remodelling than hypoxia alone [28]. We therefore aimed to both establish the murine model of SuHx and hypothesised that TRAIL−/− mice would be protected from developing PAH in this model. We found that while C57BL/6 mice developed the hallmarks of PAH including remodelling of the small pulmonary arterioles, increased right heart pressure and right ventricular hypertrophy, TRAIL−/− mice showed no significant signs of PAH.All mice were on a C57BL/6 background. TRAIL−/− mice were originally developed by Amgen/Immunex (Thousand Oaks, CA, USA.) as previously described [29,30] and obtained by material transfer agreement from Amgen Inc. (MTA # 200908042). For all procedures male C57BL/6 and TRAIL−/− mice aged 12–13 weeks of age were used (6–8 per group). For the chronic hypoxic mouse model, mice were placed in hypoxic chambers (10% v/v Oxygen) for 3 weeks to induce pulmonary hypertension. For the Sugen-hypoxic model, C57BL/6 and TRAIL−/− were exposed to hypoxia (10% v/v O2) for 3 weeks with weekly injections of 20 mg/kg Sugen5416 (Tocris) during exposure to hypoxia as previously described [28]. All animal experiments were approved by the University of Sheffield Project Review Committee and conformed to UK Home Office ethical guidelines.Echocardiography was performed using the Vevo 770 system (VisualSonics, Toronto, Canada) using the RMV707B scan head. Rodents were placed on a heated platform and covered to minimise heat loss. Rectal temperature, heart rate and respiratory rate were recorded continuously throughout the study. Echocardiography was performed and analysed as previously described [11,14].Following echocardiography, left and right ventricular catheterisation was performed using a closed chest method via the right internal carotid artery and right external jugular vein under isoflurane induced anaesthesia, as previously described [11,14]. Data were collected using either a Millar pressure-volume PVR-1045 1F catheter (mouse LV) or PVR-1030 (mouse RV) (Millar Instruments Inc., Texas, USA) coupled to a Millar MPVS 300 and a PowerLab 8/30 data acquisition system (AD Instruments, Oxfordshire, UK) and recorded using Chart v7 software (AD Instruments). Pressure volume analysis was performed (PVAN, Millar) and an estimated pulmonary vascular resistance index (ePVRi) was calculated as previously described [14]. Right ventricular hypertrophy (RVH) was measured by calculating the ratio of the right ventricular free wall over left ventricle plus septum [14].Immediately after harvest, the left lung was perfusion fixed via the trachea with 10% (v/v) formal buffered saline by inflation to 20 cm of H2O. The lungs were then processed into paraffin blocks for sectioning. Paraffin embedded sections (5 µm) of lung were histologically stained for Alcian Blue Elastin van Gieson (ABEVG) and immunohistochemically stained for α-smooth muscle actin (α-SMA, M0851, Dako, Cambridgeshire, UK) to visualize smooth muscle cells, TRAIL (TRAIL (ab2435; Abcam) was also used to localize protein expression to pulmonary vascular lesions and anti-human PCNA antibody to assess proliferation (M0879; Dako). A secondary biotinylated anti-mouse antibody (1:200) was added before an Avidin Biotin enzyme Complex (Vectastain kit;Vector Laboratories). All protocols used 3,3 diaminobenzidine as the substrate for color in the peroxidase reaction and counterstained with hematoxylin as previously described [14].Pulmonary vascular remodelling was quantified by assessing the degree of muscularization and the percentage of affected pulmonary arteries and arterioles in 3 groups based on vessel size; small pulmonary arterioles with a diameter less than 50 µm, medium pulmonary arteries with a range in diameter from 51 to 100 µm, and large pulmonary arteries with a diameter greater than 100 µm and standard immunohistochemical techniques were applied as previously described [11,14].30 µg of each sample was loaded on a 4%–12% Bis-Tris NuPage gel and run under reducing conditions in MES running buffer (Invitrogen), before transfer to a nitrocellulose membrane (Invitrogen). Trans- fer was confirmed with Ponceau S (Sigma-Aldrich) staining and the membrane was then blocked for 1 h in Odyssey® blocking buffer (LI-COR Biosciences) at room temperature. The blots were incubated with either GAPDH (1:1,000, CST) or anti–beta-Actin mouse monoclonal antibody (1:1,000, Abcam) for 1 h at room temperature and anti-TRAIL mouse monoclonal antibody (1:50, Leica Biosystems) or anti-BMPR2 mouse monoclonal antibody (1:250), overnight at 4 °C. For detection, membranes were incubated with fluorescently labeled anti-rabbit (IRDye 800CW; LI-COR Biosciences) and anti-mouse (IRDye 680LT) for 1 h at room temperature before reading at 700/800 nm on an Odyssey SA imaging system (LI-COR Biosciences).Statistical analysis was performed using two-way ANOVA followed by Dunnett’s or Tukey’s multiple comparisons test with a 95% confidence level. P < 0.05 was deemed statistically significant.To determine whether we could recapitulate molecular characteristics of the SuHx model and whether this model was suitable to investigate the role of TRAIL in PAH vascular remodelling, we first looked to determine whether BMPR2 expression was decreased, and TRAIL expression increased within the lungs. After 3 weeks exposure to Sugen5416 plus hypoxia BMPR2 protein levels were significantly reduced compared to normoxic control C57Bl/6 (wt) mice (Figure 1A). On the contrary, TRAIL protein expression was significantly increased in whole lungs lysates from hypoxia and SuHx-treated wt mice, compared with normoxic or Sugen-alone treated mice (Figure 1B). TRAIL protein expression was also detected within remodelled pulmonary arteries of hypoxic and SuHx wt mice (Figure 1C).BMPR2 expression is reduced and TRAIL expression is increased in the SuHx mouse model. Bar graph shows (A) reduced BMPR2 expression and (B) increased TRAIL protein expression in whole lung protein lysates of C57Bl/6 mice following 3-week exposure to normoxia (Nx), Sugen-alone (Su), hypoxia-alone (Hx) and Sugen plus Hypoxia (SuHx). Representative western immunoblot images are also shown. (C) Representative photomicrographs of lung sections of SuHx mice showing immunoreactivity for TRAIL. Arrows highlight TRAIL positive cells within remodelled vessels. Error bars represent mean ± SEM, * P < 0.05, ** p < 0.01; n = 3–4 animals per group. Scale bars represent 50 µm.Compared to control normoxic, Sugen-alone and hypoxia-alone treated mice, wt mice exposed to SuHx displayed outward signs of morbidity including breathlessness, consistent with a PAH phenotype. Serial echocardiography performed on wt mice exposed to SuHx displayed a PAH phenotype, as defined by a reduction in pulmonary artery acceleration time (PA-AT) (Figure 2A), cardiac output (Figure 2B), pulmonary artery velocity time integral (PA-VTi) (Figure 2E) and an increased right ventricular internal diameter at diastole (RVIDd) (Figure 2D). Hypoxia- and SuHx-treated TRAIL−/− mice however, displayed no significant change in either parameter compared to baseline or normoxic control mice. Both wt and TRAIL−/− hypoxic mice did however show a significant decrease in aortic-VTi (Figure 2F), presumably due to hypoxia mediated peripheral vasodilation [31].There were however no significant changes observed to the left ventricular wall, LV FWTs (Figure 2C) and LV FWTd (Figure 2G). The SuHx-treated wt mice were the only group to display a significant increase in RVIDd (Figure 2D). There was significant increase in RVH from both hypoxia and SuHx treated wt mice that was not observed in the TRAIL−/− mice. Although there was a trend for increased RVH between SuHx and hypoxia alone wt mice, this was not statistically significant (Figure 2H). TRAIL−/− mice show no echocardiographic signs of PAH in response to SuHx. (A–H) Bar graphs show (A) pulmonary artery acceleration time (PA-AT), (B) cardiac output, (C) left ventricle free wall thickness at systole (LV FWTs), (D) right ventricular internal diameter at diastole (RVIDd), (E) pulmonary artery velocity time integral (PA-VTi), (F) aortic velocity time integral Ao-VTi), (G) left ventricle free wall thickness at diastole (LV FWTd) and (H) right ventricular hypertrophy (RVH) in wt and TRAIL−/− mice at baseline and after 3 weeks exposure to normoxia, Sugen5416, hypoxia or SuHx. Error bars represent mean ± SEM, n = 4–6 animals in each group. * P < 0.05; ** P < 0.01; *** P < 0.001, wt mice compared to wt baseline. # P < 0.05, TRAIL−/− compared to TRAIL−/− baseline.Following echocardiography assessment, right and left ventricular catheterisation was performed. Wild-type mice exposed to hypoxia and SuHx developed significant increases in RVSP (Figure 3A), RV dP/dtmax (Figure 3C), RV P@dP/dtmax (Figure 3E) and ePVRI (Figure 3M), and reduced cardiac output (Figure 3F), RV dV/dt (Figure 3D) and LV dV/dt (Figure 3K). In contrast to wt mice, hypoxia- and SuHx-treated TRAIL−/− mice did not develop significant changes in the haemodynamic parameters associated with disease development (Figure 3A–N). Wild-type SuHx-treated mice had significantly increased RVSP (Figure 3A) and ePVRi (Figure 3M) compared to hypoxia alone, and were the only group to display a significant increase in right ventricular pressure at max dP/dt (Figure 3E) suggesting a more severe PAH phenotype similar to that reported by Ciuclan et al. [28].To examine whether the more severe PAH phenotype in SuHx wt mice, compared to hypoxia alone, was due to more extensive pulmonary vascular remodelling, and whether the TRAIL−/− mice were protected from pulmonary vascular changes, we performed quantitative analysis of serial lung sections. To assess the degree of pulmonary vascular remodelling, we measured the media cross sectional area (media/CSA) and degree of muscularization in pulmonary arteries of <50 µm, 50–100 µm and >100 µm in diameter as previously described [11,14]. Wild-type mice showed increased media/CSA (Figure 4A) and a greater percentage of muscularized arteries (Figure 4B) in pulmonary arterioles of <50 µm diameter when treated with hypoxia alone or SuHx, compared to normoxic controls. Although there was no significant difference between hypoxia and SuHx wt mice with either index, there were trends for both a higher percentage of remodelled vessels, and increased medial area. The more severe haemodynamics observed in the wt SuHx was likely due to the more progressive nature of the pulmonary vascular remodelling as demonstrated by the significant increase in media/CSA (Figure 4D), and percentage remodelling (Figure 4E) of the medium sized 50-100 µm pulmonary arteries, which was not observed in the hypoxia alone wt mice. No difference in media/CSA, or in percentage muscularization was observed in pulmonary arterioles of >100 µm (Figure 4G,H). TRAIL−/− mice displayed no significant increases in either marker of pulmonary vascular remodelling in response to either hypoxia or SuHx (Figure 4A–F). To verify that the increased vascular remodelling was driven by pulmonary vascular cell proliferation, we performed immunohistochemical analysis of proliferating cell nuclear antigen (PCNA). There was little difference between wt and TRAIL−/− mice in response to hypoxia, perhaps highlighting that the major driver of apparent muscularisation of the pulmonary arteries was more vasoconstriction, rather than aberrant proliferation (Figure 4C,F,I). Within the SuHx wt mice however, there was a marked and significant increase in the number of proliferating cells within the remodelled vessels (Figure 4 C,F,I).TRAIL−/− mice show no haemodynamic signs of PAH in response to SuHx. (A–N) Bar graphs show (A) right ventricular systolic pressure (RVSP), (B) right ventricular end-diastolic pressure (RVEDP), (C) right ventricular (RV) dP/dt, (D) right ventricular (RV) dV/dt, (E) right ventricular (RV) P@dP/dt, (F) cardiac output, (G) mean aortic blood pressure (mAoP), (H) left ventricular end-systolic pressure (LVESP), (I) left ventricular end-diastolic pressure (LVEDP), (J) left ventricular (LV) dP/dt, (K) left ventricular (LV) dV/dt, (L) left ventricular (LV) P@dP/dt, (M) estimated pulmonary vascular resistance (ePVRi) and (N) systemic vascular resistance (SVR) in wt and TRAIL−/− mice after 3 weeks exposure to normoxia, Sugen5416, hypoxia or SuHx. Error bars represent mean ± SEM, n = 3–6 animals in each group. * P < 0.05; ** P < 0.01; **** P < 0.0001, compared to wt normoxic mice, significant differences between other groups are shown.Histological and immunohistological analysis of lung tissues (Figure 5) revealed evidence of muscularization caused by increased smooth muscle actin (SMA) positive cells in small resistance pulmonary arterioles, <50 µm diameter, in hypoxic and SuHx mice compared to normoxic mice. Although more extensive pulmonary vascular remodelling was observed in the SuHx treated wt mice we did not observe any evidence of plexiform-like lesions. TRAIL−/− mice displayed reduced remodelling of the pulmonary arterioles in response to both hypoxia and SuHx that corresponded with the presence or absence of proliferating cells (PCNA, Figure 5).Previous studies in chimeric mice suggest that tissue expression of TRAIL is the predominant driver of pulmonary remodelling in the high fat diet fed ApoE−/− mouse model [14], however the contribution of bone marrow (BM)-derived cells could not be entirely ruled out. To determine whether protection from the development of a PAH phenotype was also associated with a reduced inflammatory cell infiltration, we performed immunohistochemical analysis for CD45 positive cells in SuHx wt and TRAIL−/− mice. While wt mice demonstrated a marked increase in CD45 positive cells in response to SuHx, TRAIL−/− mice had a similar expression profile to control wt mice (Figure 6). TRAIL−/− mice show reduced pulmonary vascular muscularization in response to SuHx. Bar graphs show media/CSA of pulmonary arteries (A) <50 µm diameter, (D) 50–100 µm diameter, (G) >100 µm diameter; the percentage of muscularized arteries (B) <50 µm diameter, (E) 50–100 µm diameter, (H) >100 µm diameter; percentage of proliferating cells within pulmonary arteries (C) <50 µm diameter, (F) 50–100 µm diameter, (I) >100 µm diameter in wt and TRAIL−/− mice exposed to normoxia (Nx), sugen-alone (Su), hypoxia (Hx) and Sugen plus Hypoxia (SuHx). Error bars represent mean ± SEM, n = 3–6 animals in each group. * P < 0.05; ** P < 0.01; *** P < 0.001, compared to wt normoxic mice.TRAIL−/− mice are protected against small resistance pulmonary arterial muscularization when exposed to SuHx. Representative photomicrographs of lung sections from wt and TRAIL−/− mice after 3-week exposure to normoxia, Sugen 5416, hypoxia or SuHx. Sections were stained with Alcian Blue Elastic van Gieson (ABEVG), immunostained for α-smooth muscle actin (SMA) or proliferating smooth muscle cell antigen (PCNA). Bars, 50 µm.TRAIL−/− recruit less CD45 positive cells when exposed to SuHx. Representative photomicrographs of lung sections from wt and TRAIL−/− mice after 3-week exposure to Sugen plus hypoxia (SuHx). Sections were immunostained for CD45.Rodent models, and particularly murine models of PAH, are often criticised for their limited ability to recapitulate or model human disease. Despite these caveats, the increasing availability of knockout and transgenic mice increases the importance of mouse models. The hypoxic mouse model has historically been the most common murine model [25] utilised despite the mouse being a poor responder to hypoxia, in comparison to other species, and therefore a weak model [26]. The recent adaptation of the rat Sugen-hypoxic (SuHx) [27] to mice by Ciuclan and colleagues [28] provides a more robust murine model in which to test the requirement, or importance of specific genes in disease pathogenesis.We have previously demonstrated that TRAIL−/− mice are protected from hypoxia-induced PH, and that ApoE−/−/TRAIL−/− are protected from high fat diet-induced PAH [14]. We therefore looked to test the utility of the murine SuHx model while simultaneously validating our own studies. We found that while C57BL/6 mice developed hallmarks of PAH as measured by echocardiography, cardiac catheterisation and remodelling of the small pulmonary arterioles as previously reported [28], TRAIL−/− mice showed no significant signs of PAH, as evidenced by the reduced recruitment of inflammatory cells and reduced number of proliferating cells with the pulmonary arteries. These data emphasise the utility of the murine SuHx models as a tool to screen the importance of particular genes of interest, and further support the importance of TRAIL in the pathogenesis of PAH.Funding for this study was provided by the Medical Research Council via a Doctoral Training Grant (S.D) and a Career Development Award (G0800318, A.L.). A.L. is currently supported by a British Heart Foundation Senior Basic Science Research Fellowship (FS/13/48/30453).S.D. collected and analysed data, and contributed to the writing and editing of the manuscript. N.A. collected and analysed data and helped edit the manuscript. J. P. collected and analysed data and helped edit the manuscript. S.F. obtained the TRAIL−/− mice from Amgen, and contributed to manuscript editing. A.L. Over looked all aspects of the project, collected and analysed data and contributed to manuscript writing.The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Pulmonary hypertension (PH) is a rare disease, which still carries a poor prognosis. PH is characterized by a pressure overload on the right ventricle (RV), which develops hypertrophy, followed by a progressive failure. Accordingly, recent evidence showed that RV function has an important prognostic role in patients with PH. Echocardiography, cardiac magnetic resonance (CMR), computed tomography, and nuclear imaging allow a non-invasive evaluation of the RV size and function, but only the first two are routinely used in the clinical arena. Some conventional echocardiographic parameters, such as TAPSE (tricuspid anular plane systolic excursion), have demonstrated prognostic value in patients with PH. Moreover, there are some new advanced echo techniques, which can provide a more detailed assessment of RV function. Three-dimensional (3D) echocardiography allows measurement of RV volumes and ejection fraction, and two-dimensional (2D) speckle tracking (STE), allows assessment of RV myocardial mechanics. CMR provides accurate measurement of RV volumes, ejection fraction, and mass and allows the characterization of the RV wall composition by identifying the presence of fibrosis by late gadolinium enhancement. Although CMR seems to hold promise for both initial assessment and follow-up of patients with PH, its main role has been restricted to diagnostic work-up only.Pulmonary hypertension (PH) is a rare disease which carries a poor prognosis [1,2]. Recent evidence showed that right ventricular (RV) function has an important prognostic role in patients with PH [3,4,5,6,7,8,9,10], the onset of RV failure being associated with increased mortality, independent from pulmonary vascular resistance values and the etiology of PH [1,11,12,13,14]. Therefore, accurate assessment of RV function appears to be critical in PH patients’ initial evaluation and follow-up.In this scenario non-invasive imaging techniques, such as cardiac magnetic resonance (CMR) and echocardiography [15], have been shown to be able to provide quantitative assessment of RV morphology, size, and function, covering a major role in diagnosis and follow-up of patients with PH.This review paper will summarize the current knowledge about the RV assessment by conventional and novel non-invasive imaging modalities in the setting of PH.Compared to its left counterpart, the RV shows a complex pyramidal shape, and can be divided in three main parts: the inlet, the outlet, and the apical, trabeculated part. The RV free wall myocardium is composed mainly by circumferential fibers in the superficial layer and longitudinal fibers in the subendocardial layer [16]. Usually, the circumferential fiber layer is less developed than the longitudinal one. This myocardial fiber architecture explains why, in healthy subjects, RV pump function is determined mainly by longitudinal shortening, rather than by transversal displacement [17,18].The RV contraction follows a peristaltic pattern that starts from the inlet part and ends to the outlet one 25–50 msec later [19,20]. Several distinct events contribute to overall RV pump function: (i) inward movement of the RV free wall, which produces a bellows effect; (ii) contraction of the longitudinal flbers, which draws the tricuspid annulus toward the RV apex; (iii) infundibular contraction; and (iv) contraction of the left ventricle, which assists RV contraction via mechanical transduction across the shared interventricular septum, as well as via circumferentially oriented superflcial myoflbers that are contiguous between the ventricles. Therefore, the RV presents regional contraction differences that contribute in distinct magnitude and timing to its global systolic function [21,22].In patients with RV pressure overload, with consequent RV hypertrophy, the hypertrophied fibers change their spatial orientation and become more circumferential. As a consequence, in PH patients, circumferential and radial shortening increase their contribution to the RV pump function compared to normal subjects [23,24].Conventional echocardiography provides several parameters to measure RV function, such as RV fractional area change, tricuspid anulus plane systolic escursion (TAPSE), lateral wall S wave velocity by Tissue Doppler, myocardial performance index, and longitudinal strain. Among them only TAPSE  has shown to be a strong predictor of survival [25].However, TAPSE reflects only the longitudinal shortening of the RV which is only one part of the whole complex mechanics. Longitudinal shortening is the predominant component of RV pump function in healthy subjects [26]. However, in presence of RV pressure overload, as it occurs in patients with PH, RV ejection fraction is more dependent on radial (transversal) displacement than to longitudinal shortening [27]. Particularly, Kind et al. [27] reported that transversal displacement of the RV free wall at mid-level was a more powerful predictor of lower RV ejection fraction than longitudinal shortening. This different RV mechanics between healthy subjects and PH patients can be explained by changes in RV geometry and myocardial fiber orientation. When the RV contracts against high pressure, it dilates predominantly in cross-section changing the shape of the transverse section from crescentic to more circular. Accordingly, Pettersen et al. [24] reported that, in PH patients, the mid RV free wall shortening occurs predominantly in the circumferential than in the longitudinal direction, possibly due to re-arrangement of myocardial fiber architecture in the RV free wall. Accordingly, Mauritz et al. [28] confirmed that the progression of RV function decline in PH patients is mainly related to the loss of RV transversal displacement, showing also a good correlation between prognosis and extent of residual transverse displacement. Moreover, they showed that the decline in transversal displacement is mostly due to a progressive leftward displacement of interventricular septum rather than to a further decrease of RV free wall transverse displacement [28]. They hypothesized that RV dysfunction in PH patients may start with a reduction of TAPSE, that continues to decrease until a lower limit is reached. Progression of the disease induces a further deterioration of the RV function through a loss of transversal shortening. As for longitudinal shortening, a lower limit also is reached for the RV free wall transversal displacement; thus, increased leftward septal bowing is the main explanation for a further decline in RV transverse shortening in progressive PH [28].Conventional echocardiographic measurements of RV function do not always reflect true RV pump function due to the complex geometry of the RV and its extreme dependence on loading conditions [29]. Accordingly, new echocardiographic techniques, such as 2D speckle tracking echocardiography (STE) and 3D echocardiography, have been employed to provide a more comprehensive assessment of RV function. Indeed, these new echocardiographic techniques overcome the main limitations of both two-dimensional echo (geometric assumptions about RV geometry) and M-mode and Doppler related techniques (external reference, angle-dependency) and provide a more comprehensive assessment of RV function, which is not limited to the assessment of the longitudinal excursion anymore. Deformation imaging by STE is a new echo technique that has been developed to assess left ventricular myocardial deformation. Nevertheless, recent studies have demonstrated its feasibility in measuring RV strain (Figure 1) to detect RV myocardial function changes in several pathological conditions [30,31,32,33,34,35,36,37,38]. Compared to Tissue Doppler Imaging, STE is an angle-indipendent technique based on internal reference, thus, avoiding limitations related to translational cardiac motion. Preliminary researches showed good correlations with the other standard echo parameters of RV function, in particular with TAPSE [39]. Among non-volumetric echo parameters, RV free-wall strain by STE demonstrated the closest correlation with RV ejection fraction measured by CMR [40].Right ventricular longitudinal strain by two-dimensional speckle tracking in a healthy subject. Upper right: longitudinal strain dotted curve showing the main values. Ls = longitudinal strain.RV longitudinal strain assessed by STE strain imaging is significantly impaired in patients with PH (Figure 2) and it is inversely correlated with systolic pulmonary artery pressure and RV dimensions [29,34,39,41,42].Right ventricular longitudinal strain impairment in pulmonary hypertension patient. Ls = longitudinal strain.RV longitudinal strain values demonstrated a significant prognostic role in PH which was incremental to clinical status, overcoming the other echocardiographic parameters [38,43,44]. RV longitudinal strain has been reported to be a predictor of cardiovascular events [43], all-cause mortality and complications [44]. Moreover, Fine et al. [44] demonstrated that abnormal RV strain is predictive of reduced survival, stratifying patients prognosis.However, it is important to underline that the prognostic power of RV longitudinal strain has been demonstrated in patients with pre-capillary PH, but not in patients with post-capillary PH (group 2 according to Dana Point classification) [45]. The exclusion of group 2 from these studies is justified by the different hemodynamic pattern that characterize this pathological condition [44]. Highlighted in a recent study [46], pulmonary vascular resistance and compliance demonstrated a highly predictable inverse relationship with RV loading, which is similar across different forms of pre-capillary PH. In presence of post-capillary PH, as occurs in group 2, this relationship is altered and elevated capillary wedge pressure lowers pulmonary vascular compliance for any given resistance, augmenting RV pulsatile afterload. Accordingly, Haeck et al. [38] demonstrated, in a little cohort of group 2 PH patients, that in these patients RV longitudinal strain doesn’t confirm its prognostic value.RV function is heavily load dependent and myocardial deformation is influenced by loading conditions, too. Accordingly, it has been demonstrated that in patients with RV pressure overload, both with and without loss of myocardial contractility, RV strain was significantly lower than in normal subjects [47]. These data show that RV strain can be decreased in presence of both increased afterload and normal contractility, and in presence of increased afterload and myocardial damage with decreased contractility [47], underlying the fact that the diagnostic significance of RV strain values must always be related to RV loading conditions [48,49]. However, the main determinant of RV strain impairment seems to be myocardial contractility. Indeed PH patients with RV dysfunction showed lower RV strain values in comparison to PH patients with preserved RV function [47]. Further evidences are needed to support the hypothesis that, considering its load-dependency, a mild reduction of RV strain alone should not be considered a marker of RV myocardial dysfunction in the PH setting, but a marked reduction of RV strain could be, underlying the need of cut-off values identification.Not only the absolute value of RV strain is predictive of prognosis in PH patients, but also its variation during follow-up demonstrated a prognostic role. Patients with an improvement >5% of RV strain during follow-up demonstrated better survival respect to patients without [50].Recently, Atsumi et al. [51] studied, for the first time, the application of 3D-STE to assess RV myocardial deformation. They have been able to demonstrate a good feasibility (around 80%) of the technique (feasibility close to that of 2D-STE). In comparison to 2D-STE, 3D-STE allowed to assess not only the limited part of the RV free-wall encompassed in the 2DE four-chamber view but the whole RV free wall, and to confirm the significant heterogeneity of RV contraction. Moreover, 3D-STE explores simultaneously the longitudinal and circumferential deformation of the myocardium, that are expression of the function of myocardial fibers positioned in different layers of the RV wall. The Authors found that, in presence of conditions affecting the RV, such as arrrhytmogenic RV cardiomyopathy and PH, longitudinal function was significantly reduced, whereas circumferential function was relatively preserved [51]. These findings confirm previous data showing that the motion of the base towards the apex is the first function to be impaired [28]. The importance of assessing RV radial function has been demonstrated by Di Salvo et al. [52], who found a strong correlation between the extent of radial function and exercise tolerance in RV pressure overload condition, such as in patients with transposition of great arteries who underwent atrial-switch repair. Despite all these data, the use of STE to assess RV myocardial function is not widespread because the feasibility of STE is suboptimal in the thin RV wall (better in hypertrophied RV as seen in patients with PH). Reference values for different age groups, body size, and gender have not been established yet, and standardization among the different software packages available on the market is still a pending issue [17].Three-dimensional echocardiography (Figure 3; Videos 1–2) has been demonstrated to be feasible, accurate and reproducible in measuring RV volumes and ejection fraction in adults [53,54,55,56,57,58,59,60,61], as well as in children [62,63]. Recent studies demonstrated a close correlation between 3D-echocardiography and CMR in measuring RV volumes and ejection fraction [61,64,65] (Table 1). However, 3D echocardiography appeared to underestimate RV volumes in comparison to CMR [65]. Anyway this underestimation is systematic [58], confirming the good agreement between the two techniques and the accuracy of 3D echo. 3D echocardiography confirmed the correlation between RV volumes and gender, age, and body size, previously demonstrated by CMR [58] (Figure 4). Three-dimensional echocardiographic visualization of the right ventricle in a healthy subject. (A) Multislice view showing three apical views and corresponding short axis. (B) Right ventricular beutel in end-diastole and end-systole and the corresponding time-volume curve. EDV = end-diastolic volume; ESV = end-systolic volume; RV = right ventricle.Differences between right ventricular volumes assessed by three-dimensional echocardiography and cardiac magnetic resonance (Modified from: Badano et al. [61]).Right ventricular volumes varies with gender and age, unrespect to body size. (Modified from Maffesanti et al. [58]). EDV = end-diastolic volume; ESV = end-systolic volume; RV = right ventricle.Accordingly, Morikawa et al. [68] proved a strong correlation between ≥3D echocardiography and CMR measurements of the RV volumes and ejection fraction in patients with PH. Similar results were reported in a population of patients with congenital heart disease [69].The presence of synus rhytm and the patient’s compliance for breath-holding are necessary requirements for obtaining a good quality 3D echo assessment with the multi-beat technique. However, recently Zhang et al. [70] demonstrated that the 3D-echocardiography assessment of RV geometry by a single-beat acquisition is as accurate as with multi-beat acquisition when compared with CMR. Nevertheless, the feasibility of obtaining a good quality full volume 3D data set acquisition which includes the RV anterior and apical lateral segments as well as the RV outflow tract, in patients with poor imaging windows and/or dilated RV, remains the main limitation of the technique. Accuracy tends to decrease with increasing RV size, limiting its application in patients with more dilated RV [69]. Using 3D echocardiography, it has been confirmed that in normal people the three compartments of the right ventricle provides a different contribution in RV systolic contraction, both in timing as in strength [71,72]. Inflow and outflow tracts are the most active RV compartments contributing to its pump function, whereas the apex contributes less, following a chronological order in contraction, that reflects the peristaltic pump function of the RV [71]. In pts with PH, the relative contribution of the three compartments to RV pump function seems to remain unchanged, with the loss of the timing differences. The three compartments contract simultaneously loosing the peristaltic function and making the RV behave as a single chamber. This change is coupled by changes of the RV shape, from a triangular to a cilindrical one [71]. These findings underline once more the importance of a comprehensive assessment of RV function, that could not be adequately reflected by assessing a single parameter, such as TAPSE. Indeed, the latter is representative only of the inflow tract displacement, with the consequently risk to underestimate the whole RV pump function.Three-dimensional echocardiography demonstrated to be superior in comparison to conventional echocardiography in identifying RV dysfunction in patients with PH [73] (Figure 5; video 3). Recently, RV function assessed by 3D echocardiography has been reported to be prognostically important in patients with acute pulmonary embolism [74].Three-dimensional echocardiography of the right ventricle in a pulmonary hypertension patient, showing three apical views and corresponding short axis.CMR is used to define the anatomy, to assess biventricular function and haemodynamics, and to measure blood-flow by using phase contrast technique. Compared to echocardiography, CMR has higher spatial resolution, lower temporal resolution and does not suffer from the limitations about the acoustic window [75], allowing calculation of RV volumes and ejection fraction using the Simpson’s approach. CMR-derived RV volumes have shown good correlation with in vivo standards [76]. Moreover, this technique demonstrated good accuracy, when compared to the directly measured cardiac mass in calf hearts [77], with excellent intra- and inter-observer variability [78] and good interstudy reproducibility [79] in RV volumes, mass, and function quantification. At this time, CMR is regarded as the reference standard for the assessment of RV volumes (Figure 6) and ejection fraction [17,80]. Recent data obtained by CMR have demonstrated that normal values of RV systolic and diastolic parameters vary significantly by gender, body size, and age [81,82].Right ventricular volumes obtained with cardiac magnetic resonance in a patient affected by pulmonary hypertension. (A) End-diastolic volume; (B) End-systolic volume.In PH, RV end-diastolic volume [3] and ejection fraction [14] measured by CMR have demonstrated prognostic power. In particular, RV end-diastolic volume index ≤84 mL/m2, left ventricular end-diastolic volume index ≥40 mL/m2, and a stroke volume index ≥25 mL/m2 were associated with better survival in patients with idiopathic PH [3]. RV ejection fraction has also been assessed by CMR in PH patients and a value <35% predicts increased mortality [14]. Moreover, the RV mass demonstrated a diagnostic and prognostic role in PH patients, too [3,83,84]. An open issue about quantification of RV mass by CMR is whether or not the trabeculae and papillary muscles should be included in RV mass measurements. Previous studies have variably included and excluded them [79,85,86,87,88,89,90]. The reason why this issue is controversial is that RV is highly trabeculated in normal subjects and even more in PH patients, but we do not know if hypertrophy involves all the mass components in the same extent. Recently, van de Veerdonk et al. [91] demonstrated that in PH population trabeculae and papillary muscles give a large contribution (about 35%) to the RV mass. Moreover, the trabeculae and papillary muscle mass seem to be related to changes in pulmonary pressure, closer than the RV free wall mass. According to van de Veerdonk [91], Driessen et al. [92] have recently showed that inclusion or exclusion of trabeculae has an important impact on the RV volumes and mass measurements, particularly in patients with overloaded RVs. Then, these findings highlights two main aspects: (i) in presence of RV pressure overload RV hypertrophy is more prominent in the trabeculae and papillary components, than in the compact free wall; consequently, (ii) exclusion or inclusion of trabeculae and papillary muscles measurements significantly affect RV volumes and mass measurements.The presence of late gadoliniun enhancement (LGE) pattern in the RV myocardium of patients with PH has been first described by McCann and colleagues in 2005 [93]. LGE imaging shows a characteristic retention of gadolinium at the septal insertion (junctional pattern) [94,95,96], without any retention in the free wall [94] (Figure 7). Looking for LGE deposition in RV free wall of normal subjects or in pathologies like arrhytmogenic RV cardiomyopathy is difficult, because of partial volume effects due to the fact that the RV wall is particularly thin. Conversely, PH patients show thick RV walls, which does not pose any difficulty in nulling the RV myocardium. Further, LGE junctional pattern is not specific for PH, but it has been found in other pathologies like hypertrophic cardiomyopathy [97,98]. The etiology of this gadolinium retention remains to be clarified. The first hypothesis was the presence of fibrosis. Bradlow et al. [99] examined the heart of a patient with idiopathic PH and LGE junctional pattern at autopsy. They found increased collagen and fat between fiber bundles (plexiform fibrosis) consistent with myocardial disarray, but no pathological fibrosis. Myocardial disarray is common in healthy people at the interventricular junction site, but it appears particularly exaggerated in PH patients. A recent prospective observational study [100] demonstrated a moderate correlation among the presence of LGE junctional pattern and paradoxical septal motion, assessed by echocardiography, associated to a weaker correlation with RV function parameters. This data suggest that the abnormal interventricular septal motion, rather than the elevated RV pressure and the consequent remodeling may be the mechanism underlying the appearance of LGE junctional pattern. The reported occurrence of LGE junctional pattern in PH is not univocal, it ranges from 69% [101] to 100% [94]. The amount of the LGE junctional pattern has been demonstrated to be moderately related to the amount of RV dysfunction measured as ejection fraction, stroke volume and end-systolic volume [94] and it appears to predict RV remodeling in response to increased afterload [102]. Moreover, patients with the junctional pattern of the LGE are significantly more likely to experience a worse clinical outcome than those without this marker. LGE junctional pattern has been reported to be related to worse outcome in term of death, need of lung transplantation, initiation of prostacyclin therapy, and decompensated RV heart failure, stratifying prognosis [101].CMR imaging of a PH patient showing the typical septal junctional pattern of late gadolinium enhancement (yellow arrows).Swift et al. [66] showed that changes in RV morphology assessed by CMR are able to identify PH patients quite accurately. Particularly, presence of LGE junctional pattern, RV mass index, retrograde flow and pulmonary artery relative area change predicted the presence of PH with a positive predictive value of 98%, 97%, 95%, and 94%, respectively [66]. More recently, the same research group demonstrated that CMR imaging, in addition to provide detailed functional and morphological information about the RV, can accurately estimate mean pulmonary artery pressure in patients with PH and calculate pulmonary vascular resistance by estimating all major pulmonary hemodynamic metrics measured at right heart catheterization (RHC) [67]. If these findings would be confirmed in future studies and CMR will prove to be also accurate in estimating the changes in PVR/PAP over time, the follow-up of PH patients could be managed with CMR, reducing the need of serial RHC studies.Although CMR seems to hold promise for initial assessment and follow-up of patients with PH, data are currently limited. In addition, CMR is costly, is not widely available for serial follow-up of patients with PH and cannot be performed in patients with several conditions (severe renal disease, implanted methallic devices, claustrophobia, irregular heart rhythm). Therefore, at present, its main role currently consists in providing baseline assessment in patients in whom echo cannot be performed or it is not exhaustive [103].Several authors have validated the use of multi detector cardiac tomography (MDCT), in comparison with echocardiography, scintigraphic techniques, and CMR, for RV function assessment [104,105,106]. Although MDCT cannot be regarded as a first-line imaging technique because of the radiation dose and iodinated contrast agent injection involved, MDCT evaluation of RV function is accurate and provides information on the adjacent lung parenchyma [107]. Recent improvements in temporal and spatial resolution of the technique have allowed to obtain useful information about the heart [107]. However, the use of MDCT in the setting of PH has been mainly validated for the detection of acute pulmonary embolism and for the initial evaluation of the patient with PH in order to assess the presence of chronic pulmonary embolism or lung parenchima disease. Reference values for RV volumes and ejection fraction measured by MDCT have been recently published [108]. Compared with CMR, MDCT has lower temporal resolution and tends to overestimate end-systolic and end-diastolic volumes [109].Radionuclide techniques have historically been the first imaging modalities used for assessing RV size and function [110], although they have widely been replaced by CMR and echocardiography. First pass planar equilibrium radionuclide angiocardiography (FPRNA) is currently the technique of choice because it allows temporal separation of structures that are spatially superimposed [110,111]. However it has many limitations. Gated blood-pool single photon emission computed tomography (SPECT) offers adequate 3D resolution of the cardiac chambers, without the need for multiple acquisition views; then it is the currently recommended nuclear modality for quantifying RV function [110,111,112]. Unfortunately, the absence of completely automated and clinically validated processing software limits the utilization of this promising technique for RV evaluation. Recently, Anderson and colleagues [113] demonstrated significant correlations of RV volumes and function evaluated by gated blood-pool SPECT with CMR. Further studies for validation of automatic measurement algorithms are still pending. Radionuclide techniques are of additional particular interest for assessing myocardial metabolism and perfusion. Experimental studies using SPECT have shown that acute or chronic RV pressure overload leads to a myocardial metabolic shift from fatty acid to glucose [114].In the setting of RV evaluation in PH patients, nuclear imaging and MDCT do not have a major role. Their principal applications are in patients with controindications to CMR and/or inconclusive echocardiographic exams. Conversely, CMR and echocardiography, the latter with the new techniques STE and 3D echocardiography, are promising tools. They need to be tested in multicenter longitudinal studies to have their prognostic power recognized for clinical practice [103]. In Table 2, advantages and disadvantages of the various imaging modalities are briefly summarized.Advantages and disadvantages of the different imaging modalities in right ventricle (RV) evaluation in the pulmonary hypertension (PH) setting.Right ventricular function has been recognized as the main predictor of prognosis in patients with pulmonary hypertension. According to current guidelines, prognostic parameters are obtained from right heart catheterization, or from assessment of functional capacity either by WHO functional class or six-minute walking test. In this scenario, cardiac magnetic resonance and echocardiography have demonstrated to be able to assess right ventricular function in an accurate and reproducible manner, both using with conventional and new echocardiographic techniques. However, these new parameters still need further validation to be introduced in the clinical routine.two-dimensionalthree-dimensionalcardiac magnetic resonancefirst pass planar equilibrium radionuclide angiocardiographylate gadolinium enhancementmulti detector cardiac tomographypulmonary hypertensionright heart catheterizationright ventricle/ventricularsingle photon emission computed tomographyspeckle tracking echocardiographytricuspid annular plane systolic excursionAll authors contributed to the submitted work. Particularly, DP and LPB have written the review; DM and UC revised the echocardiographic section; FT and MPM revised the cardiac magnetic resonance section; GR and SI revised the multi detector cardiac tomography and nuclear imaging section.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Marfan syndrome is the most common inherited disorder of connective tissue affecting multiple organ systems. Identification of the facial, ocular and skeletal features should prompt referral for aortic imaging since sudden death by aortic dissection and rupture remains a major cause of death in patients with unrecognized Marfan syndrome. Echocardiography is recommended as the initial imaging test, and once a dilated aortic root is identified magnetic resonance or computed tomography should be done to assess the entire aorta. Prophylactic aortic root replacement is safe and has been demonstrated to improve life expectancy in patients with Marfan syndrome. Medical therapy for Marfan syndrome includes the use of beta blockers in older children and adults with an enlarged aorta. Addition of angiotensin receptor antagonists has been shown to slow the progression of aortic root dilation compared to beta blockers alone. Lifelong and regular follow up in a center for specialized care is important for patients with Marfan syndrome. We present a case of a patient with clinical features of Marfan syndrome and discuss possible therapeutic interventions for her dilated aorta.A 59-year-old woman presents to your office with characteristic facial and ocular features of Marfan syndrome (MFS) including dolichocephaly (elongated face), down-slanting palpebral fissures, high and arched palate, dental crowding and iridodonesis (vibration of the iris with eye movement due to lens dislocation). See video (in supplementary file). She was diagnosed with Marfan at age 3. She has no prior sternotomies. Her family history is notable for father suspected of having MFS and dying suddenly in his mid 50’s while sitting at home (no autopsy performed). Although she has 3 unaffected siblings, her 2 children have the syndrome with son requiring aortic valve-sparing surgery at a young age due to progressive aortic dilation. Additional diagnostic features of MFS include aortic dilatation and bilateral ectopia lentis with multiple prior interventions. She had prior wrist and thumb signs that have disappeared with age-related reduced joint mobility and weight gain, as well as known dural ectasia on MRI. She had never been tested for fibrillin-1 gene mutation due to having typical phenotypical characteristics of Marfan. She met diagnostic criteria for MFS based on the revised Ghent nosology (positive family history, systemic score > 7, ectopia lentis and aortic criteria Z-score > 2) [1]. She has followed regularly with imaging of her dilated proximal aorta. One year ago the 2D echocardiogram demonstrated an aortic sinus dimension of 42 mm, with no significant valvular heart disease. Today, the proximal aorta measures 45 mm with normal left ventricular function and no aortic valvulopathy. An MRA is also performed demonstrating an aortic sinus diameter of 48 mm. Her Z-score measured for her aortic sinuses is 4.79 (height 195.5 cm, weight 112.6 kg, BSA 2.45 m²). Her medical management includes losartan 50 mg PO BID. She was not on beta blocker therapy due to prior drug intolerance.Do nothing. Continue annual imaging evaluation until the aortic root diameter is > 50 mm.Refer to surgery for aortic valve-sparing root replacement.Refer to surgery for a composite graft replacement (Bentall procedure).Add a dihydropyridine calcium channel blocker to decrease the progression of aortic dilation.Dilated aortic root in a patient with Marfan syndrome.Serial aortic imaging studies in this patient demonstrated progressive aortic root dilatation close to 50 mm without associated aortic valve regurgitation. Based on a family history of presumed aortic dissection in her father and need for early surgery in her son, rapid progression of her aortic root size in one year, and patient preference for earlier intervention it was recommended to proceed with surgery. She underwent elective aortic valve-sparing root replacement with hemiarch repair using a Dacron graft and reimplantation of the coronary arteries into the prosthesis. Her initial postoperative course was complicated with left vocal cord paresis which has now been resolved.Marfan syndrome (MFS) is the most common inherited disorder of connective tissue affecting multiple organ systems [2]. It is an autosomal dominant condition due to a mutation in the fibrillin-1 gene (FBN1) leading to deficiency or malformation of the fibrillin-1 protein, a glycoprotein in the extracellular matrix, as well as excessive transforming growth factor β (TGF-β) signaling, resulting in fragmentation and disarray of elastic fibers [3,4], as well as activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade implicated in aneurysm progression [5]. It has an incidence of 2–3 per 10,000 individuals without predilection for gender, race or ethnicity. Early identification and appropriate management are critical since these patients are prone to life-threatening cardiovascular complications. Identification of the facial, ocular and skeletal features should prompt referral for aortic imaging since sudden death by aortic dissection and rupture remains a major cause of death in patients with unrecognized Marfan syndrome. Mean survival of untreated patients is 40 years, and asymptomatic aortic dilatation is present in 60%–80% of affected individuals with a variable rate of progression.Prophylactic aortic root replacement with a composite graft is safe and has been demonstrated to improve life expectancy in patients with Marfan syndrome [6]. Aortic valve-sparing aortic root surgery has also been deemed safe in those patients with anatomically normal valves. Our patient’s left ventricular function was normal, but left ventricular dysfunction can occur independently of aortic root or valvular disease. Echocardiography is recommended as the initial imaging test. Once a dilated aortic root is identified, MRA or CTA should be done to assess the entire aorta. Most patients will have aneurysmal change limited to the proximal aorta and can be followed with periodic echocardiography. Patients should undergo surgery when the aortic root maximal diameter is >50 mm, but earlier repair should be done when there are indicators of increased risk, including a family history of dissection, progressive dilatation of >5 mm/year, severe aortic or mitral regurgitation, or desire for pregnancy. Medical therapy for MFS includes the use of beta blockers (BB) in children and adults regardless of the presence or absence of an enlarged aorta [7]. Addition of angiotensin receptor antagonists (ARB) to BB has been shown to slow the progression of aortic root dilation compared to BB alone [8]. The use of losartan has been shown to reduce the rate of aortic growth rates before and after surgical replacement and thus it should be continued on this patient [9,10,11]. Calcium channel blockers (CCB) have only been evaluated in small trials and are sometimes prescribed if a BB is contraindicated based on their blood pressure lowering effect properties. However, they are less useful due to the relative lack of effect on cardiac inotropic state [12]. More recent animal studies using amlodipine or placebo in MFS vs. wild-type mice showed increased ascending aortic size, aortic growth and early mortality in the CCB group, suggesting potential deleterious effects on MFS individuals [10]. There is promising research for other pharmacologic therapies in the pathogenesis of thoracic aortic aneurysm; these include an ongoing trial with children and young adults comparing atenolol versus losartan on the rate of aortic root growth over three years [13]. Lifelong and regular follow up in a center for specialized care is important for patients with MFS. Even after repair, Marfan patients remain at risk for dissection. Stable patients should have a yearly visit with associated aortic imaging.Supplementary materials can be accessed at: http://www.mdpi.com/2079-9721/2/3/296/s1.We thank B.A. for participating in the development of this manuscript.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).The decrease in the prevalence of Helicobacter pylori (Hp) infection in children in the world gave rise to a new pathological finding termed as Hp-negative gastritis. Unfortunately, the term “Hp-negative gastritis” has not been identified as a pathological process and has the status of a “second cousin”; in most publications it was never mentioned as a subject to be dealt with, but was “left over” data that was never the topic of the manuscripts’ discussions. Only recently has the topic captured the attention of the pathologists who described this phenomenon in adults, yet the pathological and/or clinical spectrum or significance of this phenomenon has not been adequately investigated. In the current manuscript we describe Hp-negative gastritis in children, summarize its clinical prevalence and touch upon the possible etiology, pathology, and/or therapeutic implication. Overall, this review has concluded that Hp-negative gastritis is a pathological phenomenon in children that needs further investigation, and to date, as the title suggests, is a new clinical enigma that needs to be considered.Gastritis is a common finding in children who undergo diagnostic upper endoscopy. Although multiple etiologies have been associated with gastritis, in most pediatric cases the etiology of gastritis is unknown (idiopathic). Indeed, various diseases can be associated with gastritis including: Helicobacter pylori (Hp) organism, inflammatory bowel disease, allergic gastroenteritis, and others; but those comprise only the minority of the etiologies for gastritis. In the last 3 decades, the Helicobacter pylori organism has been the focus of gastritis in children, and many reports have shown that this bacterium is the etiology for moderate to severe gastritis [1]. However, the rate of the Hp-associated gastritis in children has been declining in recent years, especially in the developed countries [2,3,4]. The decline of the Hp infection rate in children in our practice was previously reported, demonstrating an infection rate between 3%–7% [5,6]. In the present manuscript, we will discuss the Hp-negative gastritis defined as an “idiopathic” gastritis, a condition that involves mucosal inflammation without Hp organism or other known etiologies.Before the rate of Hp-negative gastritis is discussed, it is important to define the framework of gastritis. Until the Sydney criteria were published, gastritis was defined loosely by the practicing pathologist without any standardization. In 1991, a group of pathologists gathered and produced an important document described as “the Sydney criteria” [7]. A few years later (1994) the document was revised and the histological standard for “gastritis” was established [8]. In this document the various forms of gastritis were described and the pathologists’ language became standardized. As in many clinical guidelines, the documentation provided a practical guideline but not all pathologists follow those recommendations in their routine practices. Accordingly, when discussing the rate of Hp-negative gastritis in our medical community, we need to make sure that the local pathologists follow those guidelines. Indeed, our experience suggests that unless the report is a prospective clinical study that included “Sydney Criteria” in the methodology, the description of gastritis may not be standardized or accurate. The average rate of Hp-negative gastritis reported from our center throughout the years showed that when pathology standardization is part of the prospective studies, the rate of Hp-negative gastritis was lower compared to the rate reported in the retrospective studies [mean rate (%): 37% vs. 54%, respectively] [9]. The histopathology of Hp-associated gastritis has been well described and its association with gastric cancer, gastric pathology and/or mucosal immune changes associated with it was documented [10]. Nevertheless, as Hp-negative gastritis has not been fully appreciated, there are no specific descriptions for this entity except for a simple description of mild to moderate gastritis characterized by the Sydney criteria. As the interest of this entity increases further pathological details will be forth coming through the efforts of various investigators.The rate of Hp-negative gastritis in children has not been evaluated adequately. In most cases the data are derived from retrospective studies that did not concentrate on this subject and held no direct discussion of this diagnosis. Most of the data is usually an ad hoc analysis calculated from the data provided in the reports. Moreover, with the declining prevalence of Hp-infection in children in developed countries, the rate of “idiopathic” gastritis is now more evident. For example, we recently reviewed the charts of the first diagnostic upper endoscopic procedures performed in our practice during 2013. A total of 135 procedures were completed and a comprehensive set of biopsies (esophagus, stomach, duodenal bulb, and small intestine) were available in all children. In this cohort, gastritis was identified in 104 (75%) children, of whom 4 (3.8%) children had Hp-associated gastritis, and 10 (9.6%) children had a non-Hp etiology for their gastritis. Finally, a total of 90 (86%) children showed Hp-negative (“idiopathic”) gastritis (unpublished data). This data followed our summary report documenting the rate of Hp-negative gastritis in our patient population since the early 1990’s [9].In a recent publication, Kara et al. reviewed the histology of children who had upper endoscopy procedures performed. In a total of 358 procedures, 214 (60%) cases had Hp infection and the histological description of those patients was reported. Yet, in a total of 144 children, Hp negative gastritis was detected [11]. Of the Hp-negative patients, 98 children had corpus gastritis and 99 children had antral gastritis. As the focus of the study was Hp infection the histological description for the Hp-negative children was limited.In most pediatric publications, the data are retrieved from retrospective studies and the analysis is not directed toward Hp-negative gastritis. It is assumed that prospective studies of this topic would be more beneficial and accurate. Nonetheless, even in those reports, the specific analysis of Hp-negative gastritis was not the main topic of the discussion [12,13,14]. In a recent prospective study, a Chinese group assessed the endoscopic findings of children diagnosed with functional abdominal pain [15]. Only patients who were positive by Rome 3 criteria were included. A total of 80 children were evaluated of whom 5 had significant mucosal findings including duodenitis, gastritis, and mucosal erosion. Seventy-five children showed no significant gastric pathology. Nevertheless, the “non-significant findings” were described by the pathologists as descriptive changes such as: reactive mucosal changes and mild chronic inflammatory cell infiltrates (mild gastritis) [15]. The long term prognosis and the clinical significance of those “non-significant findings” in children have not been elucidated.The topic of Hp-negative gastritis or the lack of detection of Hp organism in the stomach of adult patients has been raised previously by Yoo et al. [16] and had an editorial response by Genta [17]. In those publications, missing Hp organism in the mucosa was attributed to atrophic gastritis/ metaplastic epithelium and to other associated clinical reasons such as: chronic use of proton pump inhibitors (PPI) and antibiotic use. In recent publications, the topic of Hp-negative gastritis in adult patients has been raised by a few investigators. In a prospective analysis of 491 adult patients, Nordenstedt et al. evaluated the rate of gastritis and its associated pathology and confounding factors [18]. In this study the authors demonstrated gastritis in 200 (40.7%) patients of whom 41 (20.5%) were Hp-negative. Of the confounding factors, alcohol consumption and smoking were significantly associated with Hp-negative gastritis but not drugs (PPI, NSAID). The authors concluded that the “causes and implications of this entity (Hp-negative gastritis) are unknown and worthy of future studies” [18]. In an editorial that followed this report the author reminded us of the other common reasons for missing the bacterium in the biopsies of adult patients including: sampling error, or chronic use of PPI and antibiotics [19]. A more innovative explanation is suggested by the author, but needs further confirmation and research, involves the gastric microorganisms (GI microbiota) that are now at the forefront of the investigators interest [19]. For the pediatric population (especially the younger age group), some of those reasons are limited (tobacco use, alcohol, NSAID, or atrophic gastritis), but others could play a role in lack of detection of the bacterium (antibiotic and PPI usage). To better understand this topic, prospective studies will need to consider those factors in their study planning.In the last few decades the human microbiome project was completed and published. Within that project, many uncultivable bacteria that inhabit the normal gastrointestinal tract were reported [20]. Indeed, the project reported that there are up to 1833 uncultivable colonies living in the normal stomach [21]. Previous reports suggested that the local gastric microbiome might interact with the H. pylori organism and may influence the development of peptic ulcer disease, or gastric cancer [22]. Other authors documented the possible interaction of the gastric microbiome with different human diseases and warned that “comprehensive knockout” of all microorganisms in the stomach may not be beneficial for the human health [23].In a recent editorial, Genta et al. suggested that the etiology for Hp-negative gastritis in adults may be related to the bacterial flora that inhabits their stomach. Indeed, the current research implicates the gastric microbiome in the pathophysiology of H. pylori infection but there is no clear evidence that describes its effect in the development of idiopathic gastritis in adults or children. Moreover, Kato et al. investigated the bacterial gastric flora in Hp negative children and adults (10 pts/group). Several microorganism species were detected in the stomach of adult patients but very few in children [24]. Unfortunately, in that report, histological description of the gastric mucosa was not available; thus, the association of the gut flora and idiopathic gastritis could not be assessed. Moreover, the repertoire of the gastric microbiota in children during normal development or during different disease states have not been adequately investigated or published. It is concluded that the gut microbiome may have an important role on the pathophysiology of many human diseases, but its relationship towards idiopathic gastritis, especially in children, has yet to be proven.In summary, the rate of Hp-negative gastritis has been reported in adults and children. The “frustration” of the pathologists described by Genta et al. [19] related to positive gastritis in the absence of Hp organism in the biopsy is well respected. It is assumed that a similar situation is part of the pediatric pathologists’ “frustrations” as well. Unfortunately, due to the lack of research, the etiology behind this finding as well as the clinical significance of Hp-negative gastritis has not been elucidated in children. As the term “gastritis” means inflammatory process by definition, many pediatric gastroenterologists in their clinical practice would treat those patients for the inflammation. The lack of correlation between patients’ symptoms and idiopathic gastritis [25,26] may suggest that those patients should not be treated. Unfortunately, we are lacking appropriate studies to direct our therapeutic decisions in either direction. Future studies should target the topic of “Hp negative gastritis” (idiopathic) to identify possible pathological factors and prospective clinical studies that should be planned in order to direct physicians to answer the Shakespearian paraphrase: “to treat or not to treat: this is the question”.The present manuscript reviews the topic of Hp-negative gastritis in children. With the decreased rate of Hp infection in children around the world it is expected that this condition will increase and become more recognized. Unfortunately, the pathophysiology and the clinical implications of this condition are unknown. As the interest in the gastrointestinal microbiome increases, new etiological factors may be discovered, advancing our knowledge towards solving this enigma.Yoram Elitsur MD: Guarantor of the article, study design, manuscript preparation, critical review, final approval of the manuscript. Deborah L Preston BS: Complete chart review, data collection, data analysis, manuscript edits.The authors declare no conflict of interest.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Our aim is to investigate the recent liver biopsy findings of autoimmune liver diseases at a university hospital located in an urban area of Japan. The study included 259 patients (mean age 56.8 ± 12.5; male/female, 46/213) who underwent a liver biopsy for primary biliary cirrhosis (PBC) or autoimmune hepatitis (AIH). We analyzed their liver biopsy findings according to age and gender. Among 127 PBC patients, Scheuer stages 1, 2, 3, and 4 were 42, 54, 18, and 13, respectively. Among 101 AIH patients, fibrosis stages F1, F2, F3, and F4 were 37, 32, 19, and 13, respectively, and inflammatory activity grades A1, A2, and A3 were 22, 25, and 54, respectively. Among PBC aged ≥65 years, Scheuer stages 1–3 and 4 patients were 27 and 6, respectively. The proportion of Scheuer stage 4 patients in PBC aged ≥65 years tended to be higher than that in PBC aged <65 years (p = 0.0659). Of interest, the proportion of AIH patients with moderate or severe activity (A2 or A3) in males was higher than in females (p = 0.0311). From the point of view of fibrosis stage or inflammatory activity grade of the liver, the proportion of AIH patients aged ≥65 years was similar to that aged <65 years. Although we identified six older cirrhotic patients with AIH, three of them were male. The progression of fibrosis and inflammatory activity of the liver should be noted when we treat older patients suffering from autoimmune liver diseases. Liver biopsy plays an important role in obtaining accurate information on autoimmune liver diseases in older patients.Primary biliary cirrhosis (PBC) is a progressive chronic cholestatic liver disease, characterized by non-suppurative cholangitis, that mainly affects middle-aged women [1,2]. The reported number of patients with PBC has increased dramatically in the United States [2], United Kingdom [3], and Japan [4,5]. Understanding of the pathophysiology and earlier diagnosis of PBC has increased its prevalence worldwide [6]. The natural history of PBC has improved greatly during the past two decades because of its diagnosis at earlier stages and the widespread use of ursodeoxycholic acid (UDCA) [7,8]. As a result, far fewer patients require liver transplantation [7].Autoimmune hepatitis (AIH) is characterized by chronic inflammatory liver disease, which, when not treated with prednisolone alone or in combination with azathioprine, leads to liver cirrhosis. It also affects mainly women [9]. AIH can occasionally have clinical, laboratory, histological, and genetic characteristics identical to those of PBC [10]. Some cases of AIH are difficult to diagnose [10,11,12,13,14]. The incidence of AIH differs in various geographic regions. The incidence of type 1 AIH among Caucasoid populations in Europe and North America ranges from 0.1 to 1.9/100,000/year [15]. A population-based epidemiological study of AIH in New Zealand demonstrated that age-standardized incidence and prevalence were 1.7 and 18.9 per 100,000, respectively [16]. In Japan, the prevalence is as high as 1.0 per 10,000 [17]. Except for atypical presentations of AIH [12,18,19], early administration of corticosteroid usually leads to a relatively better prognosis for this disease.Histological assessment of the liver is valuable for evaluation and management of patients with liver diseases even if sensitive and relatively accurate blood testing is available [20]. Liver biopsy also plays an important role in the diagnosis, staging, prognosis and management of PBC and AIH [20]. In Japan, recent opportunities to manage patients with autoimmune liver diseases such as AIH and PBC have been increasing. This tendency is especially apparent in the older population, as many more people live to an advanced age in Japan. For older patients with liver diseases, the risk of cirrhosis and hepatocellular carcinoma (HCC) is more critical than for the young [21], although there is a contrary opinion [22]. In the present study, to clarify the features of older patients with autoimmune liver diseases at a university hospital located in urban Japan, we analyzed the histological features of these diseases by reviewing the recent findings of liver biopsies.Between 2000 and 2011, 259 patients were diagnosed with AIH and PBC through liver biopsy specimens; we reviewed and re-analyzed these liver biopsy findings at the Department of Gastroenterology and Nephrology, Chiba University, Graduate School of Medicine, Japan. All subjects gave their informed consent for inclusion before participating in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Chiba University School of Medicine (No. 1841).We conducted liver biopsies as follows: a patient lay face-up on a bed, right hand resting above the head. The patient was given a local anesthetic to the body surface area where the biopsy needle would be inserted. The patient was administered 15 mg of pentazocine hydrochloride, 0.25 mg of atropine sulfate, and 25 mg of hydroxyzine pamoate as premedication. We made a small incision in the right side of the patient’s abdomen, just below the rib cage. Liver biopsy was performed with 14–20 gauge automatic or manual Tru-cut biopsy needles under ultrasound guidance. Biopsy samples were immediately placed into 10% formalin solution (Wako Pure Chemical, Osaka, Japan).Three hepatologists blindly evaluated the histopathological findings of liver biopsy samples with hematoxylin and eosin (HE) stain. Scheuer’s classification was used for evaluation of histopathological findings of PBC: a lesion characteristic of bile ducts was classified as Stage I; florid bile duct lesions were judged as Stage II; and hepatic fibrosis and cirrhosis were categorized as Stages III and IV, respectively [23]. The liver tissue samples from AIH patients were evaluated with fibrosis staging and inflammatory activity grading of the liver in chronic liver diseases [24]. We classified samples from AIH patients into no fibrosis (F0), mild fibrosis (F1), moderate fibrosis (F2), severe fibrosis (F3), and cirrhosis (F4); or minimal activity (A0), mild activity (A1), moderate activity (A2), and severe activity (A3) for staging the extent of fibrosis or grading the activity of inflammation, respectively.For all tests, two-sided P-values were calculated and the results were considered statistically significant at p < 0.05. Statistical analysis was performed using the Excel statistics program for Windows, version 7 (SSRI, Tokyo, Japan) and DA Stats software (O. Nagata, Nifty Serve: PAF01644).A total of 259 patients with autoimmune liver diseases were enrolled into the present study (Table 1). Female patients dominated (82.2%) the patient groups: 81.7% and 82.9% of PBC and AIH patients, respectively. According to our records, age was unknown in nine patients with PBC and 35 patients with AIH, although liver biopsy was still performed on these patients. The number of older patients, ≥65 years, was at least 27.4%: 25.2% and 29.7% of PBC and AIH patients, respectively. Liver biopsy findings from four PBC and 27 AIH patients were not available.Characteristics of patients with autoimmune liver diseases in the present study.Note: PBC, primary biliary cirrhosis; AIH, autoimmune hepatitis.In the present study, the total number of PBC was 131. Liver biopsy was performed in 127 of 131 patients in the years shown in Figure 1A. Between three and 15 PBC cases, including 13 with Scheuer stage 4, were biopsied in each year. Among them, males and females were 24 and 103, respectively (Figure 1B,C). Among the 13 patients with Scheuer stage 4, four were males and nine were females.PBC patients aged <65 years and ≥65 years numbered 91 and 33, respectively (Figure 2A,B). Among PBC patients aged <65 years, Scheuer stages 1–3 and stage 4 patients numbered 86 and five, respectively. Among PBC patients aged ≥65 years, Scheuer stages 1–3 and stage 4 patients numbered 27 and 6 respectively. The proportion of Scheuer stage 4 patients aged ≥65 years tended to be higher than that in those aged <65 years (p = 0.0659). The 11 PBC patients with Scheuer stage 4 are shown in Table 2A. All six patients aged ≥65 years with Scheuer stage 4 were female.Scheuer stage of primary biliary cirrhosis (PBC) patients in the present study. (A) Total patients (n = 127), Stage 1–3: Stage 4 = 114:13; (B) Male patients (n = 24), Stage 1–3: Stage 4 = 20:4; (C) Female patients (n = 103), Stage 1–3: Stage 4 = 94:9.Scheuer stage of primary biliary cirrhosis (PBC) patients according to age. (A) Age <65 (years) (n = 91); (B) Age ≥65 (years) (n = 33).Cirrhotic patients of primary biliary cirrhosis and autoimmune hepatitis in the present study. (A) Patients with Scheuer stage 4 of primary biliary cirrhosis; (B) Cirrhotic patients with autoimmune hepatitis.In the present study, the total number of AIH was 128. Table 3A shows the yearly schedule of liver biopsy of 101 of the 128 patients. Among them, males and females numbered 18 and 83, respectively (Table 3A,B). Between 2000 and 2011, the annual number of AIH was between six and 16 cases, including 13 cirrhotic patients (four males and nine females). The proportion of cirrhotic patients with AIH did not differ statistically between males and females (Table 3A). The proportion of AIH patients with moderate or severe activity (A2 or A3) was higher for males than for females (p = 0.0311) (Table 3B). Of interest, among the AIH patients, all male patients had A2 or A3 inflammatory activity grade of the liver.Histopathological findings of the liver in autoimmune hepatitis according to gender in the present study. (A) Staging of fibrosis; (B) Grading of inflammatory activity.AIH patients aged <65 years and ≥65 years numbered 57 and 36, respectively (Figure 3 and Table 4). Among those aged <65 years, fibrosis stage 1–3 and 4 patients numbered 51 and six, respectively, and among those aged ≥65 years, fibrosis stage 1–3 and 4 patients numbered 30 and six, respectively. The proportion of cirrhotic (F4) patients with AIH aged ≥65 years was the same in those aged <65 years (not statistically significant) (Table 4A). The 12 cirrhotic patients with AIH are shown in Table 2B. We identified six older cirrhotic patients with AIH, but three of them were male (Table 2B).Annual numbers of autoimmune hepatitis patients with liver biopsy in the present study. (A) Total patients (n = 128); male: female = 22:106; (B) Age ≥65 (years) (n = 38), male: female = 7:31.Histopathological findings of the liver in autoimmune hepatitis according to age in the present study. (A) Staging of fibrosis; (B) Grading of inflammatory activity.Among AIH patients aged < 65 years, patients with inflammatory activity grades A1 or A2 and A3 numbered 16 and 41, respectively. Among AIH patients aged ≥65 years, patients with inflammatory activity grades A1 or A2, and A3 numbered six and 30, respectively. The proportions of A2 and A3 patients with AIH aged ≥65 years were similar to those aged <65 years (not statistically significant) (Table 4B).In the present study, recent trends for patients with autoimmune liver diseases and liver biopsies performed at a university hospital located in an urban area of Japan were investigated. Among the patients with PBC or AIH, at least 25.2% or 29.7%, respectively, were ≥65 years old. This can likely be attributed to the fact that the opportunity to diagnose and treat older people suffering from autoimmune liver diseases has increased in line with the aging of society in Japan. As a result of the development of non-invasive serum markers of hepatic fibrosis and transient elastography [25,26,27], with a concomitant decrease in occasions for liver biopsy, these numbers might in fact be underestimated. Kim et al. [2] reported that, in the United States, the peak incidence of patient age with PBC was 60–69 years for men and 70–79 years for women. More recently, Japanese national surveillance [28] reported that the peak incidence of patient age with PBC was 60–69 years for men and 50–59 years for women, regardless of asymptomatic or symptomatic PBC.In the present study, we demonstrated that the proportion of PBC Scheuer stage 4 patients aged ≥65 years tended to be higher, and that all our six patients with stage 4 were female (Table 2A). Harada et al. [29] reported that the histological stage at the time of HCC diagnosis was an independent risk factor for HCC in females and that the time between the diagnosis of PBC and that of HCC was significantly shorter in males than in females. Attention should be paid to older PBC patients with advanced fibrosis.For AIH patients, it seems that aging has no effect on stage, indicating that this disease can appear at any age, and could be classified into three different types of AIH [30]. Miyake et al. [31] reported that there are no apparent differences in the characteristics of AIH between older and younger patients, supporting the results of the present study. Although we identified six older cirrhotic patients with AIH, three of them were males. Thus, AIH is not just a severe female disease. Among older patients with AIH, confirming the accuracy of information about the disease seems important. Interestingly, we discovered that the proportion of AIH patients with moderate or severe activity (A2 or A3) is higher for males than for females (Table 3), indicating that among male patients with liver dysfunction of unknown etiology, AIH should be considered a possible etiology of their liver diseases. Liver biopsies could be useful in male patients with liver dysfunction of unknown etiology. It was reported that an androgenic/anabolic steroid could induce toxic hepatitis [32]. Gender differences in the proportion of AIH patients with moderate or severe activity (A2 or A3) might be caused by the effects of sex steroid hormone receptors such as androgen receptors [33,34]. Further studies will be needed. The number of female patients with PBC or AIH was greater than male patients, and this may reflect the population of these autoimmune liver diseases. In spite of this tendency, the number of older cirrhotic patients with AIH was equally split between males and females (Table 2B). This may be due to the fact that male patients with AIH tend to have a more severe stage than females, or that it might be difficult to diagnose male patients with AIH using the present criteria [35,36]. Special attention should also be paid to the diagnosis and/or treatment of older male patients with AIH. In such cases, liver biopsies could be useful, and older patients with autoimmune liver diseases may have more severe cases than expected.In Japan, HCC developed in 5.1% of patients with AIH, and cirrhosis was commonly found in elderly individuals [37]. Ohira et al. [37] reported that the mean age at diagnosis of HCC was 69 years, and that the male-to-female ratio was 1:5.7. AIH patients with liver cirrhosis are at high risk for HCC. Although diagnosis of AIH has recently been performed using the AIH scoring system [35,36] in consideration of the possibility of other liver diseases, other fibrogenetic factors such as non-alcoholic fatty liver diseases and alcohol intake should also be considered [36,38]. In the present study, we found 12 cirrhotic patients with AIH (Table 2); only one patient revealed alcohol intake, but four patients were overweight or obese. In Japanese cirrhotic AIH patients, these fibrogenetic factors might have partly contributed to this condition.Previous studies have established that PBC is associated with HLA-DR8 [39] and six non-HLA loci: IL12A, IL12RB2, IRF/TNPO3, ORMDL3/IKZF3, MMEL1, and SP1B [40]. Mells et al. [40] reported that STAT4, DENND1B, CD80, IL7R, CXCR5, TNFRSF16F, and NFKB1 were new candidate genes associated with the genetic risk factors for PBC. A recent genome-wide association study revealed TNFSF15 and POU2AF1 as susceptibility loci for PBC in the Japanese population [41]. Another genome-wide association study revealed that AIH type 1 was associated with variants in the major histocompatibility complex region, and identified the variants SH2B3 and CARD10 as likely risk factors [42].In PBC patients, the aspartate aminotransferase-to-platelet ratio index (APRI) was significantly correlated with the histological degree of liver fibrosis, with a limited value of scores between 1.0 and 1.5 [43]. Although it was reported that APRI appeared to be of no value in AIH patients [44], serum surrogate markers such as hyaluronic acid level, type 4 collagen level, and APRI, as well as liver stiffness measurement, are helpful in predicting significant fibrosis of the liver in non-viral liver diseases [25]. In the near future, we will not have to perform a liver biopsy to diagnose PBC and AIH. Further studies of the differences between AIH pathology and PBC pathology might shed some new light on better management of these diseases.In Japan, opportunities to manage patients with autoimmune liver diseases such as AIH and PBC have been on the increase. In conclusion, we should pay particular attention to the progression of fibrosis and inflammatory activity of the liver when we treat older patients suffering from autoimmune liver diseases. For this reason, at the present time, liver biopsy is recommended for obtaining accurate information.This work was conducted by the Scholarship Program, Chiba University, School of Medicine, Chiba, Japan (Tatsuo Kanda, Katsuhiro Hagiwara, and Osamu Yokosuka). We would like to thank the medical staff of Chiba University Hospital for their assistance and support during this study.Yuki Haga and Tatsuo Kanda collected and analyzed data, saw the patients, and contributed to the writing and editing of the manuscript. Katsuhiro Hagiwara collected and helped analyze data. Reina Sasaki, Masato Nakamura., Shin Yasui, Makoto Arai, and Osamu Yokosuka saw the patients. Xia Jiang, Shuang Wu, and Shingo Nakamoto collected and analyzed data and helped edit the manuscript.The authors declare no conflict of interest.

Med-MDPI/diseases/diseases-03-01-00001.txt

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).The editors of Diseases would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2014:
+	  
+      Alexandraki, Krystallenia I.
+      Applemelk, Ben
+      Barry, Meagan A
+      Bauer, Natalie
+      Blaner, William
+      Bodet, Charles
+      Burger, Charles D.
+      Carrel, Thierry
+      Chan, Anthony WH
+      Chan, Stephen L.
+      Chiu, Hsin-Hui
+      De Cooman, Lien
+      D'elios, Mario M.
+      Dellegrottaglie, Santo
+      den Hollander, Wouter J.
+      Ducharme, Anique
+      Eladari, Dominique
+      Emoto, Noriaki
+      Figura, Natale
+      Fraga, Garth
+      Gaetano, Carlo
+      Genta, R. M.
+      Groepenhoff, Herman
+      Guarner, Jeannette
+      Guignabert, Christophe
+      Han, Ying
+      Harada, Kenichi
+      Houghton, Michael
+      Johnson, Martin K.
+      Karanikolos, Marina
+      Kari, Fabian A.
+      Ko, Christine
+      Lange, Tobias J.
+      Li, Wei
+      Liou, Jyh-Ming
+      Liu, Chun-Jie
+      Lu, Da-Bing
+      Markovska, Rumyana
+      Moriyama, Mitsuhiko
+      Morris, Shaine
+      Naeije, R.
+      Newbigging, Susan
+      Paulin, R.
+      Paunescu, Teodor G.
+      Piersigill, Alessandra
+      Rautelin, Hilpi
+      Robinson, Karen
+      Samoutis, George
+      Santucci, Annalisa
+      Schneider, Barbara
+      Sharma, Amar Deep
+      Song, Howard K.
+      Song, Huan
+      Sugimoto, Mitsushige
+      Takeda, Norifumi
+      van der Veerdonk, Mariëlle C
+      Wells, J. Michael
+      Wort, John
+    
+	  Alexandraki, Krystallenia I.Applemelk, BenBarry, Meagan ABauer, NatalieBlaner, WilliamBodet, CharlesBurger, Charles D.Carrel, ThierryChan, Anthony WHChan, Stephen L.Chiu, Hsin-HuiDe Cooman, LienD'elios, Mario M.Dellegrottaglie, Santoden Hollander, Wouter J.Ducharme, AniqueEladari, DominiqueEmoto, NoriakiFigura, NataleFraga, GarthGaetano, CarloGenta, R. M.Groepenhoff, HermanGuarner, JeannetteGuignabert, ChristopheHan, YingHarada, KenichiHoughton, MichaelJohnson, Martin K.Karanikolos, MarinaKari, Fabian A.Ko, ChristineLange, Tobias J.Li, WeiLiou, Jyh-MingLiu, Chun-JieLu, Da-BingMarkovska, RumyanaMoriyama, MitsuhikoMorris, ShaineNaeije, R.Newbigging, SusanPaulin, R.Paunescu, Teodor G.Piersigill, AlessandraRautelin, HilpiRobinson, KarenSamoutis, GeorgeSantucci, AnnalisaSchneider, BarbaraSharma, Amar DeepSong, Howard K.Song, HuanSugimoto, MitsushigeTakeda, Norifumivan der Veerdonk, Mariëlle CWells, J. MichaelWort, John

Med-MDPI/diseases/diseases-03-01-00002.txt

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Personalised external aortic support was first proposed in 2000 by Tal Golesworthy, an engineer with familial Marfan syndrome and an aortic root aneurysm. After putting together a research and development team, and finding a surgeon to take on the challenge to join him in this innovative approach, he was central to the manufacture of the device, custom made for his own aorta. He was the patient for the ‘first in man’ operation in 2004. Ten years later he is well and 45 other people have had their own personalised device implanted. In this account, the stepwise record of proof of principle, comparative quantification of the surgical and perioperative requirements, 10 years of results, and development and research plans for the future are presented.Personalised External Aortic Root Support (PEARS) has undergone health technology appraisal by the National Institute for Health and Care Excellence [1]. After 10 years of clinical experience since the ‘first in man’ [2] a personalised support has now been implanted in 46 patients in three English hospitals and one Belgian hospital. Twenty of these patients were operated on more than five years ago and follow-up is now more than 200 patient years for the whole experience. The purpose of this paper is to summarise the evidence available to date. The PEARS study team believe that this surgery might now be made available to a wider group of patients but in a carefully controlled fashion as befits its novel and innovative status. Such an approach has been thoroughly described under the IDEAL recommendations [3,4,5,6,7,8].This operation is intended to prevent on-going dilatation of an already morphologically abnormal aorta with the purpose of reducing the risk of death due to dissection in the aortic root. Most of the early patients have Marfan syndrome, but we see this operation as applicable to selected patients with root aneurysms due to Loeys-Dietz and Ehlers-Danlos syndrome and other genetically determined etiologies. It is the morphology of the aortic root aneurysm and the risk of dissection and rupture that are what we have to focus on in the present state of knowledge, not the genotype. PEARS might prove to be particularly applicable to patients early in their natural history.Marfan syndrome is a rare disease estimated 20 years ago in a population in north east Scotland to have a minimal incidence of about 1 in 10,000 people, a minimal prevalence of 1 in 14,000 and about a quarter of cases were new mutations [9]. A recent report from Taiwan provides an estimate of 1 in 4286 which would potentially nearly double the number of operations [10].People with Marfan syndrome are prone to aortic dissection and death predominately in their twenties and thirties. Before root replacement (effectively pre 1970) aortic dissection killed two thirds of all patients recognized as having Marfan syndrome [11,12,13]. Prophylactic root replacement potentially neutralizes that hazard and is the recommended management for patients who meet the widely accepted criteria defining them as being at high risk [14]. An estimate of the annual need for this surgery in UK is a mere 110 operations indicating the difficulty of accumulating a large series for observational studies or of finding candidates for a randomized trial (Table 1). Patients with other causes of genetically determined aortic root aneurysms which are considerably rarer than Marfan syndrome can also be considered as candidates for this surgery. To undertake a prospective comparative study, innovative study designs, of which we have experience in other fields of surgery, would be needed [15,16]. International collaboration would be required because of the relatively small numbers involved.Root replacement for congenitally determined aortic root aneurysms is available in two forms [17]. Each has advantages and limitations.First carried out in 1968 at Hammersmith Hospital in London, composite replacement of the aortic root and valve is generically known as the Bentall operation [18]. The operation as carried out now is very different to that first reported by Bentall in 1968. It has been refined during the subsequent 20 years [19] to become a highly reproducible operation, performed at low risk in expert hands. Its limitation is that a mechanical valve is usually used and there is an ensuing life-long risk of valve thrombosis. Anticoagulation is therefore mandatory and there is a consequent life-long risk of bleeding (Table 2). In an important meta-analysis by Benedetto the thromboembolism/bleeding event rate was 7% per decade. The overall rate of serious valve related events was 13% [20].Best estimates of the potential number of patients in the UK who may be candidates for aortic root surgery for congenitally determined aortic root aneurysms.† These estimates are from 20 years ago. A recent report in the Mayo Clinic Proceedings [10] revises this estimate to 1:4286 which would potentially nearly double the number of operations. * It has proved difficult to disentangle the number of these operations currently done amongst the data for root replacement for other indications.Comparative demography and outcome data from meta-analysis compared with Personalised External Aortic Root Support (PEARS).* Data for TRR and VSRR from Benedetto et al. 2011 [20]; ** for PEARS from Treasure et al. 2014 [21].In the early days in the 1970s, operative risk was considerable, as was reflected in the conservative thresholds for surgical intervention at the time. In 1981, when we as young surgeons were embarking on our careers, Johns Hopkins surgeons wrote ‘Until recently, surgical correction of Marfan defects of the aortic root has been undertaken with some hesitancy because of the high perioperative risk’ [22] but in that paper, after operating on 13 patients, they already saw the need to bring down the operative threshold to 5.5 cm. For the first 50 Bentall operations reported from Johns Hopkins in 1986 the smallest aortic size was 5.3 cm and the average size was greater than 7 cm [23]. Already the operative mortality in their hands was low; perioperative mortality approaching zero is now the expectation for composite root replacement.Forms of valve sparing root replacement were independently introduced by Yacoub in London [24,25,26] and David in Toronto [27,28,29]. The attraction of VSRR is that it spares the patient the need for life-long anticoagulation. Yacoub remodelled the aortic root with Dacron in a free-hand fashion. It is technically demanding and fell out of favour because of an unacceptable rate of progressive dilatation of the aortic annulus by the 10th postoperative year such that the valve became regurgitant. In David’s operation the aortic valve is enclosed within the tube graft to avoid this problem. It has had several iterations and has not proved consistently reproducible. Its limitation remains the technical challenge of valve conservation so the intended operation cannot always be completed and rescue valve replacement (TRR) is then done so the expected freedom from anticoagulation is not realised. In follow-up studies this is generally unreported so the intention-to-treat principle of reporting is defeated. The longer term failure rate of the conserved valves is about 13% per decade [20,30] (Table 2).It is difficult to be certain of the mortality of valve sparing root replacement by intention-to-treat because it is in the nature of this surgery that in some cases it is ‘exploratory’ and only during the operation will it be clear whether or not the valve will be conserved. The only intention-to-treat analysis of which we are aware is the Aortic Valve Operative Outcomes in Marfan Patients Study (AVOOMP) report in which there was one death among 239 patients having valve sparing surgery (upper 95% confidence interval 2%) [31]. It is impressively low but we cannot be sure of the extent to which the intention was revised during the exploratory phase of surgery and whether this excellent low mortality is reproducible outside of the high quality collaborating centres [31].There are many patients whose lives have been saved and survival extended by these operations but the perfect solution has not been reached. These are young patients, averaging around 30 at the time of surgery so nearly half are in their 20s. They may reasonably expect 50 years of life. During that time span the cumulative rates of valve related events, valve failure and reoperation have to be included ideally by intention-to-treat and should be considered in the advice and recommendations given to patients.Avoidance of thromboembolic risk and anticoagulation was the motivation for surgeons to move from the reliable and highly reproducible operation that the modern Bentall had become during the 1990s to valve sparing root replacement which began to gather momentum in the 2000s following the reports from David [28]. PEARS shares the advantage of valve sparing root replacement in avoiding anticoagulation. In addition as a non-ablative operation it ‘burns no bridges’ and so there is an argument for offering it earlier in the evolution of aortic root aneurysm. It has the potential to save patients the anxiety of repetitive aortic root monitoring which may go on for years [32].Modern methods of monitoring anticoagulation, including patient self-monitoring, have been associated with improved outcomes with respect to thromboembolism and bleeding [33,34]. However, a study from the department of Health Psychology at King’s College London (KCL) found that avoidance of anticoagulation was a strong and recurring theme for people with Marfan syndrome [32]. Daily medication and being monitored with blood tests made them feel they were in a ‘sick’ role; they wanted to be ‘normal’ people getting on with their normal lives.Having the intervention sooner than might have been justifiable for root replacement was for them a benefit because of reduction in the anxiety associated with regular monitoring. There was a quantifiable reduction in anxiety (HADS: Hospital Anxiety and Depression Scale) after PEARS [32]. Reduction in hospital attendance and medication was for them a highly desirable outcome. The study is undergoing peer review for publication. It should be noted that the participants had all had PEARS surgery and were contributing to focus groups. To verify the finding in a wider patient group a further study is ongoing at KCL.The patient’s own aortic dimensions are used to manufacture a replica in plastic of the aorta on which a bespoke external support is made of a fabric mesh (Figure 1). This can be positioned around the aortic root and ascending aorta without cardiopulmonary bypass at a much shorter operation (Table 3) [35]. There is nevertheless a risk associated with surgery in this difficult anatomical territory. The operation entails dissection over the surface of the aortic root as far proximally as the aortic annulus. It is vital, therefore, to safely circumnavigate both coronary ostia and to be familiar with the three-dimensional anatomy of the aortic root. In one case a surgical injury to the left main coronary artery led to the only perioperative death (case 36 of 46 operations) a mortality rate on intention to treat of 2.2% (95% confidence intervals 0.5% to 11%) [21]. The patient had severe pectus excavatum and with hindsight the surgical teams think it was an instance where a brief period of cardiopulmonary bypass (<20 min as was used in the first patient and has been used occasionally in other patients) would have been warranted and might have saved a life.The shape and intimate fit of the support make any movement unlikely. However, to ensure precise location the lower border of the support is folded on itself in a sutured seam. This is as inextensible as an annuloplasty ring and it is secured to the aortoventricular junction by six evenly spaced sutures.(a) The aorta before surgery. (b) The model on the computer screen after computer assisted design (CAD) modelling. (c) The soft macroporous mesh with 0.7 mm pore size mesh. (d) A depiction of the mesh surrounding the aorta and tethered to the ventricle with the coronary arteries emerging. (e) The MRI stable after 10 years.Comparison of peri-operative burdens of PEARS (first 30) with the other forms of root sparing root replacement.* Data are from a prospective study of valve sparing surgery [36] compared with PEARS published data [21].PEARS has resulted in consistent technical efficacy, stabilising the aortic root and valve. This conclusion is based on multiple measurements of images before and after surgery in the first 10 recipients of external support, two at 36 months, seven at 12 months and one at six months. These 20 paired images were interspersed with duplicate images of 37 un-operated people with Marfan syndrome. In total 94 aortic root images were presented in random sequence to a radiologist with expertise in cardiac MRI [37] (Figure 2). Further radiographic comparison has been made in the first 24 patients operated on at Royal Brompton Hospital, 8 to 101 months (interquartile range 25.5–72 months) after surgery (Table 4). The morphology of the aorta supported by the mesh is quite unchanged over time.Clinical outcomes at 1–10 years (average follow-up four years) are good. There have been no aortic or valve related events (Table 2). The PEARS operation is non-ablative, burns no bridges, and is a lesser biological insult than root replacement [38]. In two patients it has enabled a pregnancy [39].We have confirmed that the mesh is incorporated into the aortic adventitia (Figure 3). This is known from three sources. First, unknown to us at the time, a similar pliant macroporous material was used in an informal and incomplete wrapping of the aorta by a Californian team in the 1990s. Histology of samples of aorta obtained at re-operation showed the mesh to have been incorporated [40]. Second, material identical to that we have used clinically was used to sleeve the carotid artery of growing sheep. After an average of five months it was similarly incorporated with the connective tissues growing through the interstices of the mesh [41]. Third, one of the PEARS patients died in his bed at home 4.5 years after PEARS surgery [42]. There was no aortic dissection and the aortic valve was only minimally regurgitant as it had been throughout. His death was thought to be due to an arrhythmia associated Marfan cardiomyopathy, inherited from his mother. The unsupported aortic arch had the histological features of connective tissue disintegration, typical of Marfan syndrome while the supported portion of the aorta had normal histology [42] (Figure 3). This raises the intriguing possibility that the aorta, may have been able to heal when spared repetitive stress injury. What was confirmed in all three sources was that the mesh becomes incorporated in the adventitia forming a new composite vascular wall.(a) Pictorial representation of cross sectional images at the level of leaflet coaption of all MRI studies in the first 10 patients. These were anonymised and presented in random sequence among duplicate copies of 37 unidentifiable Marfan studies. (b) A Bland and Altman plot. The average of the two readings is plotted on the Y axis and the difference between them on the X axis. Before and latest after PEARS images are shown in red. The 37 read/reread control images are in black. The greatest differences were used to maximise sensitivity to any change in size of the aorta in PEARS cases. It can be seen that variation of +/− 3 mm is common in a measurements made by an experience expert when blind to the clinical significance, indicating the possibility of bias in readings when the clinical context or research hypothesis is known to them as they make each measurement. Eight of 10 PEARS cases have a reduction in size after the aorta has been mesh supported.Before and latest after PEARS aortic dimensions.N = 24 available from 27 patients. * In eight of the first 10 patients there was a reduction in size in the aortic root because the support is positioned in the anaesthetised patient with the aorta under less tension. See also [37]. The descending aorta will increase in size with the passage of time (here an average of four years) in all patients and more so in Marfan syndrome. These differences are actually below reliable resolution on individual clinical measurement [37].Per operation PEARS is likely to save money because of the reduced procedural costs and avoidance of anticoagulation [43]. Fewer complications would also favour cost-effectiveness. However, intervening earlier would lead to more operations and inevitably increase the number needed to treat and thus the proportion of patients operated on who were never destined to dissect—but then earlier intervention would save lives now lost while waiting. Patients continue to die of dissection while knowing that the aorta is expanding. We have explored these issues elsewhere [43]. Further evaluation would be incorporated in a future prospective study.Histological appearances of the aorta post mortem 4.5 years after surgery. The images above: on the left the unsupported arch and on the right the supported root. (Magnification ×2.5) Below are the corresponding appearances of the media (×10). The arrows mark the mesh. Collagen fibres (stained red) pass through the interstices of the mesh which is incorporated in the adventitia. The histology of the arch (left) shows fragmentation while on the right the histological appearances are normal.For those whose valves cannot be spared, or who are operated in an emergency, PEARS is by definition, not an option. For elective prophylactic surgery we believe that this operation is sufficiently established in Britain [1] for a reasonable patient to expect to be informed. In Britain there is a duty to fully inform the patient. How this is interpreted will depend on all the circumstances but if a patient had an ablative operation, a decision to not inform the patient of this option might become questionable.We proposed a prospective cohort study modelled on the Aortic Valve Operative Outcomes in Marfan Patients study [44] but with the inclusion of PEARS alongside TRR and VSRR. Such a study could theoretically provide decision making points amenable to randomization. The first is for patients who have not reached the criteria for root replacement. They could be offered random allocation between continued monitoring and PEARS, which can be justified in our opinion at lower aortic size. For patients who have reached the size criterion and have no more than trivial aortic valve regurgitation and are candidates for VSRR, the assignment to VSRR or PEARS could be made by randomization. This would require international collaboration to achieve the necessary numbers and wider acceptance of PEARS than is at present the case.We know full well the difficulties of performing randomized studies in surgery [45] but they can and should be done if we are to have evidence rather than clinical judgment alone to help our patients towards the best course of action [46,47]. The proposed randomization points could be nested within a cohort study with a protocol based on prospective evaluation, with independent verification of outcomes, as was done in AVOOMP [31].The core members of the PEARS development team were Tal Golesworthy (Design engineer, inventor and first patient), Tom Treasure (Clinical Operational Research Unit, UCL) John Pepper and Raad Mohiaddin (Royal Brompton Hospital BRU, Imperial College). The nucleus of the research team for the proposed evaluation are JP, TT, JJM Takkenberg and Mohammed Mokhles of Erasmus, Rotterdam and David Taggart, Oxford.This article is based upon presentations to the European Society of Cardiology ‘Marfan Syndrome: Novel Insights; Barcelona 2nd September 2014 and the Marfan Foundation 9th International Research Symposium, Marfan Syndrome and related disorders, Paris 25th September 2014.Tom Treasure developed the concept from 2000 and has led on analysis and dissemination and provided the first draft. John Pepper performed the first PEARS operation in 2004 and the majority of subsequent operations and has led on clinical development. Both authors have contributed equally to subsequent editing.The authors declare no conflict of interest.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Introduction: The submaximal exercise test (SET), which gives both a measure of exercise tolerance, as well as disease severity, should be a more robust functional and prognostic marker than the six-minute walk test (6MWT). This study aimed to determine the prognostic value of SET as predicted by the validated REVEAL (Registry to Evaluate Early and Long-Term Pulmonary Artery Hypertension Disease Management) registry risk score (RRRS). Methods: Sixty-five consecutive patients with idiopathic and associated pulmonary arterial hypertension (PAH) underwent right-heart catheterization, echocardiogram, 6MWT and a three-minute SET (Shape-HF™). Analyses explored the association between SET variables and prognosis predicted by the RRRS. Results: Although multiple SET variables correlated with the RRRS on univariate analyses, only VE/VCO2 (ρ = 0.57, p < 0.0001) remained an independent predictor in multivariate analysis (β = 0.05, p = 0.0371). Additionally, the VE/VCO2 was the most discriminatory (area under receiver operating characteristic curve, 0.84) in identifying the highest-risk category (RRRS ≥ 10), with an optimal cut-off of 40.6, resulting in a high sensitivity (92%) and negative-predictive value (97%), but a lower specificity (67%). Conclusion: SETs, particularly the VE/VCO2, appear to have prognostic value when compared to the RRRS. If validated in prospective trials, SET should prove superior to the 6MWT or the RRRS, with significant implications for both future clinical trials and clinical practice.Identification of prognostic factors that affect survival has been a key goal in the clinical care of patients with pulmonary arterial hypertension (PAH). The REVEAL (Registry to Evaluate Early and Long-Term Pulmonary Artery Hypertension Disease Management) cohort helped identify some important independent predictors, as well as a composite scoring system—the REVEAL registry PAH risk score (RRRS)—to help in prognosticating these patients [1,2]. The REVEAL study confirmed the increased risk of mortality in patients with World Health Organization (WHO) Group I PAH, including those with portal hypertension [3] and connective tissue diseases [4,5].The elements of the RRRS are readily available and include the patient’s WHO functional class (WHO-FC), echocardiographic and hemodynamic parameters, as well as the six-minute walk test (6MWT) as a marker of exercise tolerance. Although the 6MWT is considered a simple, noninvasive, inexpensive marker of functional status and exercise tolerance, it suffers from several limitations, including learning effect, day-to-day variability and anthropomorphic/demographic variables [6]. Additionally, the factors that go into the 6MWT include non-cardiopulmonary factors that have little to do with PAH or right ventricular function (e.g., neurologic disorders, musculoskeletal issues, peripheral arterial disease, conditioning, effort). Despite these and other limitations, the 6MWT has been widely used as an important clinical endpoint of most PAH treatment trials and, thus, has been used regularly in the clinical monitoring and management of PAH patients. More recently, a meta-analysis showed that changes in the six-minute walk distance (6MWD) may not correctly predict favorable clinical outcomes [7].In comparison, the cardiopulmonary exercise test (CPX) provides more comprehensive evaluation of exercise tolerance, but has more specific measures to evaluate ventilation, gas exchange, cardiac function, as well as muscle physiology and, therefore, could be more pertinent in the management of patients with PAH. Specific to PAH, both peak oxygen consumption (VO2) and ventilatory equivalent of carbon dioxide (VE/VCO2) obtained from a CPX appear to be important predictors of survival [8,9,10]. Some investigators have suggested using CPX variables as target goals of therapies (e.g., VO2 > 15 mL/min/kg and VE/VCO2 > 55) [6]; however, the routine use of CPX in PAH patients clinically is limited by the added equipment, time and expertise required, and it may not be suitable for the more severe PAH patients with right heart failure at risk for syncope and arrhythmias.A novel compromise on the benefits and drawbacks of both the 6MWT and the formal CPX is the submaximal exercise test (SET), which is a low-intensity, three-minute exercise test that is easier to perform than a CPX test, yet unlike a 6MWT, is able to acquire some of the key ventilatory variables that can additionally inform the clinician on the status of the cardiopulmonary system [11]. In particular, some of the key SET variables that can be acquired and that have been shown to be perturbed in PAH include lower partial pressures in the end tidal CO2 (PETCO2), a greater VE/VCO2, a reduced oxygen saturation and VO2 efficiency slope [10,12,13,14,15]. Our center has extensive experience with this tool clinically and has shown that the SET was a more robust marker of PAH severity than the 6MWT [16].Such a tool that can measure functional status, exercise tolerance and more specific hemodynamic/ventilatory markers may not only help in prognostication, but may also potentially be an important surrogate endpoint in and of itself in clinical trials and, eventually, in clinical practice, to guide the management of PAH patients. Towards this eventual goal, we first sought to determine whether the SET variables would correlate with the validated RRRS as a prognostic marker, particularly in identifying high risk patients for whom management would be escalated.Consecutive WHO Group 1 PAH patients seen by the Pulmonary Hypertension Clinic between March, 2011, and May, 2013, were eligible for the study. All subjects had received a right heart catheterization, an echocardiogram a 6-minute walk test with its distance (6MWD), WHO-FC, as well as a SET.The Shape-HF™ (Shape Medical Systems, Inc.) was utilized for SET in this study. Specific measures able to be obtained by this test include: (1) VE/VCO2, a measure of breathing efficiency defined as the linear slope of the amount of air expired per minute (VE) versus the amount of carbon dioxide produced per minute (VCO2); (2) the oxygen uptake efficiency slope (VO2/log VE), indicating the amount of oxygen that is used per unit ventilation; (3) PETCO2, reflecting the alveolar CO2 partial pressure at the end of expiration; (4) the heart rate decay during the first minute of exercise recovery; and (5) the chronotropic response index, a measure of the patient’s heart rate response to dynamic exercise. In this study, we specifically examined whether the peak VO2 (mL/kg/min), VE/VCO2 and the partial pressure of carbon dioxide at baseline and at the end of exercise (PETCO2-b, PETCO2-ex) would correlate with the RRRS.Baseline characteristics are provided with descriptive statistics. For consistency, medians with their interquartile ranges (IQR) are reported for continuous variables. Non-parametric Spearman’s correlation (ρ) was computed between the raw numeric RRRS and the SET variables. A multivariate analysis to predict the RRRS from the SET variables of age, sex, VE/VCO2, PETCO2-b and the PETCO2-ex was also performed to identify significant predictors of the prognostic risk based on the RRRS. Finally, to identify a potential SET variable to distinguish those with the worst prognosis (RRRS ≥ 10), we performed univariate analyses with non-parametric assumptions (Mann–Whitney test) comparing each of the SET variables between those RRRS ≥ 10 to those with RRRS < 10. We then explored potential cut-offs for the SET variable VE/VCO2 (which was the most discriminatory) by constructing a receiver-operating characteristic (ROC) curve and its area under the curve (AUC), as well as determining the sensitivity, specificity, predictive values and likelihood ratios (LHR) for the optimal cutoff. All statistical analyses were performed using JMP® Pro 9.0.1 (SAS, Cary, NC, USA). The study was approved and overseen by the Mayo Clinic Institutional Review Board (IRB 12-003335).Sixty-five subjects were enrolled and available for analysis. The majority were older women, with an even separation of associated PAH (APAH) from idiopathic PAH (IPAH). On average, most had moderate to severe pulmonary hypertension and were at least WHO-FC III. The specific baseline characteristics are presented in Table 1. There were no adverse events noted during any of the SET.Baseline characteristics.IPAH, idiopathic pulmonary arterial hypertension (PAH); APAH, associated PAH; mPAP, mean pulmonary artery pressure; PVR, pulmonary vascular resistance; RAP, right atrial pressure; PAOP, pulmonary artery occlusion pressure; CI, cardiac index; 6MWD, six-minute walk distance; BNP, brain natriuretic peptide; DLCO, diffusion capacity of lungs for carbon monoxide; PETCO2, pressure in the endtidal CO2.In univariate analyses, all variables obtained from the SET were significantly correlated with the RRRS; see Table 2. The strongest correlation was modest and seen with VE/VCO2 (ρ = 0.57, p < 0.0001). In a multivariate model with the covariates of age, sex, VO2, VE/VCO2, PETCO2-b and PETCO2-ex, the only SET variable that remained an independent predictor was VE/VCO2 with an effect size of a 0.5 increase in the RRRS for every 10 increase in the VE/VCO2 (β = 0.045, p = 0.0371).Of the 65 patients, 13 (20%) had the worst prognosis as defined by a REVEAL registry score ≥10. The SET variables were compared between these two risk categories. All SET variables proved significantly worse for those in the higher risk category; Table 3. VE/VCO2 was the most discriminatory among the SET variables. An ROC curve was generated revealing modest discriminatory ability with an AUC of 0.84; see Figure 1. An optimal VE/VCO2 cut-off of 40.6 revealed a sensitivity of 92% and a specificity of 67%. The positive likelihood ratio or the positive predictive value is poor, but the VE/VCO2 appeared to have a strong negative predictive value or a negative likelihood ratio; see Table 4.Correlation between submaximal exercise test (SET) parameters and the REVEAL registry risk score (RRRS).* Difference from exercise to baseline. Units are otherwise defined in the text and Table 1.SET parameters comparing the highest risk group (RRRS ≥ 10) versus the lowest risk group.Units are as defined in the text and Table 1.Receiver operating characteristic curve for VE/VCO2 in predicting the highest risk category (RRRS ≥ 10). The AUC was 0.84.Operating characteristics of VE/VCO2 in predicting the highest risk category (RRRS ≥ 10). LHR, likelihood ratio.The current study showed a statistically significant correlation among SET and PAH prognosis as predicted by the RRRS. On univariate analysis, VE/VCO2 showed the strongest correlation with RRRS and was the only independent parameter that remained significant in multivariate analysis. VE/VCO2 also had excellent discriminatory power at identifying patients with the highest mortality risk (i.e., RRRS ≥ 10). In particular, the VE/VCO2 demonstrated excellent sensitivity with high negative predictive value, such that a normal or low VE/VCO2 value (<40.6) would exclude those least likely to suffer disease progression and death.Formal CPX testing is a powerful diagnostic and prognostic tool for a variety of cardiopulmonary disorders, including PAH [17,18]. Exercise limitation in PAH is characterized by impairment of oxygen transport and inefficient gas exchange with very high ventilatory demands [19]. Typically, the oxygen transport abnormalities are reflected by a moderate to severe reduction in the peak VO2, work rate (WR), anaerobic threshold (AT), VO2/WR slope, oxygen (O2) pulse and a steep heart rate (HR)-VO2 response, combined, on occasion, with a submaximal HR response. The gas exchange response is more conspicuous: high ventilatory equivalents (VE/VCO2 and VE/VO2) and a low PETCO2, along with exercise-induced hypoxia with advancing disease [15]. Of these parameters, most studies have evaluated, in particular, the prognostic value of the peak VO2 [8,9,10,20,21] and, to a lesser extent, the VE/VCO2 at AT [10,13]. Both of these values require that the patient exercise to the point of achieving AT and maximal exercise. However, in the routine management of a PAH patient, despite being an informative non-invasive test, CPX is infrequently used diagnostically, and rarely repeatedly, to track disease and guide management. This may be, in part, because of the generally sicker PAH population, as well as the additional expertise, time and equipment needed to perform this reliably and safely. Although peak VO2 and the VE/VCO2 at AT may not be obtained during a submaximal test, an elevated breathing efficiency (VE/VCO2 slope), which can be determined from submaximal data [22] (thus, independent of patient effort and motivation), has been shown to be an important prognostic marker [2,23,24]. As such, SET is an attractive compromise to the limitations of both the CPX and the 6MWT, by providing information on both disease severity and exercise tolerance and, thus, prognosis.Although previous studies have not specifically evaluated the role of SET in PAH prognosis, there have been few and limited investigations illustrating the potential role of SET in PAH. Woods et al. [11,15] recently evaluated the clinical utility of SET in PAH patients and concluded that the various gas exchange variables obtained during the test could differentiate PAH patients from healthy controls and among different severities of PAH. The authors concluded that SET gas exchange may be a useful end point measure in PAH patients.Adding to this limited body of literature, we found in this study that when compared to the validated RRRS, the SET also provides prognostic value. Given the advantages of the SET over the 6MWT, this supports the need for further exploring SET as an objective tool to be used in clinical trials (for example, as an endpoint in lieu of the 6MWT) or clinically, to guide the clinician in helping to decide who requires treatment or escalation of therapies among the growing complexity of PAH medications now available.There are significant limitations to consider when interpreting the results of this study. This was an observational, single center study at a tertiary academic center with a specific PAH clinic. Generalizability is thus limited. Furthermore, although there were no adverse events, this may be limited by the small sample size and the selection bias introduced by the clinical selection of PAH undergoing SET testing. However, by practice protocol, all suspected or diagnosed PAH patients undergo SET testing. More importantly, the ability of the SET to prognosticate was obtained, not prospectively, but through a cross-sectional comparison of the SET to a validated prognostic score, the RRRS. We did not have enough long-term follow-up to directly compare the two tools, but our findings have encouraged us to prospectively collect data (ongoing) to more directly determine whether SET will prove a superior marker of both prognosis and disease severity. Despite some of these and other limitations, the biologic plausibility, that a tool that is able to measure both exercise tolerance and disease severity should be a powerful prognostic marker, supports that the findings in this observational study is real. The consecutive collection of hemodynamically confirmed PAH and the systematic characterization of our PAH cohort are additional strengths of this study.In summary, we are encouraged to further explore prospectively the value of SET in both predicting prognosis, as well as in guiding the management of patients affected by PAH. Furthermore, given the intrinsic advantages of the SET over the 6MWT, we hope future studies will also consider its role as a principle outcome as potentially a superior surrogate biomarker than the 6MWT. If it proves to be a more robust prognostic marker, it may have significant implications, not just for clinical practice, but on the conduct, cost and efficiency of completing clinical trials.In this hypothesis generating study, SET variables correlated significantly with the RRRS. Further prospective investigations should be considered to confirm whether SET does have significant prognostic information and to test whether it might prove to be a superior surrogate marker to the 6MWT and/or the RRRS. VK contributed to the analysis, interpretation of the data and drafting and critical revision of the article. JN contributed to the experiments, collection, analysis and interpretation of the data. CDB contributed to the conception and design of the study and the drafting and critical revision of the article. ASL contributed to the conception and design of the study, collection, analysis and interpretation of the data, the collection/generation of the figures and the drafting and critical revision of the article. All authors gave final approval of the article.The authors have no conflicts of interest to disclose.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Due to age dependent organ manifestation, diagnosis of Marfan syndrome (MFS) is a challenge, especially in childhood. It is important to identify children at risk of MFS as soon as possible to direct those to appropriate treatment but also to avoid stigmatization due to false diagnosis. We published the Kid-Short Marfan Score (Kid-SMS) in 2012 to stratify the pre-test probability of MFS in childhood. Hence we now evaluate the predictive performance of Kid-SMS in a new cohort of children. We prospectively investigated 106 patients who were suspected of having MFS. At baseline, children were examined according to Kid-SMS. At baseline and follow-up visit, diagnosis of MFS was established or rejected using standard current diagnostic criteria according to the revised Ghent Criteria (Ghent-2). At baseline 43 patients were identified with a risk of MFS according to Kid-SMS whereas 21 patients had Ghent-2 diagnosis of MFS. Sensitivity was 100%, specificity 77%, negative predictive value 100% and Likelihood ratio of Kid-SMS 4.3. During follow-up period, three other patients with a stratified risk for MFS were diagnosed according to Ghent-2. We confirm very good predictive performance of Kid-SMS with excellent sensitivity and negative predictive value but restricted specificity. Kid-SMS avoids stigmatization due to diagnosis of MFS and thus restriction to quality of life. Especially outpatient pediatricians and pediatric cardiologists can use it for primary assessment.Marfan syndrome (MFS) is an inherited connective tissue disorder with multifaceted phenotype involving cardiac, ophthalmologic and skeletal symptoms as well as skin, lung and dura abnormalities [1,2]. Due to age dependent organ manifestation, diagnosis of Marfan syndrome, especially in children, is sophisticated [3,4]. A major aspect in care of patients who are suspected of Marfan syndrome is to assure correct diagnosis of MFS as soon as possible but also to strongly avoid false diagnosis to prevent stigmatization with chronic disease, which can cause various restrictions in quality of life. Decreased quality of life, challenges of education, work and family life, depression and anxiety in patients with diagnosed MFS could be shown before [5]. Therefore, especially in childhood, where organ manifestations may not be fully present, a safe follow-up regime for patients who are suspected of MFS is necessary until definite diagnosis of MFS is possible.Since 1996, diagnosis of MFS according to Ghent Criteria has been established, including genetic analysis with detection of FBN1 mutation [6,7]. Loeys et al. published revised Ghent Criteria (Ghent-2) (Table 1: Revised Ghent Criteria) in 2010 to improve diagnosis [8]. Ghent-2 is still the gold standard for diagnosis of MFS. It includes organ manifestations of MFS and allows diagnosis in different constellation of clinical symptoms. Although Ghent-2 is a major step forward, its utility in children is still restricted with expensive and technically advanced diagnosis and due to age dependent organ manifestation.In 2012 we published the Kid-Short Marfan score (Kid-SMS) (Table 2: Kid-Short Marfan Score) to stratify the pre-test probability of MFS in early childhood, especially upon first presentation of patients who are suspected of MFS [9]. Kid-SMS allows early risk stratification without stigmatization of children with chronic disease too early, but enables a safe follow-up regime and flexibility in stratification class and diagnosis, which is indispensable concerning age dependent onset of organ manifestations in MFS.Due to the life limiting aspect of cardiac pathologies in MFS, and the possibility of early medical treatment to avoid surgery or even life-threatening events like dissection or aortic rupture, Kid-SMS concentrates on those symptoms [10,11]. In addition we included ectopia lentis, skeletal features and family history in the score. According to resulting pre-test probability class Kid-SMS recommends further diagnostic steps and follow-up strategies. This study now re-evaluates the predictive performance of Kid-SMS for MFS. We also want to estimate the advantage and disadvantage of using Kid-SMS in clinical life. Finally we review its usefulness and importance in the diagnosis of MFS in childhood.Revised Ghent Criteria (Ghent-2) for diagnosis of MFS [7].MFS, Marfan syndrome.Kid-Short Marfan Score (Kid-SMS) [8].SV, dilatation of sinus of valsalvae; EL, ectopia lentis; MVP, mitral valve prolapse; TVP, tricuspid valve prolapse; PA, dilatation of pulmonary artery; 3 skeletal features, at least 3 skeletal features of the systemic score of the revised Ghent Criteria; Ghent-2, revised Ghent Criteria; MFS, Marfan syndrome.Between January 2012 and August 2014 we prospectively investigated 106 pediatric patients who were suspected of having MFS (39 female, 67 male) in the Marfan clinic at the University Heart Center Hamburg. All patients had complete assessment of manifestations of MFS with echocardiography and clinical examination. If necessary and reasonable we also accomplished magnetic resonance imaging (MRI) and genetic analysis.At baseline, children were examined according to Kid-SMS. At baseline and follow-up visit, diagnosis of MFS was established or rejected using current standard diagnostic criteria according to Ghent-2.We performed echocardiography with General Electric Vivid 7 with 10, 5 and 3 MHz probes. To obtain measurement of aortic root diameters we operated in parasternal long axis view on 2D and M-Mode images using leading edge to leading edge technique at end diastole. We evaluated dilatation of sinus of valsalvae according to Roman et al. [12]. For the measurement of the pulmonary artery, we operated in parasternal short axis view and evaluated using Z-score [13]. We estimated mitral valve prolapse and tricuspid valve prolapse in four-chamber view and parasternal long axis. An experienced pediatric cardiologist performed the examination.We collected data using Filemaker software V.10 pro advanced. To perform statistical analysis, we used SPSS V16.0 and for tables and figures we used Microsoft Excel 2003 and SPSS V16.0.We expressed quantitative variables as means with standard deviation and qualitative data as numbers. For comparison of quantitative data between groups we used unpaired t test and considered p values < 0.05 as significant. We compared qualitative data by Fisher’s exact test. We also determined positive likelihood ratio for Kid-SMS. Likelihood ratio larger than ten was considered as extremely reliable to identify patients with MFS, whereas values between three and ten were useful and values below three were not useful. Finally, we used Kaplan Meier analysis and log-rank test to display age of diagnosis with Kid-SMS and Ghent-2 [14]. Age of diagnosis of MFS concerning Ghent-2 and age of risk stratification with Kid-SMS were defined as endpoint of surveillance. Kid-SMS was considered as positive as soon as moderate risk for MFS was stratified. Negative Kid-SMS was considered as negative test. Again we considered p values < 0.05 as significant.To assess clinical data and samples, we obtained informed consent of patients or parents of patients. The study was approved by the Hamburg ethical board (Project identification code: PV 4005).Mean age of first presentation to Marfan clinic was 12.18 ± 5.49 years (2 patients 1–12 months, 14 patients 1.01–6.00 years, 33 patients 6.01–13.00 years, 53 patients 13.01–18.00 years, 4 patients > 18 years) (Figure 1).At baseline, Kid-SMS stratified 43 children at risk of having MFS; 20 of those patients also had Ghent-2 diagnosis of MFS (Table 3). During follow-up period, four patients were diagnosed according to Ghent-2, whereas Kid-SMS stratified a risk of MFS in those patients at baseline. Another five patients showed FBN1 mutation without Ghent-2 diagnosis of MFS, whereas Kid-SMS identified two at a risk of MFS. Besides, 14 patients were identified at risk of MFS without current diagnosis according to Ghent-2 or FBN1 mutation (Table 4).Age at first presentation to Marfan clinic.Fourfold table of risk stratification with Kid-SMS and diagnosis with Ghent-2 at baseline (first presentation). (Ghent-2 Pos, revised Ghent Criteria positive; Ghent-2 Neg, revised Ghent Criteria negative; Kid-SMS Pos, Kid-Short Marfan Score positive; Kid-SMS Neg, Kid-Short Marfan Score negative).Fourfold table of risk stratification with Kid-SMS and diagnosis with Ghent-2 at follow-up visit, p < 0.05. (Ghent-2 Pos, revised Ghent Criteria positive; Ghent-2 Neg, revised Ghent Criteria negative; Kid-SMS Pos, Kid-Short Marfan Score positive, Kid-SMS Neg, Kid-Short Marfan Score negative).Sensitivity of Kid-SMS was 100% (CI 95% 0.86 to 1.00), whereas specificity was 77% (CI 95% 0.66 to 0.85). Positive predictive value was 56% (CI 95% 0.40 to 0.71) and negative predictive value 100% (CI 95% 0.94 to 1.00). Likelihood ratio was 4.3, which reveals good predictive performance of Kid-SMS. Fisher’s exact test showed statistic significance (p value < 0.05).At follow-up visit, detailed analysis of risk stratification at baseline with Kid-SMS in comparison with diagnosis of MFS according to Ghent-2 showed “very high risk” in two patients with MFS and none in patients without diagnosis according to Ghent-2. “High risk” was graded in 20 patients with diagnosis according to Ghent-2 and two patients without diagnosis according to Ghent-2 or FBN1 mutation. “Moderate risk” was graded in five patients with MFS, two with FBN1 mutation, and 12 patients without MFS (Table 5).Comparison of risk stratification with Kid-SMS and diagnosis according to Ghent-2 at follow-up visit. (Ghent-2 Pos, revised Ghent Criteria positive; Ghent-2 Neg, revised Ghent Criteria negative; FBN1 Pos, FBN1 mutation positive; FBN1 Neg, FBN1 mutation negative; SV, dilatation of sinus of valsalvae; PA, dilatation of pulmonary artery; MVP, mitral valve prolapse; TVP, tricuspid valve prolapse; 3Skel, at least 3 skeletal features of the systemic score of the revised Ghent Criteria; EL, ectopia lentis; FH, family history).Mean age of risk stratification with Kid-SMS was 10.63 ± 1.23 years, whereas age of diagnosis with Ghent-2 was 12.07 ± 1.16 years. In Kaplan-Meier analysis the endpoint of presented curves is age of diagnosis of MFS concerning Ghent-2 (dashed line) and age of declared risk stratification with Kid-SMS, which may be very high risk, high risk or moderate risk (continuous line). Analysis did not show significant difference concerning age of diagnosis or risk stratification (p = 0.2, ns) (Figure 2).Kaplan-Meier analysis of age of diagnosis with Ghent-2 (Revised Ghent Criteria, dashed line) and risk stratification of MFS (very high risk, high risk, moderate risk) with Kid-SMS (Kid-Short Marfan Score, continuous line), p = 0.2, ns.In this study, all patients with diagnosed MFS according to Ghent-2 were stratified for risk of MFS with Kid-SMS, which is shown by perfect negative predictive value. Kid-SMS also defines a group where diagnosis cannot be assured yet but Kid-SMS recommends a safe follow-up regime. This avoids stigmatization and unnecessary restrictions in quality of life due to false diagnosis of chronic disease. Especially children should be protected from stigmatization to support psychosocial development und encourage setting of resources for psychosocial coping in life.At first presentation (baseline), Kid-SMS identified a distinctly larger group of patients with a risk of MFS than patients with diagnosis according to Ghent-2. During follow-up period, three patients were diagnosed according to Ghent-2, whereas Kid-SMS already stratified a risk of MFS in those patients at baseline. Nevertheless, there is a group of children with stratified risk of MFS where Ghent-2 was negative at final visit. Some of those (2) showed FBN1 mutation without diagnosis with Ghent-2. Others (14) were neither diagnosed with Ghent-2 nor showed FBN1 mutation.Both patients with FBN1 mutation were at a moderate risk of MFS due to positive family history of MFS. Those patients were aged 1.1 and 8.4 years at final presentation. Due to age dependent organ manifestation in MFS symptoms may not be present especially in early childhood. Those patients need a strict follow-up regime to evaluate upcoming manifestations of MFS later on. They should be treated as patients with MFS and may subsequently be diagnosed according to Ghent-2.In three patients with FBN1 mutation, Kid-SMS did not predict a risk of MFS. One showed FBN1 mutation with isolated mitral and tricuspid valve prolapse. Thus, this patient would of course be followed up by cardiological evaluation. Consequently, in case of aortic enlargement or other cardiac pathologies, further diagnostic tools and necessary treatment would be induced. Both of the others showed a FBN1 mutation with a positive systemic score and no vascular involvement aged 15.8 and 17.6 years. Those children had no vascular symptoms, which are life-limiting in MFS. Even though MFS is unlikely in those patients they may have another connective-tissue disorders like MASS phenotype associated with FBN1 mutation.Prediction of risk of MFS with Kid-SMS in patients without diagnosis according to Ghent-2 or genetic analysis was established in two patients who showed dilatation of sinus of valsalvae in combination with skeletal symptoms.Another five patients were at moderate risk of MFS because of dilatation of sinus of valsalvae. Even though there were no other symptoms of MFS those patients will be followed up in a specialized center and may be diagnosed later on with another disease involving thoracic aortic aneurysm [15]. Seven showed moderate risk of MFS because of family history. After additional examinations like genetic analysis and MRI, MFS was very unlikely.Although Kid-SMS also predicts a risk of MFS in some patients without disease, it will probably cover patients with other syndromes, including thoracic aortic aneurysms like Loeys-Dietz syndrome or Shprintzen-Goldberg syndrome, that also need regular follow-up. The main criteria in Kid-SMS are cardiac and vascular involvement of MFS. Dilatation of sinus of valsalvae is the most common feature in children with MFS [16]. And it is the symptom that is most important for medical and surgical treatment. Therapies with beta-blocker as well as with AT-1 antagonists avoid progression of aortic root dilatation, surgery and, in the end, life-threatening events [17,18]. Because of interference with other connective tissue diseases where those vascular symptoms occur, differentiation with Kid-SMS is difficult and specificity is restricted. To at least optimize power of discrimination dilatation of sinus of valsalvae is not included in the score as an isolated symptom but in combination with other features, which are typical for MFS like dilatation of pulmonary artery and skeletal features.We were not able to examine larger groups of children with other syndromes, including thoracic aortic aneurysms, to analyze exact power of discrimination of Kid-SMS. Although improvement of differential diagnosis was one reason to revise the Ghent criteria, this is important for Kid-SMS but not the main focus [8]. Consideration of differential diagnosis should attend patients in follow-up regimes until definite diagnosis is assured.In total, at risk stratification for MFS with Kid-SMS, children were younger at diagnosis than according to Ghent-2. Early identification of patients with a risk of MFS enables their inclusion in a safe follow-up regime and can identify the possible need for prophylactic medical treatment. Indeed, first presentation to a Marfan clinic is not the time of incidence of symptoms and thus not the time of detection of Kid-SMS, which may be the reason for absent significance. To evaluate a better reliable age of pre-test probability with Kid-SMS use of the score in outpatient children with reasonable suspicion of MFS is necessary. In our experience we see many children where Kid-SMS is the first diagnostic tool that correctly supports or excludes diagnosis of MFS.According to level of risk stratification with Kid-SMS, further examinations are recommended. Patients with very high risk of MFS, which is equivalent to diagnosis according Ghent-2, must be presented to a specialized center of MFS. In patients with a high risk or moderate risk with dilatation of SV, full examination concerning Ghent-2 has to be completed urgently, even though risk of MFS is lower in moderate risk patients. In areas where access to MRI and genetic analysis is limited, further examinations can be delayed as long as regular echocardiographic follow-up is assured. Also, performance of MRI may not be possible sometimes due to low age of patients where quality of MRI data is reduced due to fast heart rate and children would need anesthesia for examination. Patients with moderate risk of MFS due to positive family history further examinations are recommended to include or exclude them in a follow-up regime. Due to age dependent onset of organ manifestation, many children of our study group changed level of risk stratification during follow-up, which represents flexibility of the score. Except the change from negative to positive risk stratification, we only comment on first level of risk stratification of MFS in results and discussion. A restriction of the application of Kid-SMS, especially in outpatient care, is the performance of echocardiography, which requires expert experience. Nevertheless, this is much more easily available than a specialized pediatric center for MFS.Unfortunately, this study does not represent the use of Kid-SMS in outpatient children, which is indeed one of the most important skills of the score. To evaluate use of Kid-SMS in those children, further analysis and studies are necessary. But even though our clinic is a specialized center, we used Kid-SMS to give parents first advice for probability of MFS and to decide how urgent further investigations were. Kid-SMS involves symptoms that are also examined to assure diagnosis according to Ghent-2. Thus, performance of the score is not time-consuming and does not require additional examinations, which would limit its usefulness, especially in outpatient care. In summary, practice of Kid-SMS in this patient group was valuable for children with assumed MFS. Kid-SMS showed very good sensitivity and restricted specificity. Likelihood ratio shows good predictive performance of stratifying risk of MFS with Kid-SMS. It was easily executable and not time-consuming. After all, Kid-SMS is not supposed to replace Ghent-2 diagnosis of MFS. Instead, it should be used in addition to Ghent-2 to improve diagnosis in childhood and assess early pre-test probability of MFS. Patients without a definite diagnosis or exclusion of disease are thus not stigmatized with the diagnosis of MFS but in a safe follow-up regime.Especially pediatricians and pediatric cardiologists not working in specialized centers can use it for a risk assessment and to estimate whether or not a patient requires transfer to a specialized center. Particularly for them, Kid-SMS is an easily executable diagnostic tool for first screening of MFS in childhood.Whereas diagnosis of MFS is sophisticated, Kid-SMS is a useful tool to assess early pre-test probability of MFS in childhood, especially for pediatricians and pediatric cardiologists in out-patient care.There was no funding received for this work. We would like to thank Claudia Schlesner for documentation and Ruth Cork for editing the manuscript.V.C.S. conceptualized the study, performed data collection, designed data collection instruments, performed initial analysis, drafted the initial manuscript and approved final manuscript. G.C.M. supervised data collection, performed initial analysis and critically reviewed and revised the manuscript and approved the final manuscript. K.J.S. and G.H. coordinated and performed data collection, designed data collection instruments, critically reviewed the manuscript and approved the final manuscript. F.A. performed data collection, critically reviewed the manuscript and approved the final manuscript. Y.V.K. and R.K. reviewed and revised the manuscript and approved the final manuscript. T.S.M. conceptualized the study, coordinated and supervised data collection, reviewed, revised and approved the final manuscript.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).A large number of studies link H. pylori infection with a reduced risk of developing extra-gastric conditions such as allergy, asthma, inflammatory bowel disease, coeliac disease and multiple sclerosis. The strength of the evidence for these protective associations is quite variable, and published studies often do not agree. This review article discusses some of the reasons for these discrepancies, and the difficulties faced when designing studies. Examples of some protective disease associations are described in detail, where the evidence is most abundant and thought to be more reliable. The most convincing of these are supported by published mechanistic data, for example with animal models, or incidence of disease exacerbation in humans following H. pylori eradication. Although controversial, this field is very important as the prevalence of H. pylori is decreasing throughout the world whilst many chronic diseases are becoming more common. These trends are likely to continue in the future, therefore it is important that we fully understand if and how H. pylori confers protection.Helicobacter pylori is a very common bacterial pathogen, which colonizes the mucosa of the human stomach. It is being intensively investigated as the main cause of peptic ulcer disease and gastric cancer, however these disease outcomes occur in a relatively small proportion of those infected (approximately 10%–20%) [1,2,3]. For the vast majority of people, there are no symptomatic indications that the bacteria are present in the stomach. This is despite gastric colonization generally persisting life-long from a very young age [4].The prevalence of H. pylori is declining around the world, as fewer children are becoming infected [4,5]. The infection is virtually ubiquitous in developing countries (such as India and Mexico), but in many developed parts of the world (such as the USA, Scandinavia, Australia and the UK), the prevalence of the infection is now below 20%. As fewer than 5% of children in such regions are colonized, the decline will continue [2,3,6]. This is perceived as an unintended public health benefit arising from antibiotic usage in childhood since, as the prevalence of infection declines, cases of gastric and duodenal ulcers are also becoming less common [3]. Evidence is emerging, however, that adverse consequences may arise from a lack of exposure to H. pylori. This is hypothesised to be because humans have co-evolved with the bacterium over the last 60,000 years, and our physiology has developed taking account of its continual presence in the stomach [5,7]. When interactions with H. pylori are missing, this may lead to effects, such as defective development of the immune system, that increase the risk of developing diseases outside the stomach and duodenum. Many of these, such as asthma, inflammatory bowel disease, and autoimmune conditions, are becoming much more common as the prevalence of H. pylori has dropped [8,9]. They cause a great deal of pain and suffering, as well as becoming very substantial financial burdens on health care expenditure [10]. It is therefore extremely important to fully assess whether H. pylori infection plays a protective role, and determine how antibiotic eradication strategies should then ideally be managed in the light of this. Obviously it is necessary to eradicate the infection when patients have gastric symptoms, ulcers, or signs of pre-cancerous pathology. If H. pylori confers protection against other life-threatening diseases, however, it will be important to prevent exposure to this infection being completely lost from populations.A huge number of published studies have now reported a correlation of H. pylori infection with lowered risk of developing extra-gastric diseases, and there is also some evidence for an influence on disease severity. These include gastro-oesophageal reflux disease and oesophagitis [5,11,12], asthma and allergy [13,14], several autoimmune disorders (including coeliac disease, systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis [15,16,17,18]), inflammatory bowel disease and irritable bowel syndrome [11,19]. It is intriguing to find so many reported protective associations against such widely varying conditions, and this raises questions about how these effects could possibly arise. It is suggested that the mechanisms might involve the reduction of gastric acid output [20]; H. pylori-mediated suppression of immune and inflammatory responses [14]; or modifications of the intestinal microbiota [5,21].The idea that exposure to infectious organisms, particularly during childhood, is beneficial for health has been discussed for many years. Such exposure is thought to be important for the development of a healthy immune system, preventing the occurrence of conditions associated with an overactive immune response. The term “hygiene hypothesis” was first coined by David Strachan, in his paper showing that hay fever and eczema were less common in children from larger families. He proposed that the declining family size would result in a lack of protective unhygienic contact and cross-infection from other siblings [22]. This has recently been revised as the “Old Friends Hypothesis”, with further understanding that modernization has depleted access to many of the immunoregulatory stimuli that humans have co-evolved with [23]. Infections proposed to play a role in this include intestinal parasites, ectoparasites (such as ticks and lice), environmental bacteria, gut commensal organisms and also H. pylori [23,24,25,26,27,28]. Since exposure to some of these “Old Friends” and H. pylori is similarly associated with factors such as socioeconomic status, it is difficult to distinguish whether H. pylori itself could be a protective agent, or if it is merely a marker for some other as-yet unidentified factor.Factors such as gender, age and social class influence H. pylori infection rates and gastro-duodenal disease risk [3]. These also affect the risk of developing many of the extra-gastric immune and inflammatory conditions listed above [29,30,31]. H. pylori is more common in those of older age, in males, and in populations of a lower socioeconomic status [3]. In contrast, many of the conditions that H. pylori is reportedly protective against are becoming more common in developed countries, especially those of higher socioeconomic status, and some are more common in females. Studies must therefore carefully control for such confounding influences and bias. Some associations are reported to be stronger with more virulent strains of H. pylori, for example those that possess the cag pathogenicity island (cagPAI) [12,32]. This provides some helpful clues about causal relationships because this H. pylori-specific virulence marker is not present in all strains. The best way to prove whether H. pylori is protective would perhaps be to administer an infection to volunteers and monitor them for systemic effects, e.g., by measuring inflammatory markers. It is only just becoming possible to deliberately infect humans with virulent H. pylori strains, and such studies have been restricted to healthy volunteers rather than those with underlying disease [33]. Many of the protective effects attributed to H. pylori could require infection from an early age, or over many years, and this makes such experimentation extremely difficult. Instead the literature to date are mostly based on epidemiological associations and, since H. pylori eradication therapy also affects other colonising bacteria, animal model studies currently provide the most reliable means to show cause and effect.Unfortunately, in many cases the evidence for protective associations in humans is weak, contradictory and/or inconclusive. This tends to be because of constraints in study design. Most studies are cross-sectional, comparing the sero-prevalence of H. pylori in groups with and without disease. Wide variations in population size, and demographic differences between studies, mean that there are lots of conflicting reports. In general there is a real lack of longitudinal and mechanistic data. A systematic review published in 2014 by Smyk et al. [18] reported that 95 autoimmune conditions have been investigated as being influenced by H. pylori. Based on the strength of the evidence however, only four of these could be classified as having “probable” associations at best. All four were causative rather than protective associations.Having discussed the barriers and difficulties in testing protective associations of H. pylori infection, the following sections will discuss the diseases for which the evidence is most abundant and more reliable.Gastro-oesophageal reflux disease (GORD) is a disorder arising from the retrograde flow of acidic stomach contents into the oesophagus. The development of gastro-oesophageal reflux is linked with several factors including obesity, gastric acid output, and consumption of fatty foods or drugs which may weaken the lower oesophageal sphincter [34,35]. Chronic mucosal damage from acid reflux can lead to a spectrum of symptoms, histopathological features and endoscopic findings, ranging from non-erosive to erosive oesophagitis, Barrett’s oesophagus, and oesophageal adenocarcinoma [35,36,37]. The incidence of GORD is increasing world-wide, in an inverse trend to the prevalence of H. pylori infection [38]. In Europe and the United States, where H. pylori is less common, GORD incidence is fairly stable but at the highest level (11%–23% and 17%–29% respectively). East Asia currently has a very low incidence of GORD (2%–9%), but this is increasing [34].A relationship between H. pylori infection and GORD was first reported in 1998 [39], where the prevalence of H. pylori infection was significantly lower amongst patients with GORD. There were also negative associations with Barrett’s oesophagus and oesophageal adenocarcinoma. A number of subsequent studies supported the inverse relationship between H. pylori infection and GORD, showing that symptoms and endoscopic features developed or worsened after eradication of the infection [40,41]. They have also confirmed the protective associations with Barrett’s oesophagus and oesophageal adenocarcinoma [11,42]. It was proposed that a reduced acid output arising from H. pylori-induced corpus-predominant gastritis or pan-gastritis, plays a role in preventing damage to the oesophageal mucosa [43]. CagA positive strains, which cause more severe inflammation and are stronger acid suppressants, appeared have a greater protective effect [42]. In addition, eradication of H. pylori infection has been reported to result in increased production of ghrelin, a peptide hormone which stimulates appetite and promotes weight gain. Since obesity is a known risk factor for GORD and oesophageal cancer, suppression of ghrelin could be another mechanism by which H. pylori exerts an influence on the disease [35,44].Although several studies provided evidence of H. pylori having an influence on GORD, others have refuted this and generated long-standing controversy within the field [45,46,47]. In particular, not all studies have shown an increased risk of GORD development in patients following H. pylori eradication therapy [41,48,49]. A number of issues could have contributed to the discrepant findings. Some studies, where all participants (both disease and control groups) were under clinical investigation for upper GI symptoms, may have unintentionally incorporated an element of selection bias. There has previously been a lack of agreement on the definition of GORD, and there are also differences in whether patients were assigned GORD status on the basis of symptoms and/or endoscopic findings. Another important source of discrepancy comes from failure to control for the pattern of H. pylori-mediated gastritis, or basing studies on populations where one gastritis pattern is more common [11,42,50]. In Western populations, infections are more commonly antral-predominant, which is associated with increased acid output [43,51]. In some Asian countries (including Japan), patients tend to have corpus-predominant gastritis and are consequently more likely to have a reduced gastric acid output [52]. When H. pylori is eradicated from these patients, gastric contents are likely to become more acidic and more damaging upon reflux into the oesophagus.Awareness of these issues has been taken into account in a number of large-scale well-controlled multi-centre clinical studies and meta-analyses. These have confirmed that H. pylori eradication leads to increased risk of GORD, especially in Asian populations [50,53]. Others have successfully shown a negative association between the infection and Barrett’s oesophagus, as well as a stronger impact of CagA+ strains [11]. The controversies remain however, and in weighing the evidence, the recent Maastricht IV/Florence Consensus Report on the management of H. pylori acknowledges that GORD is less common amongst those who are infected, but concludes that eradication of H. pylori does not influence the severity of GORD [54].Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), are chronic debilitating inflammatory disorders [55]. Potent immunosuppressant drugs are necessary to relieve the symptoms and control against severe recurrences, however these frequently fail to keep IBD in remission. CD can affect the entire length of the digestive tract (including the stomach), but is most commonly observed in the distal small bowel and the first section of the colon. In contrast, UC only affects the colon. Genetic susceptibility plays an important part in determining the risk of CD and UC development [56], however the prevalence of both is increasing and the reasons for this remain unclear. A large number of studies on IBD in adults and children have fairly consistently shown that H. pylori infection and its associated gastritis are less common in IBD cases (reviewed recently in [57]). A meta-analysis showed a significantly reduced risk of IBD when infected with H. pylori (relative risk 0.63) [58]. If H. pylori is really protective against IBD, then one might expect to find IBD development occurring after eradication of the infection. There are just two published reports on this, and both are concerned with CD. In 2001, Jovanovic et al. [59] reported that a patient given H. pylori eradication therapy due to dyspeptic symptoms developed Crohn’s disease three months later. Tursi [60] subsequently reported on two further patients who developed CD after receiving triple therapy for duodenal ulceration. There have been no further publications about this, and no reports at all on the development of UC following H. pylori eradication. Obviously it is possible that these patients might have developed CD regardless of any treatment for H. pylori. Several papers reporting the reduced incidence of H. pylori infection in IBD patients however, have warned of possible exacerbation following eradication therapy [61,62].The possible mechanisms behind such associations are complex. There is published evidence for H. pylori being protective against IBD, as well as H. pylori colonization being inhibited by factors associated with the damaged mucosa and drug treatments for IBD [63,64]. It has been documented that amongst patients with CD-related gastritis, an unusually small proportion are H. pylori-positive [63]. Several mechanisms have been suggested for H. pylori-mediated protection against IBD. One possibility is that H. pylori infection of the gastric mucosa results in modification of the intestinal microflora, and immune responses to these organisms stimulate markedly less inflammation in the gut [65]. There is evidence from the Mongolian gerbil model that H. pylori infection causes alterations in the microbiota of the stomach and duodenum [66]. A long-term H. pylori colonization study in gerbils also showed that there was a change in the microbiota of the large intestine [21].A few animal model studies have demonstrated that H. pylori exerts protective immunological effects against experimental colitis. In one study it was shown that oral doses of H. pylori DNA could substantially reduce the severity of dextran sulphate sodium induced colitis [67]. The protective mechanisms were proposed to be mediated via effects on dendritic cells, which were inhibited from producing proinflammatory cytokines after treatment with H. pylori DNA in vitro. In a second study from the same group [68], mice infected with H. pylori prior to induction of Salmonella typhimurium colitis, had markedly reduced levels of colonic inflammation compared to control animals that were not Helicobacter-infected. This was thought to be due to increased expression of interleukin-10 (IL-10), an anti-inflammatory and immunomodulatory cytokine, and reduced expression of proinflammatory IL-17, in the draining lymph nodes and mucosal tissues.There is broad agreement that regulatory T cells (Tregs), a suppressive subtype of CD4+ T cells, play an important role both in IBD and in H. pylori infection. These cells can act in a bystander manner by secreting immunosuppressive cytokines such as IL-10 and transforming growth factor beta (TGFβ) to modulate inflammation, or they may act in an antigen-specific manner by a myriad of mechanisms (reviewed in [69]). H. pylori stimulates an enhanced Treg response, both in the gastric mucosa and peripheral blood. Tregs are thought to be important for maintaining persistent H. pylori colonization, via suppression of protective immunity, as well for limiting the severity of gastric inflammation so that disease outcomes do not usually result [70,71,72,73]. IBD patients (both CD and UC) tend to exhibit a marked deficiency in Tregs during relapses [74,75,76]. In a similar way to the suppressive effects on gastritis to prevent peptic ulceration, these cells are essential for controlling immune-mediated intestinal pathology [69]. IBD patients with the most severe disease, requiring surgical intervention, have the lowest numbers of Tregs in their peripheral blood [74]. Interventions to enhance the Treg response in CD patients have been tested in clinical trials, including the adoptive transfer of Treg cells [77,78], and administration of Treg-inducing infections (e.g., intestinal parasites [79]). It remains to be seen whether therapies can be developed and advanced, based on deliberate infection with H. pylori, or formulations of Treg-inducing H. pylori components.Coeliac disease is an autoimmune condition that affects up to 1% of people in the developed world, but this is increasing [80,81,82]. Cross-reactive immune responses to a gluten protein found in wheat, result in inflammation and damage to the mucosa of the small intestine. This causes flattening of the villi and impaired absorption of nutrients, and is manifest in a variety of symptoms such as abdominal pain, mouth ulcers, anaemia, muscle cramps, joint pain and growth impairment. There is a very strong genetic component to disease risk [83,84]. Over the last few decades there have been a number of conflicting reports on associations with H. pylori infection. Some studies have shown that the prevalence of H. pylori is reduced in patients with coeliac disease [85,86,87,88], whereas others have found no differences [89,90,91] or even that it is increased [92]. These differing results could be explained by many of the same reasons stated in the previous sections: small sample sizes, determination of H. pylori status via different methodologies, and variable control for age, gender and socioeconomic status. This is important as coeliac disease is more common in females and in those of higher socioeconomic status, whereas H. pylori is more common in the opposite groupings [83,86]. A recent large-scale and well-controlled study compared the prevalence of H. pylori amongst 2,689 patients with coeliac disease and 127,619 patients with normal duodenal histology [17]. They found that the proportion infected amongst the coeliac disease patients was half that of the control group (OR 0.48; p < 0.0001).The mechanisms by which H. pylori might protect against coeliac disease remain unknown and unexplored. It is possible that presence of the infection could affect the antigenicity of gliadin (via effects on gastric acid), or perhaps the Treg response induced by H. pylori modulates the autoimmune reaction. Animal models for coeliac disease do exist (reviewed in [93]), however these have not so far been used to investigate protective effects of H. pylori. There is no evidence for coeliac disease diagnosis arising directly after H. pylori eradication, and no published data show whether H. pylori infection status influences disease severity.Multiple sclerosis (MS) is an inflammatory demyelinating immune-mediated disorder which affects the central nervous system (CNS). Development of autoreactive T cell responses against CNS-derived antigens leads to an influx of Th1 and Th17 cells into the spinal cord and CNS [94,95]. These cells cause damage to the myelin sheath of neural axons, along with inflammation and degeneration of nerves [96]. Several epidemiological studies have reported a significantly lower prevalence of H. pylori infection amongst MS patients [97,98,99,100,101]. Additionally, two case control studies found that amongst MS patients, neurological disability was reduced in those with H. pylori [99,100]. In contrast, other studies have failed to find any association between H. pylori infection and MS [102], perhaps because of a positive association between H. pylori and a severe variant of MS, neuromyelitis optica (NMO) (reviewed in [18]).To date, there is very little mechanistic evidence for a protective association between MS and H. pylori. There are no data concerning the impact of H. pylori eradication therapy on MS, and only one animal model study has been reported so far. Recently our group showed that prior H. pylori infection of mice inhibited the severity of experimental autoimmune encephalomyelitis (EAE), an animal model of MS [101], the most commonly used model for investigating human MS [103]. EAE was induced by immunization with the MOG35-55 myelin peptide, leading to an autoimmune response that mimics MS [103,104].The hypothesis that H. pylori may be protective against MS and EAE is perhaps counterintuitive, since Th1 and Th17 cells, which play a role in disease pathogenesis [105], are also induced by the infection [106]. It has been shown that injection of mice with heat killed H. pylori bacteria and Freund’s incomplete adjuvant, however, is not sufficient to trigger EAE [107]. Our data showed that the numbers of CD4 cells in the spinal cords of infected animals was approximately half that of the uninfected controls. There was an extremely pronounced reduction in Th1 and Th17 cells, both in the spleen and in the spinal cord [101]. The balance between pro-inflammatory (Th1 and Th17) and anti-inflammatory (Treg) T cell subset responses is known to be important in the development and progression of MS [108,109]. Humans and animals infected with H. pylori have elevated IL-10-secreting Treg populations, therefore this suppressive T cell response may provide protective activity in a bystander fashion. In patients, H. pylori infection is associated with alterations in the profile of homing receptors expressed by peripheral blood T cells, directing their migration towards the inflamed gastric mucosa [70,110]. This includes an increase in the proportion of Tregs that express the chemokine receptor CCR6 [70]. CCR6 is a marker for Th17 cells and is important in modulating the balance between Treg and Th17 populations [111]. CCR6 plays an important role in EAE, since CCR6-deficient mice develop less severe disease [112] and are also less able to control EAE when it develops [113]. The infection may therefore alter the expression of chemokine receptors and integrins by T-effector or regulatory T cells, resulting in fewer T cells entering the CNS and inhibiting EAE development. Further work is necessary to confirm these findings and determine the mechanisms behind them.The best evidence for H. pylori-mediated protection against extra-gastric disease comes from research on allergy and asthma. Although genetic predisposition is important, developing atopy is frequently associated with exposures in early life [114]. Protection against atopic disease may be mediated via a number of infectious organisms in the context of a diverse microbial microflora, rather than one simple component [115]. However, as the rate of H. pylori infection in childhood has declined in many developed countries, the prevalence of atopic disease in developed countries has increased markedly [116,117]. In one Finnish study for example, a 3-fold increase in the incidence of allergy was accompanied by a 30% decrease in H. pylori prevalence between 1973 and 1994 [118].Multiple groups have shown that H. pylori infection is less common amongst those with atopy, and that H. pylori-infected adults and children are less likely to suffer from allergic asthma, rhinitis, food allergy, or have skin-prick allergen test sensitivity (reviewed in Table 1). Not all the studies agree, however the overwhelming evidence (supported by very large studies and meta-analyses) is supportive of a protective association. One of the key papers reported findings from a large population-based study of 7663 adults in the US National Health and Nutrition Examination Survey [32]. This confirmed a link between H. pylori negative infection status and having an early-life asthma diagnosis. People infected with CagA+ strains were even less likely to have had childhood asthma. Since not all strains express this virulence factor, this provided the first evidence that H. pylori could be driving these associations.The strongest protective associations appear to be against childhood asthma [119,120], and age of the population being studied may be one of the reasons for contradictory findings. H. pylori infections are usually acquired in early childhood, a common age for onset of asthma [121]. Unfortunately current studies are limited by a paucity of data concerning the age of H. pylori acquisition and its relationship with age of asthma development. Other issues include the fact that asthma is not a uniform disease, being a manifest airway hyperresponsiveness to a variety of triggers including inhaled allergens, but also air pollutants, respiratory viruses, bacterial infections, and medications [25,122]. Asthma may also be worsened by aspiration of acidic gastric juice into the lungs [123,124]. The presence of GORD could therefore have an impact in some H. pylori studies.H. pylori infections are usually established in early childhood [125], when the immune system is developing. The infection is known to stimulate a Th1 response in the gastric mucosa and also in peripheral blood [126,127]. Cytokines secreted by Th1 cells can counterbalance and suppress a Th2 response, which is the predominant T-helper subset associated with allergy [25]. In addition, infection with CagA+ H. pylori strains is reported to result in even greater Th1 responses [128] and reduced Th2 responses [129]. The H. pylori neutrophil-activating protein (HP-NAP) is an important Th1-promoting virulence factor, which has been shown to modulate Th2 responses in humans and mice [130,131,132,133]. When HP-NAP was administered via systemic or mucosal delivery to mice undergoing ovalbumin (OVA) allergen sensitization, this inhibited the development of lung eosinophilia, markedly reduced serum IgE levels, and there were lower bronchoalveolar Th2 cytokine concentrations [130]. H. pylori infection also influences the Th1/Th2 balance via effects on gastric hormones. When levels of somatostatin are reduced and gastrin production is increased, this also inhibits Th2 cytokine release and promotes Th1 responses [134].The main immunological mechanism being investigated, however, is the H. pylori-mediated stimulation of Tregs. As previously mentioned (Section 3.2), these can act in a bystander manner to dampen immune responses such as those in asthma and allergy [135]. Th2 type inflammation in asthma is usually suppressed by Treg cells, and many current asthma therapies act by enhancing Treg responses. IL-10 is thought to play a major role in this, as it suppresses Th2 cell activity. It suppresses mast cell activation and cytokine production by mast cells and eosinophils. IL-10 also inhibits IgE production and promotes IgG4 production, an immunoglobulin balance thought to be protective against allergic responses. The role of the suppressive cytokine transforming growth factor β (TGF-β) is more complex, as apart from immunomodulation it is also involved in fibrosis and tissue remodeling in the airways [136,137,138].Increased numbers of Tregs are present in the gastric mucosa and peripheral blood of H. pylori-infected patients [70,71,72,73,127]. H. pylori infection is also known to induce Treg responses in the gastric mucosa, peripheral blood and spleens of mice [72,139]. Stronger IL-10 and Treg responses are present in people with cagA+ strains [71,128], perhaps explaining the stronger protective associations between asthma and CagA+ infections [32]. CagA-dependent T cell priming in infected mice is also important for inducing Treg differentiation [140]. The fact that H. pylori stimulates a systemic Treg response supports the idea that such cells could have a more general immunoregulatory role.In 2011, a paper from the group of Anne Müller provided the first mechanistic evidence that H. pylori infection could protect against allergic asthma in a mouse model [141]. Infected animals had significantly reduced airway hyperresponsiveness, measured by methacholine resistance, compared to uninfected controls. This was accompanied by a marked reduction in markers of asthma, including allergen-specific serum IgE, pulmonary infiltration of Th2 cells, Th17 cells and eosinophils, IL-5 and IL-13 in the lung lavage fluids, as well as a reduction in goblet cell metaplasia. These protective effects were strongest in mice that had been infected with a cagPAI+ strain of H. pylori as neonates, thus mimicking the human data where there was greater protection against childhood asthma, and in those infected with CagA+ strains [119]. The protection against asthma in mice was conferred by Tregs, since it could be induced by adoptive transfer of mesenteric lymph node cells from neonatally-infected donors, but not when the cells were depleted of Tregs prior to transfer. It was subsequently shown that H. pylori reprogrammes the differentiation of dendritic cells (DCs) to a tolerogenic phenotype, and these cells promote the differentiation of naïve T cells into Tregs. Such immature DCs (DC-SIGN+ HLA-DRhi CD80lo CD86lo) were also found in the infected human gastric mucosa [142]. More recently the group has shown that the regulatory cytokine IL-10 and CD103+CD11b− dendritic cells are necessary to successfully protect against asthma with H. pylori in mice.Epidemiological evidence for and against H. pylori-mediated protection against allergy and asthma.The H. pylori factors gamma-glutamyl transpeptidase (GGT) and vacuolating cytotoxin A (VacA) play an important role in the development of the Treg response. Intraperitoneal or intragastric delivery of purified VacA or recombinant GGT in mice could induce similar levels of protection against asthma as observed with the infection [159,160]. The effects of these components in humans must now be characterized to ensure that the mechanisms are clinically relevant, prior to investigating them as possible therapeutic agents.Investigating the impact of H. pylori on extra-gastric diseases is extremely challenging. The majority of the current evidence is based on cross-sectional epidemiological data, some of which may be flawed, and there is no way to assess cause and effect. Discrepancies between studies frequently arise from the use of different diagnostic methods, which may or may not be able to distinguish current from previous H. pylori infections. There is also a lack of adjustment for important confounding factors such as socioeconomic status, smoking, obesity and the effectiveness of eradication therapy. The genetic characteristics of both the bacterium and its host play an important role in determining the consequences of host-pathogen interactions, however this is extremely difficult to control for.There are many remaining unknowns (see Table 2), and it is possible that some protective associations may ultimately not turn out to be driven by H. pylori. Perhaps H. pylori is merely a marker for other protective exposures. Alternatively, its observed effects could be mediated by modifying the complex microbiota of the gastro-intestinal tract rather than acting directly. To advance this field further, better-designed human studies must be carried out and ideally these should incorporate some way to assess cause and effect. This might be achieved by monitoring the long-term impact of H. pylori eradication, or with longitudinal studies, comparing disease severity over time in H. pylori-infected and uninfected patients. Such an approach cannot exclude the role of potential confounding exposures, however. The most direct way forward would be to perform clinical trials, monitoring effects arising from administering a H. pylori infection to volunteers. There have already been several human infection trials, however these have involved short-term periods of colonization and were not designed to investigate extra-gastric effects [33,161,162]. Data showing that particular strain types confer different levels of protection will be very informative, and we should establish whether immune-mediated protective effects in childhood can persist to later life, even when the infection has been eradicated. More animal model studies are needed to determine if the infection has a protective effect, and also to provide an understanding of the mechanisms. The results of these experiments should also be confirmed using human cells or tissue, to ensure that they are relevant to the clinical condition rather than mouse-specific mechanisms.This field is difficult, but the benefits are potentially very far-reaching and rewarding. H. pylori is becoming less prevalent, and therefore the impact of this trend on the development of chronic immune and inflammatory disease must be assessed. As developing countries become more industrialised, loss of exposure to H. pylori could trigger a substantial increase in the prevalence of these debilitating diseases. In the future it may become possible to develop therapies based on H. pylori components. In the meantime however, strategies for H. pylori eradication should consider the possible health benefits conferred by this infection.Evidence gathered for H. pylori-mediated protection against extra-gastric diseases.Research in the laboratory of Karen Robinson is supported by the University of Nottingham, the Medical Research Council, and the National Institute for Health Research (NIHR), through the NIHR Biomedical Research Unit in Gastrointestinal and Liver Diseases at Nottingham University Hospitals NHS Trust and the University of Nottingham. The views expressed are those of the author and not necessarily those of the NHS, the NIHR or the Department of Health.The author declares no conflicts of interest. 

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Prader-Willi syndrome (PWS) is characterized by hyperphagia, obesity if food intake is not strictly controlled, abnormal body composition with decreased lean body mass and increased fat mass, decreased basal metabolic rate, short stature, low muscle tone, cognitive disability, and hypogonadism. In addition to improvements in linear growth, the benefits of growth hormone therapy on body composition and motor function in children with PWS are well established. Evidence is now emerging on the benefits of growth hormone therapy in adults with PWS. This review summarizes the current literature on growth hormone status and the use of growth hormone therapy in adults with PWS. The benefits of growth hormone therapy on body composition, muscle strength, exercise capacity, certain measures of sleep-disordered breathing, metabolic parameters, quality of life, and cognition are covered in detail along with potential adverse effects and guidelines for initiating and monitoring therapy.Prader-Willi syndrome (PWS) is a complex genetic disorder caused by lack of expression of genes on the paternally inherited chromosome 15q11.2-q13. Approximately 70% are due to deletion of this region, 25% are due to maternal uniparental disomy (UPD) of chromosome 15, and most of the remaining 5% are due to an imprinting center defect [1]. In adulthood, clinical manifestations include hyperphagia, obesity if food intake is not strictly controlled, decreased basal metabolic rate, sleep-disordered breathing, cognitive disability, short stature, and hypogonadism. The following are also characteristic of the syndrome: low muscle tone and abnormal body composition with decreased lean body mass and increased fat mass. Hypothalamic dysfunction has been implicated in many manifestations of this syndrome, including growth hormone (GH) insufficiency. The benefits of recombinant growth hormone therapy (hGH) on body composition and motor function, in addition to linear growth, in children with PWS are well established [2,3]. Evidence is emerging on the benefits of growth hormone therapy in adults with PWS as well. This review summarizes the current literature on growth hormone status and use of hGH in adults with PWS. We discuss the benefits of growth hormone therapy on body composition, muscle strength, exercise capacity, certain measures of sleep-disordered breathing, metabolic parameters, quality of life, and cognition. We also discuss potential adverse effects and guidelines for initiating and monitoring therapy.Evidence of growth hormone insufficiency is present in the great majority of children with PWS [4,5]. There is also evidence of growth hormone insufficiency in many adults with PWS. Clinical evidence includes the abnormal body composition, which is also present in adults with growth hormone deficiency not affected by PWS [6]. Biochemically, adults with PWS have a diminished response to growth hormone provocative testing and lower insulin-like growth factor 1 (IGF-1) levels. Anatomically, anterior, but not posterior, pituitary size was found in one study to be significantly smaller in adults with PWS compared to healthy controls [7]. Another study found differences in cardiac anatomy and function, specifically decreased left ventricular mass and end diastolic diameter, in adults with PWS compared to obese controls, consistent with the hypotrophic hypokinetic syndrome that is associated with growth hormone deficiency [8].Compared to obese controls, adults with PWS have a significantly lower response to growth hormone provocative testing with growth hormone releasing hormone (GHRH) plus arginine, not only for peak growth hormone values, but also the more functional determination of instantaneous secretion rates [9,10]. In studies using GHRH + arginine, 8%–55% of adults with PWS met diagnostic criteria for severe growth hormone deficiency using body mass index (BMI)-specific cut-off values [7,9,11,12,13]. However, some have questioned the validity of the GHRH + arginine test in those with hypothalamic disorders, given that GHRH is of hypothalamic origin [14]. With arginine as a provocative agent, 67% met diagnostic criteria for severe growth hormone deficiency, and 40% met criteria with insulin-induced hypoglycemia in one study [15]. In several studies, those with the UPD genotype had a lower response to stimulation testing than those with deletion [9,13,16]. IGF-1 levels are significantly lower in adults with PWS compared to obese controls [9]. In several studies, 75%–91% of adults with PWS had IGF-1 levels below the normal range [9,13,15]. Insulin-like growth factor binding protein-3 (IGFBP-3) levels were below the 5th percentile in 27% of adults with PWS [15]. It is important to note however that sex steroids promote growth hormone secretion and affect IGF-1 levels [17]. Hypogonadism is prominent in PWS [1]. Less than half of the subjects in the above studies were receiving sex steroid replacement therapy, which may have affected growth hormone secretion and IGF-1 levels.Studies have shown benefits of growth hormone therapy in adults with PWS on body composition, muscle characteristics, motor function, exercise capacity, peak expiratory flow, certain measures of sleep-disordered breathing, metabolic parameters, quality of life, and cognition.The Scandinavian study is the largest published randomized placebo-controlled trial of growth hormone therapy in adults with PWS. Forty-six patients were randomized to hGH therapy with 0.6 or 0.8 mg daily after the first month depending on body weight, or placebo for the first year. Following this period of randomized treatment, all patients were treated with hGH for a total of 2 years. Thirty-nine patients completed both phases of the study. Lean body mass (LBM) increased in the hGH treated group by 2.3 kg as measured by duel-energy x-ray absorptiometry (DXA), and did not change in the placebo group. Fat mass decreased in the hGH group but increased in the placebo group with a difference of 4.2 kg. Subcutaneous and visceral fat, measured by targeted computed tomography (CT), also decreased in the hGH treated group while it increased in the placebo group with the greatest difference seen in subcutaneous fat. These benefits on body composition were sustained after 2 years of hGH therapy. Body mass index (BMI) and waist circumference did not change significantly [18,19,20]. Other studies have shown similar effects of growth hormone therapy on body composition. Increases in LBM of 2.3–3.7 kg and decreases in percent fat mass (FM%) of 2.2%–3.8% have been reported after 1 year of hGH therapy at mean doses of 0.53–1 mg daily [21,22,23,24,25]. One cohort was followed for 4 years with hGH therapy and these benefits persisted [26]. Subcutaneous and visceral fat measured by targeted CT in this cohort decreased significantly after 1 year of hGH therapy and visceral fat remained markedly decreased after 4 years of therapy [22,26]. Another group reported an increase in LBM of 2.8 kg and decrease of 1.9% in FM% after 2 years of hGH therapy [27]. In a study of six adults with PWS who had been on growth hormone 0.2–0.5 mg daily for a median of 5.1 years, median LBM increased by 4.9 kg and median FM% decreased by 4.7%, with the changes occurring gradually over time [28]. Consistent with the Scandinavian study, there was no significant change in BMI in any of these studies. A recent study of 10 males with PWS treated with hGH at a mean dose of 0.35 mg/day in adulthood for a mean of 15.5 years reported maintenance of a higher fat free mass than fat mass in all [29].Two studies investigated changes in BMI and body composition after growth hormone therapy was stopped. In one study of 11 adults with PWS, the improvements in LBM and FM% after 1 year of hGH therapy at a mean dose of about 1 mg/day reverted back to baseline after therapy was withdrawn during the 2nd year of the study [24]. In another study, BMI standard deviation score (SDS) increased significantly in the 2 years after cessation of growth hormone therapy in late adolescence [30].Several studies have shown improvements with hGH in skeletal muscle characteristics and function. There was a significant increase in thigh muscle volume by targeted CT after 2 years of hGH therapy in the Scandinavian study [19]. Another group confirmed a significant increase in thigh muscle size by CT imaging in 15 adults with PWS after 2 years of hGH at a mean dose of 0.4 mg/day, as well as a significant increase in lumbar muscle size and change in muscle tissue attenuation, reflecting a decrease in lipid accumulation. Strength measured by handgrip dynamometer also increased significantly and correlated with the increase in lumbar muscle size [27]. Growth hormone therapy also positively affects exercise capacity in adults with PWS. With hGH therapy, participants in the above study were able to continue a treadmill exercise test significantly longer before reaching exhaustion [27]. The same group also reported a metabolic equivalents (MET) increase of 19% on a treadmill exercise test after 1 year of hGH therapy at a mean dose of 0.9 mg daily in 12 adults with PWS [25]. In another study, the number of bouts of moderate-vigorous physical activity per day increased significantly after 1 year of therapy at a mean dose of about 1 mg/day and reverted back to baseline after hGH was withdrawn during the 2nd year of the study [24]. Left ventricular mass improved after 1 year of hGH therapy at a mean dose of 0.96 mg/day without negative effects on cardiac function in a study of 13 adults with PWS [22]. Nine of these individuals were followed for 4 years on hGH and these cardiac benefits persisted [26]. Furthermore, peak expiratory flow improved by 33 l/min after 2 years of hGH therapy in the Scandinavian study [19].A few studies showed improvements in metabolic parameters with hGH therapy. The inflammatory marker, C-reactive protein (CRP), decreased significantly in 2 studies after 1 year of hGH and remained decreased after 4 years of therapy in one cohort [22,26,27]. One study reported normalization of low baseline triiodothyronine (T3) levels with 1 year of hGH therapy [23]. In the Scandinavian study, low-density lipoprotein (LDL) cholesterol decreased significantly in the hGH versus placebo group with 1 year of therapy. However, there was no change in LDL levels after 2 years of therapy [18]. One study reported an increase in HDL cholesterol after 1 year of hGH therapy that decreased significantly when growth hormone was withdrawn during the 2nd year of the study [24]. Other studies, however, showed no change in lipids after 1 year of hGH [22,23,27].Quality of life improved in adults with PWS after 2 years of growth hormone therapy as measured by two validated instruments, the 36-Items Short Form Health Survey (SF-36), and the Psychological General Well-Being Index (PGWBI). Improvements were seen in anxiety, depression, general health, and total scores, reported by both the PWS adults and their parents. Improvements reported by the PWS adults however were greater than those reported by their parents [31]. In another study, participants spontaneously reported improvements in psychological well-being after 1 year of hGH therapy [21]. An additional study reported improvements after 1 year of hGH in a 20-item behavioral assessment completed by the participants’ caregiver [23]. These studies were not controlled.On neuropsychological testing during a 6 month double-blind randomized placebo-controlled trial of hGH, followed by a 12 month open label treatment period of all participants, significant improvements were noted at 6 and 18 months in the hGH treated group in mental speed and flexibility, and motor performance. No improvements were seen at 6 months in the untreated group. However, only 13 of the 19 patients in this study were genetically confirmed to have PWS [32].Two years of hGH therapy in the Scandinavian study did not show improvement in bone mineral density [33]. Similarly, a more recent study showed no effect of long-term hGH on bone density but did show a positive effect on bone size and strength, measured by bending breaking resistance index [34].Despite the significant benefits of hGH on body composition, quality of life, muscle strength, exercise capacity, and pulmonary function in adults with PWS, there remains concern for potential adverse effects of this treatment. Due to the obesity that is commonly present in adults with PWS, the concern for adverse metabolic sequelae from growth hormone therapy, especially impairment in glucose homeostasis, deserves careful exploration. Type 2 diabetes mellitus has been reported in 25% of adults with PWS with a mean age of onset of 20 years [35]. The association of hGH treatment with insulin resistance is well documented. Although the mechanism of action for this association is not well defined, changes in free fatty acid metabolism, non-physiologic levels of IGF-1, and chronic basal hyperinsulinemia represent potential causes [36]. A recent meta-analysis of seven studies evaluating efficacy and safety of hGH for at least 12 months in adults with PWS showed a small increase in fasting glucose, and trends toward higher fasting insulin and insulin resistance. No significant changes in hemoglobin A1c or development of diabetes mellitus were noted. The longest study included in this meta-analysis involved 6 patients treated over a mean of 5 years [37]. More recently, the Scandinavian study group investigated glucose homeostasis in relation to BMI in 39 adults with PWS before and after 12 months of hGH therapy. They found that hGH treatment at a mean dose 0.6 mg was associated with a small but significant increase in fasting glucose, 2 h glucose after 75 gm oral glucose tolerance test (OGTT), and homeostatic model assessment of insulin resistance (HOMA-IR), irrespective of BMI [38]. In a recent report of 10 adult males with PWS treated with hGH for a mean of 15.5 years, 3 (30%) developed diabetes while on therapy. All 3 also had significant associated weight gain [29]. Therefore, close monitoring of glucose homeostasis is warranted in adults with PWS treated with hGH and additional longer term studies are needed.The most common adverse event associated with hGH therapy in adults with PWS is lower extremity edema. Edema occurred in approximately 15% of those studied [37]. The edema often resolved after decreasing the dose [39]. A patient in one study stopped the hGH due to myalgias associated with lower extremity edema [23]. Other adverse events noted in the largest group of PWS patients studied were headache and nausea. Of note, one patient in the Scandinavian study was reported to have carpal tunnel surgery during hGH therapy [20]. Significantly, in the recent meta-analysis study, sudden death was not reported as an adverse event in any of the 7 cohorts [37].Individuals with PWS have a high incidence of both central and obstructive sleep apnea [40,41,42]. Factors contributing to sleep-disordered breathing include obesity, restrictive lung disease due to muscle weakness or scoliosis, reduced ventilatory response to hypercapnia, and hypoxia during sleep and wakefulness [43]. Therapy with hGH potentially worsens sleep-disordered breathing because increased IGF-1 levels lead to lymphoid hyperplasia [44,45]. Only one study of relatively short duration has examined the effects of hGH on sleep-related breathing disorders in adults with PWS. This study evaluated sleep related breathing disorders at baseline and 6 weeks after the initiation of hGH therapy in 15 children and 10 adults with PWS. Nine of the 10 adults showed improvement in the apnea/hypopnea index (AHI) and decrease in the frequency of central events. The one adult who showed worsening of the AHI and frequency of obstructive events had a concurrent respiratory infection and tonsillar hypertrophy [44]. Until further data more clearly define the relationship between hGH and sleep-disordered breathing in PWS, current guidelines recommend polysomnography, with treatment of significant findings, prior to starting hGH therapy, and repeat polysomnography within 3-6 months of hGH initiation [46]. In addition, it is prudent to titrate hGH dosing to keep IGF-1 levels in the normal range to minimize the possibility of lymphoid hyperplasia and, while on therapy screen for signs and symptoms of worsening sleep apnea.After an extensive review of the literature, consensus guidelines for recombinant human growth hormone therapy in PWS were published in 2013 [46]. These guidelines are summarized in this section. For adults with genetically confirmed PWS, hGH therapy should be considered after an expert multidisciplinary evaluation. Guidelines recommend considering severe obesity (BMI > 40 kg/m2), uncontrolled diabetes mellitus, untreated severe obstructive sleep apnea, active cancer and psychosis as contraindications to hGH therapy. Cognitive impairment is not considered an adequate barrier to treatment and it is strongly stated that informed consent/assent should include a discussion of the known risks and benefits of therapy. Therapy with hGH should be used in conjunction with dietary, environmental and lifestyle interventions. It should not be viewed as a weight loss medication. Therapy should be continued as long as the benefits outweigh the risks.Prior to initiating hGH therapy in adults with PWS, the following evaluation is recommended (Table 1). A baseline IGF-1 level should be measured and growth hormone provocative testing considered. Many regulatory agencies require provocative testing in order to diagnose growth hormone deficiency prior to allowing insurance coverage of hGH treatment for adults with PWS. However, many participants in the studies showing benefit of hGH therapy did not meet the cut-off definition of growth hormone deficiency. For example, only 14.6% of those in the Scandinavian study tested as growth hormone deficient with GHRH + arginine [11,20]. Other studies have reported improvements with hGH in body composition, muscle size, strength, and exercise capacity that were independent of growth hormone secretory status [25,27]. Therefore, relying on provocative testing results may deny treatment to those who would benefit.It is also important to carefully document anthropometric status including weight, height, BMI, waist circumference, and skinfold thickness if possible. Screening for hypothyroidism and appropriate treatment is warranted. As central adrenal insufficiency has been associated with PWS, providers should consider evaluation of adrenal function on an individual basis [47,48,49]. Growth hormone inhibits 11β-hydroxysteroid dehydrogenase type 1 (11βHSD-1), resulting in less cortisone conversion to active cortisol. Thus any underlying adrenal insufficiency could be exacerbated by hGH initiation.Metabolic status should be assessed with a hemoglobin A1c and fasting insulin and glucose levels. An oral glucose tolerance test should be considered for individuals with a strong family history of diabetes, acanthosis nigricans, or ethnic risk factors. For obese individuals, fasting lipids and liver transaminases (AST, ALT) should be assessed. Sleep oximetry is felt to be mandatory before starting hGH in all patients, preferably by polysomnographic evaluation. Other helpful evaluations include body composition by dual energy x-ray photon absorptiometry (DXA) or bioelectrical impedance, assessment of cognitive status, and assessment of motor function (consider physiotherapy or occupational therapy referral).Recommended evaluation prior to initiation of growth hormone therapy (hGH) in adults with Prader-Willi syndrome (PWS) [46].Adults with PWS should be started on hGH therapy at a dose of 0.1–0.2 mg/day. Starting dose should take into consideration age, presence of edema, prior hGH exposure and sensitivity, and concomitant oral estrogen use. Once therapy is started, the hGH dose should be titrated based on clinical response with a goal of maintaining IGF-1 levels in the 0 to +2 SDS range for age and gender.Once started on hGH, monitoring should address specific benefits and risks of the treatment and impact on other potential hormone deficiencies (Table 2). IGF-1 levels should be checked at least annually and as needed for dose titration. IGF-1 levels checked for titration purposes should occur no less than six weeks after the dose change [14]. We recommend checking thyroid function within 3–6 months of initiation of hGH therapy and at least annually. Continued monitoring of glucose homeostasis should occur at least annually. For obese patients, routine monitoring of obesity co-morbidities such as hypertension, hyperlipidemia, and hepatic steatosis should occur as recommended by obesity guidelines [50]. Monitoring for other side effects of growth hormone therapy should take place on a routine basis. Evaluation for lower extremity edema should be done at each visit as well as assessment for signs or symptoms of sleep apnea. As one of the most well established benefits of hGH therapy in adults with PWS is improved body composition, repeat evaluation by DXA or biochemical impedance should be performed every 2 years [14]. Formal measurements of cognitive function, quality of life or exercise tolerance should be considered on an individual basis.Recommended monitoring during hGH therapy in adults with PWS [46].WeightBMIWaist circumferenceSkin fold thickness if possibleHemoglobin A1cFasting glucose and insulinOGTT if high riskBlood pressureFasting lipidsConsider AST and ALTCurrent evidence supports that adults with PWS have lower baseline IGF-1 levels and a diminished response to GH provocative testing when compared to obese controls. Depending on the cohort studied and the provocative agents used, 8%–55% of adults with PWS met criteria for severe GH deficiency based on BMI specific cut-offs. Adults with PWS treated with hGH experience many benefits of therapy, even when GH provocative testing is normal. The most consistently reported benefit is a beneficial change in body composition with increased LBM and decreased fat mass. Other benefits include improved skeletal muscle function, increased exercise tolerance, increased left ventricular mass, increased peak expiratory flow, improvement in cognition and quality of life, and a decrease in the apnea/hypoxia index. Studies of relatively short term use of hGH in adults with PWS show relatively few side effects, with lower extremity edema and increased insulin resistance being the side effects most consistently reported. There is a dearth of information regarding the effects of hGH on sleep disordered breathing in adults with PWS and further study of this relationship is warranted. Monitoring for known side effects of therapy is crucial during treatment. Finally, given that most of the current studies are uncontrolled and of short duration, and the effect size of many reported benefits is small, further longer-term controlled studies on the benefits and risks of hGH therapy in this patient population are necessary.The views expressed in this publication are those of the authors and do not necessarily reflect the official policy or position of the Departments of the Army or Navy nor the US Government.We thank Kirk Jensen and Merrily Poth for their critical appraisal and comments that contributed to the final version of this manuscript.The authors declare no conflict of interest.

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+Members of the Saga Liver Cancer Study Group. The Saga Liver Cancer Study Group is composed of tertiary-care hospitals with specialists in liver cancer treatment in Saga Prefecture, Japan.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Sorafenib exerts modest antitumor activity in patients with advanced hepatocellular carcinoma (HCC), and radiological progressive disease (rPD) does not always correspond to so-called clinical progressive disease (cPD). We evaluated 101 patients who initiated sorafenib treatment for HCC and assessed post-progression survival (PPS) using the Cox proportional hazards model. PPS was calculated from the date of the first rPD until the date of death or the last follow-up. Using Cox model analysis of the 76 patients who experienced first rPD, we identified the Child-Pugh class, Eastern Cooperative Oncology Group performance status, the best antitumor response during treatment (using Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1) and α-fetoprotein levels as independent factors affecting PPS. When these factors were used to define scores ranging from zero to five with a cutoff value of two, PPS of patients who received best supportive care (BSC) after rPD was not statistically significantly different from that of patients who received post-rPD therapy with scores ≥2 (p = 0.220). In contrast, the PPS for the post-rPD therapy group was significantly longer compared with the BSC patients with scores <2 (p < 0.001). Patients who scored ≥2 at their first rPD were judged cPD and as candidates for BSC.Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide [1,2]. The prognosis of HCC depends on its stage at diagnosis. Although the prognosis is favorable for patients with early HCC who receive radical therapy, it is poor for those with advanced HCC. Sorafenib is the first targeted agent with significant clinical activity for advanced HCC. In previous multicenter, double-blind, randomized phase 3 studies, the SHARP [3] and Asia-Pacific [4] studies, sorafenib provided statistically-significant survival benefits compared with placebo in patients with advanced HCC. Because survival is influenced by second- and beyond-line therapies, reviews of other cancers demonstrate the requirement for post-progression survival (PPS) analysis [5,6,7].However, sorafenib only induces modest tumor shrinkage. Cytotoxic therapy is most often discontinued or changed when radiological progressive disease (rPD) occurs; however, the optimal termination point for targeted therapy can be difficult to determine according to rPD [3,4,8]. So-called clinical PD (cPD) does not correspond to rPD during sorafenib therapy for HCC, because we experienced a few patients who continued sorafenib after diagnosis of rPD retaining disease control over a long period in clinical practice. The Response Evaluation Criteria in Solid Tumors (RECIST) was originally developed to assess responses to cytotoxic agents and may not be appropriate for targeted agents [9,10].This study analyzed prognostic factors for PPS using patient parameters at rPD. We then attempted to develop an indicator for judging cPD as an adjunct to RECIST using these prognostic factors for patients with advanced HCC administered sorafenib as first-line therapy.The Saga Liver Cancer Study Group (SALC) comprises tertiary-care hospitals in Saga, Japan, with specialists in liver cancer treatment. A retrospective analysis of all patients with HCC treated with sorafenib in Saga Prefecture was performed using the unified database system of the SALC. The patients received 400 mg of sorafenib twice daily; however, initial dose reduction considering each patient’s condition was allowed. All other aspects of sorafenib therapy, including dose adjustment or interruption, treatment schedule, supportive therapies and post-rPD therapies, were determined by a physician. The institutional review board or ethics committee of each institution approved the SALC protocol.All patients had histologically- or radiologically-confirmed HCC that was diagnosed as advanced, ineligible for resection or locoregional treatment or refractory to chemoembolization. Patients’ conditions when sorafenib treatment was initiated were defined according to the Eastern Cooperative Oncology Group performance status (ECOG PS) as scores ranging from 0 to 2, Child–Pugh scores ≤8 and adequate hematologic and liver function. Adequate hematologic functions were defined as follows: hemoglobin concentration ≥8.5 g/dL, neutrophil count >1500/µL and platelet count >75,000/µL. Adequate liver functions were defined as alanine aminotransferase and aspartate aminotransferase levels lower by a factor of 5 than the normal upper limit and total bilirubin level <3.0 mg/dL. Patients requiring hemodialysis were not included. Patients were also considered ineligible if they received concomitant systemic therapy, including any targeted agents. All patients provided written informed consent before receiving sorafenib therapy.The physician determined the first rPD. Radiologic evaluations were conducted every 4–8 weeks using enhanced computed tomography or magnetic resonance imaging, according to RECIST Version 1.1 [11]. Time to progression (TTP) was calculated from the date of initial sorafenib administration to the date of first rPD, or was censored at the last follow-up, or at the time of death without rPD. PPS was the primary endpoint and was calculated from the date of first rPD until the date of death or last follow-up.For this analysis, patients with first rPD were classified into three groups according to post-rPD management as follows: patients who continued to receive sorafenib beyond rPD for at least 1 month (Group A), patients who received other second-line therapies after rPD (Group B) and patients who received best supportive care (BSC) (Group C). Groups A and B were integrated into one post-rPD therapy group (Group A + B) and compared with Group C. Post-rPD management was decided by the physician in accordance with the patient’s condition. Distributions of variables among the groups were compared using the χ2 test for categorical data and the Mann–Whitney U test for continuous data. TTP and PPS were estimated using the Kaplan–Meier method, and the curves were compared using the log-rank test. Cox proportional hazards models were used to identify factors associated with PPS. The models were also used to devise the scoring system for judging the first rPD. Univariate analyses were performed to assess potential factors related to PPS. If the factors attained a significance level of p < 0.01, a multivariate proportional hazard model for these factors was constructed to estimate each coefficient (β) and hazards ratio. Scores were defined according to the weighted sum of those factors, with weights defined simply as the estimated coefficients. Cutoff value, sensitivity and specificity for PPS were assessed using the time-dependent receiver-operating characteristic (ROC) curve [12] and area under ROC (AUROC) curves. ROC curves were plotted with 1-specificity and sensitivity measured along the horizontal and vertical axes, respectively. All statistical analyses were performed using R Version 3.0.1 (The R Foundation for Statistical Computing, Vienna, Austria).Sorafenib was administered to 101 patients from July, 2008–April, 2012, at five SALC institutions. Seventy-six patients experienced their first rPD, and the remaining 25 patients were either alive without rPD (n = 17) or discontinued sorafenib caused by adverse events before rPD (n = 8). Among the patients documented with rPD, 26 were in Group A, 29 in Group B and 21 in Group C. Group A (beyond rPD) and Group B (second-line therapy) were integrated into one group (Group A + B), and the patient characteristics for the A + B and C groups at the first rPD are listed in Table 1. At the first rPD, ECOG PS, Child–Pugh class and the level of α-fetoprotein (AFP) of Group C were significantly worse than those of Group A + B. Dose reduction or interruption of sorafenib were required for 45 patients (81.8%) in Group A + B and 12 patients (57.1%) in Group C (p = 0.027). The median TTP was significantly longer in Group A + B than in Group C (2.5 vs. 1.8 months, respectively; p = 0.012). Similarly, median PPS was also significantly longer in Group A + B than in Group C (9.5 vs. 2.1 months, respectively; p < 0.001).Patient characteristics.† According to the Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1 until the first rPD. Abbreviations: AEs, adverse events; AFP, α-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; ECOG PS, Eastern Cooperative Oncology Group performance status; HBV, hepatitis B virus; HCV, hepatitis C virus; rPD, radiological progressive disease.The following variables at the first rPD based on previous reports [3,4,13,14,15,16,17,18,19,20,21,22] and clinical experiences were selected for analysis as follows: age, gender, ECOG PS, Child–Pugh class, Barcelona Clinic Liver Cancer stage, best antitumor response until first rPD using RECIST Version 1.1, tumor shrinkage, contrast enhancement disappeared lesion, adverse events caused by sorafenib, course of treatment and AFP level. In univariate analyses, ECOG PS ≥2 points (p < 0.001), Child-Pugh B (p < 0.001), Child-Pugh C (p < 0.001), PD as the best antitumor response (p = 0.004) and AFP level >1000 ng/mL (p = 0.002) were statistically-significant factors for PPS. These factors were selected as independent factors affecting PPS in multivariate analysis (Table 2).Cox proportional hazard model analysis of post-progression survival (PPS).Abbreviations: AEs, adverse events; AFP, α-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; CI, confidence interval; ECOG PS, Eastern Cooperative Oncology Group performance status; post-progression survival; HR, hazard ratio; PD, progression disease; PPS, post-progression survival.To determine an indicator for judging cPD at the first rPD, the scoring system for PPS was designed using the factors described above (Table 3). The median score was significantly lower for Group A + B than Group C (one vs. three points, respectively; p < 0.001). The AUROC was 0.856 in time-dependent ROC for PPS according to Group C. The cutoff value for the scoring system of two points yielded a theoretical sensitivity of 71.5% and a theoretical specificity of 87.5%. When scores were limited to ≥2 points, no statistically-significant difference in PPS was observed between Groups A + B and C (median 4.1 vs. 1.9 months, respectively; p = 0.220). In contrast, the PPS of Group A + B was significantly longer than that of Group C in patients with scores of zero or one (median 14.0 vs. 3.3 months, respectively; p < 0.001) (Figure 1), although there were only three patients in Group C with scores of zero or one.Scoring system for PPS.† According to the RECIST Version 1.1 until the first rPD. Abbreviations: AFP, α-fetoprotein; CR, complete response; ECOG PS, Eastern Cooperative Oncology Group performance status; PPS, post-progression survival; PR, partial response; RECIST, Response Evaluation Criteria in Solid Tumors; rPD, radiological progressive disease; SD, stable disease.Kaplan–Meier analysis of post-progression survival (PPS). In patients with scores ≥2 points, no statistically-significant difference in PPS was observed between the A + B and C groups (median 4.1 vs. 1.9 months, respectively; p = 0.220). In contrast, the PPS of Group A + B was significantly longer than that of Group C in patients with scores <2 points (median 14.0 vs. 3.3 months, respectively; p < 0.001).When patient characteristics were compared between Groups A and B, there were no statistically-significant differences with respect to the variables at baseline or first rPD as described above in comparisons between the A + B and C groups. With reference to treatment duration of sorafenib after first rPD in Group A, median time to sorafenib withdrawal was 2.7 months. Post-rPD therapies of Group B were as follows: transarterial chemoembolization (n = 14), systemic chemotherapy with fluoropyrimidines (n = 7), hepatic arterial infusion chemotherapy (n = 4), other targeted therapy (n = 3) and radiofrequency ablation (n = 1).The median PPS was 8.0 months for Group A and 10.0 months for Group B (p = 0.310). Sixteen and 18 patients in Group A and B, respectively, scored between zero and one. Their prognoses were better, and median PPS was comparable between the two groups (both median 11.6 months; p = 0.537). In contrast, patients with scores ≥2 points, PPS was poorer compared with those with scores of zero or one (median 3.9 vs. 11.6 months, respectively; p < 0.001).The present study identifies prognostic factors for PPS in patients treated with sorafenib, and we propose a numerical indicator for cPD. Prognoses for patients with scores ≥2 were very poor; PPS did not vary significantly between Groups A + B and C. Therefore, these patients could be judged as cPD, and BSC would be recommended for treating these patients after the first rPD.Although tumor response has been used in clinical research as a surrogate indicator of survival, the survival benefit of sorafenib for advanced HCC does not correlate with decreased tumor size [3,4]. Therefore, distinguishing disease progression using conventional response criteria can be difficult, and vigorous research has been conducted to identify other surrogate indicators or biomarkers to assess the effects of sorafenib on patients with HCC. For example, AFP levels and AFP response correlate with survival outcomes [3,16,17]. Similarly, ECOG PS and Child-Pugh class correlated with the prognosis of patients with advanced HCC who were treated with sorafenib [3,19,20]. The modified RECIST (mRECIST) is widely used to assess the effect of treatment on patients with HCC [22]. The data of the present study were also evaluated with mRECIST, and the same results were obtained.PPS was comparable between patients in Groups A and B who continued sorafenib beyond rPD, as well as for patients treated using other therapies after rPD. For patients with metastatic colorectal cancer treated with first-line therapy that included bevacizumab, continuing bevacizumab beyond progressive disease increased survival, showing the efficacy of continuous inhibition of angiogenesis for cancer therapy [23,24,25]. Moreover, tumor rebound occurs after discontinuing anti-angiogenic therapy [26,27,28]. Second-line therapy after sorafenib failure is not well established for patients with advanced HCC, although several randomized trials are in progress to define salvage therapy [29,30,31]. A randomized phase 2 study showed that increasing the dose of sorafenib beyond progressive disease resulted in a trend, although not statistically significant, toward improved progression-free and overall survival, compared with BSC administered to patients with progressive disease previously treated with sorafenib [32]. Our present results demonstrate that continuation of sorafenib beyond rPD may provide a therapeutic option for patients with scores of zero or one until promising second-line regimens become available. However, we were unable to identify patients more likely to respond to continued sorafenib treatment beyond rPD. The number of patients in our study was limited. Moreover, it is possible that the lower score in Group A + B might be reflected by a selection bias because post-rPD management relied on physician’s decision in this retrospective study. To overcome these limitations, further studies are required to define the role of post-rPD therapy. We are conducting a prospective study to validate the efficacy of sorafenib therapy beyond rPD and the reliability of the score.Child-Pugh class, ECOG PS, the best antitumor response during treatment and AFP levels were identified as independent factors affecting PPS. We propose a useful scoring system as an adjunct to RECIST for predicting rPD in patients with advanced HCC. Because prognoses for patients with scores ≥2 at first rPD were very poor, BSC may be recommended in these patients with cPD until promising second-line therapy becomes available.T.O. designed the study, analyzed and interpreted the data and wrote the manuscript. T.O. and S.N. collected the data. Y.E. and T.M. supervised the study and edited the manuscript. All authors treated patients and gave final approval of the article.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by the absence of paternally expressed, imprinted genes on chromosome 15q11-13. Individuals with PWS characteristically have poor feeding and lack of appetite in infancy, followed by the development of weight gain and then uncontrolled appetite and lack of satiety, sometime after the age of two. The overwhelming drive to eat is coupled with reduced energy expenditure and decreased caloric requirements, thus, individuals with PWS will become severely obese unless their food intake is strictly controlled. The mechanisms underlying hyperphagia in PWS remain incompletely understood, and to date no drugs have proven effective in controlling appetite. However, clinical trials have started for several medications, which may provide therapeutic options for those with PWS. These medication trials may also provide insight into potential treatments for obesity in the general population. Ideally, these treatments will help alleviate the complex metabolic issues that are part of this syndrome.Prader-Willi syndrome (PWS) is a complex multisystem syndrome that occurs with a frequency of approximately 1/15,000 to 1/30,000 and has significant morbidity, including central hypotonia, developmental disabilities, behavioral issues, growth hormone deficiency and hypogonadism, and obesity compounded by a centrally driven excessive appetite (hyperphagia). It is the most commonly known genetic cause of obesity. PWS results from the loss of expression of paternally derived genes by a variety of mechanisms, which include large deletions (70%–75%), maternal uniparental disomy (UPD, 20%–30%), and imprinting defects (2%–5%) [1,2,3]. The body composition of individuals with PWS is significantly different from that of individuals in the general population. Patients with PWS have decreased lean body mass and increased adiposity that is apparent throughout life.PWS is a spectrum disorder, which affects multiple body systems. The underlying pathophysiological basis of the PWS phenotype remains unclear. Current therapies for PWS show limited efficacy and are directed to various symptoms associated with the disease process. Growth hormone (GH) deficiency is present in almost all children, and many adults, with PWS. Growth hormone replacement therapy is standard of care for children with PWS and increasingly prescribed in infants and adults. GH replacement therapy is effective in increasing height, improving body composition, increasing bone mineral density, improving exercise endurance and quality of life, and favorably impacting development and behavior [4]. However, it does not affect hyperphagia. Intellectual disability and neuropsychiatric issues are present to some degree in all individuals with PWS [5,6]. Intellectual quotient (IQ) is generally in the mild range, with some individuals demonstrating low normal IQ and others exhibiting moderate intellectual disability. Behavioral difficulties include cognitive rigidity, emotional liability, and obsessive-compulsive type symptoms, with autistic symptomatology also frequently reported [7]. Additional clinical complications frequently found in PWS include excessive daytime sleepiness, scoliosis, osteopenia/osteoporosis, decreased gastrointestinal motility, sleep disturbances, and reduced pain sensitivity [1].Individuals with PWS experience a characteristic change in food intake over time. Infants with PWS typically exhibit hypotonia, lethargy, a weak or discoordinated suck and swallow mechanism, and failure to thrive [8]. Infants with PWS do not exhibit signs of hunger and typically require force-feeding via gastrostomy tube, nasogastric tube, or cross-cut nipples on the bottle. Hypotonia in infants with PWS slowly resolves over time, although adults with PWS continue to exhibit some muscle hypotonia and weakness throughout their lifetime. Nutritional support is critical to optimize development in infancy, but feeding typically improves in young children. Excessive weight gain begins typically before the appetite increases, and then hyperphagia commences sometime during childhood. As adolescents and adults, all individuals with PWS will become morbidly obese if strict environmental controls are not implemented.The underlying molecular mechanisms that contribute to excessive weight gain and hyperphagia in PWS remain incompletely understood. Most gut-related appetite regulating hormones are normally expressed in PWS, with the most notable exception being ghrelin, a growth hormone secretagogue that is high in fasting states and decreases with eating. Highly elevated levels of ghrelin have been identified in individuals with PWS. Ghrelin levels decrease after intake of a meal, as is normal, but still remain comparatively high. Thus, the relationship between hyperphagia and ghrelin remains unclear, and it has been proposed that the hyperghrelinemia may be the result of hypoinsulinemia [9]. Leptin levels are appropriately elevated for degree of obesity, indicating that leptin deficiency is not a cause of the appetite issues.Appetite and satiety issues are regulated by a complex interplay between gut hormones and hypothalamic neuropeptides. With regards to the neuropeptides that regulate hunger/satiety circuits, Swaab and colleagues [10] reported a deficit in the oxytocin (OT)-producing neurons of the paraventricular nucleus (PVN) in the brain of these patients and Bittel and colleagues [11] reported decreased oxytocin receptor gene function in PWS. OT is a well-known satiety hormone that is also involved in establishing and maintaining social standards. It is thought that perhaps the low levels of OT in the brain of individuals with PWS may contribute to the hyperphagia. Other studies have suggested that the hyperphagia may be caused by abnormalities in another hypothalamic hormone, hypocretin/orexin. Orexin regulates the sleep/wake cycle, metabolism, and appetite, and so is a viable candidate hormone to explain several of the clinical findings in PWS. It is known that the sleep/wake cycle is abnormal in individuals with PWS, many of whom suffer from excessive daytime sleepiness and narcolepsy, a disorder caused by abnormally low hypocretin/orexin. Within the PWS region on Chromosome 15, the SNORD116 host gene transcript has been reported to regulate expression of diurnal and circadian genes in the brain, suggesting a potential role in energy expenditure and hyperphagia in PWS [12]. However, despite considerable investigation, at this time there are no consistently identified hormone abnormalities to fully explain the hyperphagia in PWS, and the metabolic correlates of hyperphagia are still unclear.To date, no medication has proven effective in regulating appetite in PWS. Bariatric surgery is not accompanied by a decrease in appetite that occurs in typical individuals and is contraindicated in this population [13]. Other medications evaluated to date in PWS have proven ineffective [14,15] or have been associated with unacceptable side effects [16].The inability to control food intake is often the biggest obstacle keeping those with PWS from living independently, participating in normal social events, and working in unrestricted settings. The lack of satiety associated with PWS has chronic consequences (morbid obesity) as well as potentially acute consequences. Without supervision, individuals with PWS will die prematurely as a result choking (especially during episodes of voracious eating), stomach rupture or tissue necrosis, or from complications caused by morbid obesity [17,18]. In addition to limiting living arrangements, opportunities for community engagement and social activities are highly constrained by food access issues. Similarly, given need for 24/7 supervision regarding food, hyperphagia strictly limits employment opportunities, even for those individuals with higher intellectual function. Finally, extreme behaviors such as anxiety, aggression, perseveration, are common in PWS, and frequently revolve around food issues. Thus, this is a population to whom an effective therapy for appetite and obesity has the potential for a profound positive effect on lifestyle opportunities and quality of life.Families and individuals with PWS are fortunate to have significant community support in the form of parent-run foundations. These groups—the Prader-Willi Syndrome Association of the United States (PWSA-USA), the Foundation for Prader-Willi Research (FPWR), and the International Prader-Willi Syndrome Organisation (IPWSO)—have lobbied for years to try to get pharmaceutical companies interested in PWS as a model for obesity, with the idea that if the appetite in PWS can be treated, the appetite of anyone with obesity can be treated. The motto that “PWS is a window into the house of obesity” has finally begun to resonate with pharmaceutical companies, and currently there are several medication trials which are ongoing, with more yet to come.Current medication trials in PWS are summarized in Table 1. The first company to express an interest in research for treatment of hyperphagia in PWS was Zafgen, which manufactures a medication called beloranib. Beloranib is a methionine aminopeptidase 2 (MetAP2) inhibitor that decreases fat biosynthesis and enhances fat oxidation and lipolysis [19]. In studies of animal models with diet-induced obesity or hypothalamic obesity, beloranib resulted in substantial and persistent weight loss, decreased appetite, and improvements in metabolic and inflammatory biomarkers [20]. Additionally, in studies of adults with exogenous obesity, beloranib caused substantial weight loss and a significant reduction in hunger [21]. Therefore, the benefits of this medication seem uniquely suited to the needs of individuals with PWS. The phase 2 trial of beloranib was completed in 2013 and demonstrated a good safety and efficacy profile in this population. Phase 3 trials are currently ongoing.Rhythm Pharmaceuticals has developed a medication called RM-493 which is a melanocortin 4 receptor (MC4R) agonist for trial in individuals with PWS. The basis for this medication is that the pro-orexigenic pathways in the hypothalamus, which are apparently normally expressed in the brains of individuals with PWS, can potentially be overcome by activation of the MC4R, thus controlling appetite and improving metabolism. RM-493, cardiovascularly safe and efficacious MC4R agonist, is a potent activator of the MC4R, thus making it a target for potential treatment of both the hyperphagia and low metabolism in individuals with PWS. Phase 2a trials are currently in progress to evaluate the safety and efficacy of this medication in PWS.Oxytocin (OT) has been of interest as a potential treatment for PWS since the abnormalities in OT neurons were identified in 1995. Oxytocin is involved in establishing and maintaining social standards. Indeed, it has recently been shown in a double blind placebo study, that OT administration to adults with PWS significantly decreased depressive mood tendencies and tantrums while increasing trust in others, with data supporting a trend to decrease appetite with higher satiety [22]. Moreover, in a PWS knock-out mouse model for the Magel2 gene, a single OT injection at 5 hours of life prevented early death observed in 50% of new-born mice by recovering a normal suck [23]. However, a study of adolescents and adults with PWS treated with OT twice daily failed to show any improvements in appetite, and there was even some worsening of behaviors [24]. This medication needs further investigation to determine if it is helpful in ameliorating the excessive appetite in PWS and if there is potentially an overdose effect that could worsen behaviors. Ferring Pharmaceuticals has developed an intranasal oxytocin analogue (Intranasal FE 992097), which exerts its effects on the OT receptors in the brain. Phase 2a studies have been completed on this medication in individuals with PWS.Current Medication Trials in Prader-Willi syndrome (PWS).Diazoxide (Essentialis) is a medication which has been used for many years for individuals with hyperinsulinism, so has a well-characterized safety profile. Diazoxide is a potent K+-ATP channel agonist which is hypothesized to potentially address many of the abnormalities observed in patients with Prader Willi Syndrome by hyperpolarizing hypothalamic neurons whose activity is otherwise impaired by a defective leptin signaling pathway. Phase 2 studies are almost completed in children with PWS.Alize has developed an unacylated ghrelin analog (AZP-531) which has been shown in phase 1 studies to improve blood glucose levels and weight with once daily administration in individuals with obesity and type 2 diabetes. This company is preparing for phase 2 trials targeting treatment for individuals with PWS and those with type 2 diabetes.Exanatide and Liraglutide are glucagon-like peptide-1 (GLP-1) receptor agonists which were originally developed for treatment of Type II Diabetes. This class of drugs has been shown to suppress appetite and to induce weight loss in both diabetic and non-diabetic individuals. A small 2011 placebo controlled crossover study reported that a single injection of exanatide improved satiety and lowered circulating levels of both glucose, insulin, PYY, and GLP-1 in both individuals with PWS and those with exogenous obesity [25]. Ghrelin levels and energy expenditure were not affected and there were minimal side effects in the PWS group. Two case reports of liraglutide for treatment of diabetes in individuals with PWS suggested improvement in hyperphagia during the treatment period [26,27]. These results warrant larger, longer-term studies. A 6-month study of exanatide was just completed in individuals with PWS. A significant concern with respect to the long term use of GLP-1 receptor agonists in the PWS population is that these medications delay stomach emptying. Delayed stomach emptying, decreased intestinal motility and gastroparesis are common findings in PWS, and pilot studies (unpublished) indicate that GLP-1 receptor agonists further exacerbate abnormally slow gut movement in PWS.There are also some other FDA-approved medications which may potentially improve satiety or help ameliorate obesity in individuals with PWS, including lorcaserin, naltrexone HCl/bupropion HCl (Contrave®), and phenteramine/topiramate (qsymia®). There are also some devices in testing which may also improve appetite and behavior in individuals with PWS, including transcranial direct magnetic stimulation and vagal nerve stimulators.This is a tremendous time of hope for potential treatment of the appetite issues in PWS. Given the fact that the great majority of patients are now diagnosed in infancy, the combination of early intervention and early growth hormone therapy, along with a medication to decrease the appetite issues could result in a much improved quality of life, and increased independence, for individuals with Prader-Willi syndrome.The authors would like to thank Daniel Driscoll for his support and mentorship (JLM).Jennifer Miller: Primary author ��� wrote the manuscript. Theresa Strong: Editing and assistance with manuscript preparation. Janalee Heinemann: Editing and assistance with manuscript preparation.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Celiac Disease (CD) affects at least 1% of the population and evidence suggests that prevalence is increasing. The diagnosis of CD depends on providers being alert to both typical and atypical presentations and those situations in which patients are at high risk for the disease. Because of variable presentation, physicians need to have a low threshold for celiac testing. Robust knowledge of the pathogenesis of this autoimmune disease has served as a catalyst for the development of novel diagnostic tools. Highly sensitive and specific serological assays including Endomysial Antibody (EMA), tissue transglutaminase (tTG), and Deamidated Gliadin Peptide (DGP) have greatly simplified testing for CD and serve as the foundation for celiac diagnosis. In addition, genetic testing for HLA DQ2 and DQ8 has become more widely available and there has been refinement of the gluten challenge for use in diagnostic algorithms. While diagnosis is usually straightforward, in special conditions including IgA deficiency, very young children, discrepant histology and serology, and adoption of a gluten free diet prior to testing, CD can be difficult to diagnose. In this review, we provide an overview of the history and current state of celiac disease diagnosis and provide guidance for evaluation of CD in difficult diagnostic circumstances.Celiac disease (CD) is a chronic immune-mediated enteropathy triggered by exposure to gluten in genetically predisposed individual [1] and is a common autoimmune disorder, affecting ~1% of the population in many regions of the world [2,3]. CD is genetically based and prevalence is enriched in patients with family history of CD or a personal history of autoimmune disease, including thyroid, liver, and type 1 diabetes mellitus [4]. Symptoms of undiagnosed CD can range from subclinical to severe malabsorption, known as celiac crisis [5].The most important reason for the relatively low rate of CD diagnosis [3] is failure to consider testing. CD is often not considered due to its wide range of clinical presentation. CD diagnosis rate is increasing due to both increased true prevalence [6] and improved awareness of its variable clinical presentation [7]. Through the 1950s, diagnosis was based on malabsorptive features and clinical observation. Development of intestinal biopsy, initially by Crosby capsule and later by endoscopy, subsequently became the gold standard for confirmation of CD diagnosis, a position it has kept to this day [8]. Serologic tests are generally recommended as the first step when there is suspicion of CD in order to identify patients who should undergo intestinal biopsy. Recommendations are now available to help clinicians decide which patients should be tested for CD (Table 1) [9] and how to proceed with evaluation of potential CD. (Figure 1) [7].Celiac disease (CD) diagnostic algorithm. DGP: deamidated gliadin peptide; HLA: human leukocyte antigen; Ig: immunoglobulin; TTGA: tissue transglutaminase antibody [7]. Reproduced with permission from Kelly C.P. [7], (ACG clinical guidelines: Diagnosis and management of celiac disease-3).Who should be tested for CD [9].Reproduced with permission from Leffler D.A. Celiac disease diagnosis and management: A 46-year-old woman with anemia. JAMA 2011, 306, 1582–1592 [9].Four guidelines on CD diagnosis have been published by gastrointestinal organizations since 2012. All guidelines include the combined use of biopsy and serologic analyses for diagnosis. According to American College of Gastroenterology (ACG) 2013 CD guidelines combination of both small intestinal biopsy and serologic tests (anti-tissue transglutaminase (tTG) or anti-deamidated gliadin peptide (DGP)) are recommended for diagnosis of CD. This guideline also recommends that children age <2 years should have IgG DGP test due to the lower sensitivity of tTG in this population, discussed in detail below [7]. A consensus guideline in 2012 by the European Society of Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) proposed a non-invasive method of diagnosing CD in select pediatric patients. The ESPGHAN algorithm suggested that, in pediatric patients who have symptoms consistent with CD can be diagnosed without biopsy confirmation if they have an IgA tTG titer >10-fold above the upper limit of normal, a positive endomysial antibody (EMA) in a separate blood sample, and carry the HLA DQ2 or DQ8 haplotype [10]. The British Society of Gastroenterology recommendations for adult CD diagnosis suggest that serologic tests, either tTG, EMA, or DGP should be done as the first step in diagnosis, followed by small intestinal biopsy is a definitive test to diagnose CD. This guidelines suggests that duodenal biopsy cannot currently be replaced by lone serologic testing [11]. Recent guidelines for adult CD patients from the World Gastroenterological Association recommend serologic tests including anti-tTG and/or anti-EMA, or anti-DGP for diagnosis and biopsy suggested but not considered mandatory for CD diagnosis which is appropriate for countries with limited healthcare resources [12]. The guidelines overlap substantially with the major difference between all four guidelines being that ACG and BSG mandates intestinal biopsy to confirm the diagnosis of CD, while ESPGHAN and WGO allow diagnosis of CD without biopsy in certain conditions.A new era in CD diagnosis began in 1980s with the identification of IgG and IgA anti-gliadin antibodies (AGAs) circulating in plasma of untreated patients [13]. This was a major step forward as prior to this development, CD could be diagnosed only on the basis of clinical suspicion and intestinal biopsy. Binding of Immunoglobulin A (IgA) and IgG antibodies to native gliadin (AGAs), were found to be associated with CD and were the first serologic tests for CD used to inform the need for duodenal biopsy. However, as antibodies to foreign proteins, at least at low titers, are common in healthy individuals, the accuracy of AGA tests are limited, with sensitivity and specificity generally less than 90% and positive predictive values less than 50% in many populations [14]. AGA testing largely fell out of favor in the 1990s as more accurate testing became widely available [15].In early 1990s, immunofluroscent assays of IgA binding in active CD serum against monkey esophagus endomysium (EMA) was found to be a highly specific and sensitive marker of CD [14,16,17]. Although EMA has a specificity and sensitivity of >95% [18], EMA has thus been suggested to be less sensitive, especially among celiac children under two years of age [19], as well as in elderly patients [18]. Moreover, it has also been reported that false negative EMA results may be associated with milder small-bowel mucosal lesions [18,20]. Despite the high accuracy of EMA, it has the disadvantages that testing is expensive, subjective, and labor-intensive, requiring experienced personnel to perform [2]. While in a reference lab, EMA remains the most sensitive CD test, the technical disadvantages which result in significant inter-observer and inter-site variability have led to EMA being largely replaced by newer enzyme-linked immunosorbent assay (ELISA) based assays [16,17].In 1997, research studies identified the ubiquitous enzyme tTG as the auto antigen, which reacts with EMA, leading to the development of ELISAs that detect antibodies against tTG [16]. tTG assays quickly demonstrated high sensitivity and specificity with lower cost and greater reproducibility than immunofloroscence assays. Although performance characteristics of assays vary, overall tTG testing is reliable and inexpensive and for these reasons, has become the most common test for celiac diagnosis and monitoring [16,21,22].Most recently, testing for antibodies against DGP has become clinically available. AGAs are produced in response to gliadin, a prolamin found in wheat. DGP is based on the conversion of gluten peptides to deamidated peptides by the action of intestinal tTG [23,24]. These peptides bind with high affinity to human leukocyte antigen DQ2 or DQ8 on celiac patients’ antigen-presenting cells which stimulate the inflammatory T-cell response observed in the intestinal mucosa of patients with CD [14]. This results in an antibody response to DGP that displays a higher specificity and specificity for CD than antibodies to native gluten (AGAs) [23], and are especially useful in children younger than two years of age [20]. In patients over two years of age IgA-tTG antibody is the preferred serological diagnostic test for diagnosing CD [25]. On the other hand, IgG anti-tTG testing has disappointing sensitivity [24], and IgG anti-DGP and the composite IgA/IgG anti-DGP reach sensitivities above 80% and, importantly, specificities above 95% [14,16,21]. For this reason, IgG-DGP tests are the most accurate available assays for patients with selective IgA deficiency. According to the study high concentration of DGP antibody found to be correlated with severity of intestinal damage in infants and may also help assess dietary adherence [26,27].Most recently point of care (POC) finger-stick [28,29] blood tests have been introduced for rapid detection of CD. POC tests based on transglutaminase 2 (TG2) auto-antibodies are now available and may be of particular use when laboratory based testing is not readily available [13,29,30]. The newest generation of POC test is based on a combination of DGP and tTG together and has a reported sensitivity of 93.1% and specificity of 95% [31,32]. A combination of immunoassays is particularly useful as an addition to detection of patients with CD who are IgA deficient, however IgG-DGP was able to detect a small number of IgA-sufficient patients who are seronegative for IgA-tTG [20].Serologic tests in CD should be done as first step in patients with symptoms suggestive of CD and those with CD-associated diseases. However, when clinical suspicion for CD is high, small intestinal biopsies should be considered even in patients with negative serology results.Intestinal fatty acid binding protein (I-FABP) is a cytosolic protein, which is released by necrotic enterocytes, first described as a marker of intestinal ischemia. I-FABP has been studied as possible marker to evaluate mucosal damage and it is proposed as a sensitive diagnostic test in the evaluation of ischemia in mechanical small bowel obstruction [33,34]. The expression of I-FABP is primarily limited to epithelial cells of the ileum of small intestine. I-FABP is abundantly present in enterocytes and has been reported to be a sensitive marker for damage to the intestinal epithelium [33]. In children with a positive serological test for CD I-FABP has demonstrated positive predictive value of 98% [35]. If validated and clinically available, serum I-FABP might be useful in clinical practice for identifying noncompliance and unintentionally gluten intake, evaluating new therapeutic options, as a non-invasive marker for detection of ongoing mucosal architectural abnormalities without the need of endoscopy and as an aid for celiac diagnosis during gluten challenge [33,34].Nearly all individuals with CD carry the type II class Human Leukocyte Antigen (HLA) DQ2 and/or DQ8 haplotypes. These molecules are requisite for the high affinity binding of deamidated gluten peptides necessary to generate an immune response [36,37]. Less than 1% of the CD population carries half of the HLA-DQ heterodimer [36]. However, HLA DQ2 and DQ8 are highly prevalent, and can be found in 20%–40% of the general population [37,38]. For this reason, it is estimated that only 3% of individuals with these haplotypes will go on to develop CD giving this test a very low positive predictive value [39,40]. At the same time, HLA typing has a negative predictive value of >99% and is useful for ruling out CD in patients on a gluten free diet (GFD), for patients with an uncertain diagnosis of CD as recommended by ESPGHAN [10], and for risk stratifying CD in at risk family members. Although only one third of family members will be spared repeated testing, particular combinations (e.g., homozygocity for DQ2) increase risk for CD to up to 40% [38,41,42]. Overall however, HLA testing has a positive predictive value generally under 50% depending on the population tested [36].The diagnosis of CD is based on patient’s symptoms, CD-specific antibody levels, the presence of HLA-DQ2 and/or HLA-DQ8, and characteristic histological changes (villous atrophy and crypt hyperplasia) in the duodenal biopsy. Despite the existence of highly sensitive serological assays, small-bowel mucosal biopsy is still considered the definitive method for diagnosis of CD [43].Classical CD histologic findings include crypt hyperplasia, blunted or atrophic villi, and increased number of intra-epithelial lymphocytes (IELs), especially at the villous tips (Table 2 Marsh Classification). In addition to these findings, additional histopathological changes are common in CD including neutrophilic and eosinophilic infiltrates, subepithelial collagen thickening and associated lymphocytic gastritis [43,44,45].Comparison of Marsh modified (Oberhuber) Histological classification and Villanacci classification of Celiac Disease.While histologic changes may be obvious, the location, number, and size and orientation of biopsies can affect diagnostic yield. As 70% of cases have patchy mucosal damage [46], it is important to maximize diagnostic accuracy by collecting ≥5 duodenal biopsies with two samples from the duodenal bulb. According to recent studies as many as 13% of patients demonstrated characteristic enteropathy only located to duodenal bulb [46,47]. Endoscopic staining using dye like indigo carmine or methylene blue and water immersion as been suggested to allow visualization of villi and identification of patchy areas however the benefit of this beyond the standard five or more biopsies is unclear  [48,49]. Biopsies from the duodenal bulb should be carefully interpreted, because peptic injury can damage villi and be mistaken for CD in some cases [44]. While CD does account for at least 90% of enteropathy in western countries [6], histological features of CD are not specific and are also associated with disorders like giardia infection, common variable immune deficiency, Crohn’s disease, and Helicobacter pylori infection [50]. This issue is discussed further below in the section on diagnostic dilemmas.A simplified histologic classification is also proposed by Villanacci et al. [51], based on villous morphology and IEL count. Type A represents the non-atrophic type which is defined as villous crypt ration 3:1, >25 IELs × 100 epithelial cells. Type B is the Atrophic type which is defined as villous crypt ration <3:1, >25 IELs × 100 epithelial cells.Scalloping folds, mosaic pattern and decrease of duodenal folds are typical endoscopic markers of villous atrophy however should not be relied upon in practice. Studies suggest that the overall specificity and sensitivity of gross endoscopic findings ranges from 83% to 100%, and from 6% to 94%, respectively [50,51] and normal appearance of the duodenum should not preclude biopsy [51,52]. On the other hand, the scalloped feature (Figure 2) of duodenal folds has a positive predictive value of 69% for CD and 96% for enteropathy and should always prompt biopsy when endoscopy is performed for other indications [53].Classical scalloping of duodenal mucosa seen in Celiac disease at endoscopy.Video Capsule Endoscopy (VCE) has also been evaluated for use in CD diagnosis. VCE is a good alternative in patients who refuse upper endoscopy, for cases with negative duodenal biopsy and positive serology to assess for distal enteropathy, and to evaluate patients with non-responsive disease, to investigate for complications, such as ulcerative jejunitis or neoplasia. The lack of ability to biopsy is the main limitation of VCE [54].In general, CD diagnosis can be readily established with currently available tests and diagnostic procedures. However, in some patients, the diagnosis is not straightforward and presents a challenge to clinicians. Potential dilemmas include those with positive serology but normal histology, negative serology but abnormal duodenal mucosal histology, and adoption of the GFD prior to testing. In the following section we discuss common dilemmas in diagnosis of CD.Positive IgA tTG serology may be seen in in patients with normal small intestinal histology. False positive tTG results are rare but do occur and are usually low titer (less than twice the upper limit of normal). Hypergammaglobuliniemia, chronic liver disease, congestive heart failure and enteric infections have been associated with false positive results [14]. This was a more frequent issue with early generations of tTG assays, which were more liable to cross-react with other antigens.We suggest that the initial step in evaluation of an individual with elevated tTG titers and normal histology should be review of the biopsies by a gastrointestinal pathologist familiar with CD to look for subtle abnormalities and to confirm that the biopsies are of sufficient number and orientation. If the histology is convincingly normal, we confirm that the patient was on a full gluten containing diet at the time of endoscopy. If the patient was on a low gluten diet, we recommend gluten challenge, discussed below. If these steps do not reconcile results HLA typing should be considered, though, in our experience, patients with high titer IgA-tTG are nearly always positive for HLA DQ2 or DQ8. We also check DGP and/or EMA antibodies in these cases. If more than one celiac serologic test is positive, it strengthens the argument that the patient has a true, if mild, form of CD. After evaluation many of these patients will be found to have potential CD. Potential CD is defined as positive serologic titers with a normal biopsy [1]. It is controversial whether these patients benefit from the gluten free diet. Limited data suggest that patients with mild villous atrophy appear to benefit from a gluten-free diet, but many of these patients are minimally symptomatic [54,55]. We typically recommend a six-month trial of a GFD in this scenario and if there is any clinical benefit, or if there are other clinical signs of CD, such as osteopenia or nutritional deficiencies, the GFD should be continued. If there is no clinical benefit from the GFD, patients may be reasonably followed on a regular diet and reevaluated if tTG titer increases or symptoms change. It has been recently shown that CD markers disappear in many symptomless children at family risk of CD with potential CD left on an normal diet [56]. Spontaneous normalization of tTG has been also demonstrated in children with type 1 diabetes mellitus [57]. Therefore, in symptomless children with positive celiac-type serology, the decision to perform the biopsy may be preceded by repeat serological testing at three to six months when the antibody titer is not very high.Although increased intraepithelial lymphocytes (IELs), defined as >25 IELs/100 enterocytes, is recognized as potentially consistent with CD, by itself, this finding lacks specificity. This histological finding can be found in CD, but more commonly with other disorders and medications that cause small intestinal inflammation. Reported etiologies of lymphocytic infiltration of the intestinal epithelium in the absence of villous atrophy include non-steroidal anti-inflammatories, proton pump inhibitors, small intestinal bacterial over growth, helicobacter pylori infection, inflammatory bowel disease, and eosinophilic gastroenteritis [58]. About 2.5% of proximal small intestinal mucosal biopsies [59] display increased IELs in the absence of villous architectural change [60]. Determining the etiology of increased intraepithelial lymphocytosis can be challenging and relies on assessment of clinical, serological, and histopathological data [56,57,61].In general, patients with this finding should have celiac serology tested but if these are negative, CD can be confidently ruled out in most cases.While modern celiac serologies are highly sensitive, a small percentage of patients with CD will be seronegative at diagnosis [14] and others may show an extremely slow resolution of histological findings, despite a gluten free diet [62], making the diagnosis uncertain. Most patients with villous atrophy on duodenal biopsy will have a serologic test consistent with CD. Differentiation of seronegative CD from alternate causes of enteropathy is a clinical challenge and requires integration of clinical, genetic, and histopathologic criteria [63]. The major possible etiologies in patients with villous atrophy but negative celiac serologies, include patients on a GFD at the time of testing, IgA deficiency, seronegative CD and non-celiac enteropathy(NCE). Celiac-like histological features can be seen with multiple other gastrointestinal disorders including peptic duodenitis [64], Common Variable Immune deficiency (CVID) with malabsorption or anemia [65], and food allergies or eosinophilic gastroenteritis [43,49,62]. Other etiologies include post-viral enteropathy, autoimmune enteropathy, tropical sprue, NSAID enteropathy, and immune mediated enteropathy, all of which may present with negative celiac serology and histology consistent with CD [50].The histopathological changes of small intestinal Crohn’s disease are heterogeneous and involve varying degrees of acute and chronic inflammation similar to CD [66]. Inflammatory bowel disease has been reported as the second most common cause of villous atrophy after CD [67]. In patients where the diagnosis is uncertain or findings are atypical, intestinal biopsies should be reviewed by a skilled gastrointestinal pathologist. If the initial biopsies are unavailable or prove to be non diagnostic after re-evaluation, repeat endoscopy with biopsy should be performed.In patients with villous atrophy and negative celiac serology (tTG-IgA) our typical next step is to review the pathology, obtain HLA DQ2/DQ8 and DGP testing and consider causes of NCE [36,50]. If this is non-diagnostic for CD we will check serum immunoglobulins. If serum immunoglobulin reveals low IgG and IgA in setting of normal albumin this suggests CVID [65]. As a part of serologic assessment, IgG-based DGP testing should be considered in all patients with Ig A deficiency because they have a 10–20 times greater risk of developing CD [25,64].One of the most common and vexing clinical issues in celiac diagnosis is the evaluation of patients who adopt the GFD prior to formal testing. In this scenario, the best approach is to start the diagnostic process with serologic tests (anti-tTG and/or DGP) and HLA typing. Seropositivity indicates that the patient is eating sufficient gluten to have active CD and duodenal biopsy is the next step in these cases. However if the serologic tests are negative, this should be followed by genetic testing for HLA DQ2/DQ8, which, if negative, excludes current or future CD.Only once the patient is found to be seronegative and HLA DQ2 and/or DQ8 positive, should gluten challenge for diagnosis be undertaken. (Figure 3) [36,42]. In the past, gluten challenge guidelines suggested intake of at least 10 g of gluten per day for a period of eight weeks or longer. Recent studies have helped to refine the timing, doses, and duration of the gluten challenge [38,66,67]. According to these studies two weeks of gluten challenge can result in mucosal damage. Additionally, a lower level of gliadin (2–3 g/day, equivalent to one to two slices of bread) appears better tolerated and sufficient for diagnosis. (Figure 3) [68]. While this reduced dose and duration gluten challenge significantly reduces the burden of testing, it is still difficult for many patients to complete, and may carry some risk in children and patients with celiac-related neurological manifestations. Further, extended exposure of six months or more may be necessary in a minority of patients. For these reasons, work is being done on alternate methods of diagnosis including in vitro gluten challenge [69] and HLA tetramer testing [36] , however none are clinically available at this time.Modified Gluten Challenge Algorithm [68]; Reproduced with permission from Leffler D.A. and Kelly C.P. (Kinetics of the histological, serological and symptomatic responses to gluten challenge in adults with coeliac disease. Gut 2012).CD can present at any age where there is exposure to gluten, including infancy. In early childhood, classic gastrointestinal symptoms of CD are most common, but other presentations including asymptomatic growth delay are also recognized [10,68]. As the immune system is not fully mature, serologic tests are not as sensitive in young children as they are in older children and adults. For many years, AGA testing was considered to be the best test for children under the age of two years [27]. However, in this population DGP antibodies have been found to be more accurate than both AGA and tTG testing [7,42,70]. In young children suspected of having CD, it is reasonable to check both tTG and DGP. If titers are very high and the child otherwise meets ESPGHAN criteria described above, the diagnosis of CD can be confidently made. However, if there are any atypical manifestations, if serologic tests are normal but the child is considered at high risk, or if serologic titers are positive but not highly elevated, endoscopy should be considered.CD is a chronic systemic immune-mediated disorder associated with variable small-intestinal mucosal injury triggered by gluten ingestion in genetically predisposed individuals. It is a common disorder, affecting individuals worldwide, with recent studies suggesting an increase in prevalence. Small-bowel biopsy currently remains the gold standard for diagnosis of CD. The improved sensitivity and specificity of serologic testing and growing awareness among physicians should increase the diagnosis of CD. Due to the changing presentation of disease, as well as the recognition of a number of potential histopathologic mimics, communication between pathologists and gastroenterologists is essential for appropriate interpretation of small-bowel biopsy specimens. The clinical, histologic, and laboratory data need to be assessed to provide an explanation for atypical manifestations of CD and possible differential diagnoses. While CD remains a morbid disease, awareness of disease risk factors, current diagnostic testing recommendations and a framework for the evaluation of common diagnostic issues can result in earlier and more accurate diagnosis.Dharmesh Kaswala and Gopal Veeraraghavan: None.Ciaran Kelly: Consultant and scientific advisor related to Celiac disease for Alba, Alvine, Immunosan T and Pfizer.Daniel Leffler: Consultant for/research support from: Alba Therapeutics, Alvine Pharmaceuticals, INOVA diagnostics, Genzyme, Coronado Biosciences, Pfizer, GI Supply.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).In the present paper, we discuss the change in celiac disease (CD) awareness and perception through patients’ concerns and the most recent literature. Nowadays CD has moved in the public awareness (both doctors and population) from a rare disease to a common one and the gluten free diet (GFD) is no longer the exclusive therapy for CD patients but is becoming a popular health choice for everybody. Gluten-free food, once hard to find and requiring home preparation, is now available at restaurants and grocery stores. However, the quality of life of those affected by CD seems to be still compromised and this is particularly true for those who find it difficult to adhere to a GFD and those who were asymptomatic at the time of diagnosis. Intervention at diagnosis and follow-up to improve the patients’ adaptation to the condition and its limitations should be implemented.In the last decades, the celiac disease (CD) epidemiology [1,2] and awareness [3,4] have been changing. CD is now considered a common disease and the gluten free diet (GFD) is no longer the exclusive therapy for CD patients but is becoming a popular health choice for everybody [5,6].In the past, CD was usually diagnosed in the presence of the “classical triad”: chronic diarrhea, abdominal distention, and failure to thrive [7]. A celiac patient was then likely a child and the family felt lonely to face this “strange” disease with an odd dietetic therapy. In the latest decades, CD has been recognized as a worldwide problem, affecting people of all ages and characterized by a wide spectrum of clinical presentations [8,9]. We learnt that CD has many faces and screening programs with sensible and specific serological markers showed that CD is present in different conditions and also in apparently healthy people [8]. We also learned that CD is not only more easily recognized but also increasing in its prevalence [2,10]. All over the world we are facing what can be defined as a celiac epidemic.CD has moved in the public awareness (both doctors and population) from a rare to a common disease. Gluten-free food, once hard to find and often requiring home preparation, is now readily available at restaurant and grocery stores.Typing the words celiac disease in Google generates more than 6,800,000 results in few seconds. Millions of news articles and non-scientific papers provide large quantities of information, which influence healthcare policies more than scientific papers alone can do [11].The perception of CD has changed, it is no longer a “disease” but a condition, id est a factor affecting the way people live or work, especially with regard to their well-being. The GFD is not only the sole therapy for CD patients but also a choice for non-CD patients [6].In this paper, we examined the following factors involved in the perceived quality of life (QoL) of a CD patient:
+      
+        Awareness of the disease among caregivers;
+        Social burden;
+        Quality of life (QoL) and coping strategies.
+      
+	  Awareness of the disease among caregivers;Social burden;Quality of life (QoL) and coping strategies.We selected some of the comments made by the patients during outpatients visit as an example of the most challenging situations and used these as a guide in the literature search. We used the PUBMED database and the search terms “celiac disease” AND “quality of life” (240 hits) “burden” (68 hits), “awareness” (165 hits), with the final search performed in May 2015. Systematic reviews, case series, case-control studies, and randomized controlled clinical trials were analyzed and chosen as a support of this opinion paper.“I feel more reassured if my doctor knows well my disease”To our knowledge, there is no paper that directly deals with physician knowledge of CD. The following studies, however, suggest that active case-finding, presupposing a good knowledge of the disease presentation, is not a common practice among physicians, A study from 2005 showed the need for education among Californian GPs, as only 35% of them had ever diagnosed CD [12]. In Europe the picture is variable. While in France CD awareness is quite low among GPs and the diagnosis is much better recognized by gastroenterologists and pediatricians [13], in Finland there is an increasing awareness among GPs who are now in charge of the diagnosis. The passage from tertiary centers to GPs has been shown to be a good strategy to reduce the time lapse between onset of symptoms and diagnosis [14]. Case–finding strategies conducted by GPs in USA and Italy have showed that awareness campaigns among doctors improve the knowledge of CD and its diagnosis [15,16]. Catassi et al. [17] showed in a large screening study that the CD diagnoses were only the tip of an iceberg and the majority of CD cases were under the water level. Recently, Zingone et al. [18], after more than ten years from the Catassi’s study, have showed that CD incidence in Benevento, a rural area of Campania, is lower compared to other nearby provinces, assuming that residents there tend to visit their doctors only in case of severe clinical conditions making it more difficult to capture subclinical and non-classic forms of CD. Similarly, a study conducted using GP datasets in the UK found that children living in less socioeconomically deprived areas were about twice as likely to be diagnosed as those from more deprived areas [19]. This latter finding may be due to the fact that underprivileged people may be less likely to seek medical care or consultation in general and thus be potentially less likely to be tested for CD. However, another possibility is that underprivileged people have an inferior hygienic environment that may protect against celiac disease [20]. After diagnosis is made, however, the follow-up is made in most cases by a specialized Celiac Center in which the CD awareness is assured. Follow-up should include a dietitian consultation, as patients report better satisfaction when they are referred to dieticians [21,22] and psychological support when required [23]. The importance of a psychological support has been recently reported by Sainsbury et al. [24] who found that psychological symptoms and the GFD adherence were more strongly related to reduce QoL than gastrointestinal symptoms. Despite the large mass of information from media, the CD awareness among doctors can be improved to enable early diagnosis and to support the patients once the diagnosis is made.“When I go out with my friends I feel different from them… there is no restaurant in my little town that serves gluten free food”Patients with CD feel cautious when eating food not prepared at home. The unavailability of gluten-free food outside home and the low CD awareness among food workers have been a problem in the past years, although they are rapidly changing. A survey conducted in 2003 among chefs in the UK showed that they knew less about CD than the general public [25]. The authors conducted the same survey in 2013 [26], which showed a significant increase in chefs’ awareness of gluten-related disorders (both CD and non-celiac gluten sensitivity) and a similar level of recognition of the gluten-free symbols among the public and chefs (about 40%).A 2006 survey conducted on 2681 adult members of the Canadian Celiac Association showed that 44% of them found difficulties following the GFD which included: determining if foods were gluten free (85%), finding gluten free foods in stores (83%), avoiding restaurants (79%), and avoiding travel (38%) [27]. Another survey conducted in Canada seven years later among the same population showed that difficulties and negative emotions were experienced less frequently by those on the diet for >5 years, although food labeling and eating away from home remained very problematic [28].Similarly, a survey aimed at evaluating the adherence to GFD in children and adolescents showed that participants reported good adherence at home and school, but low adherence at social events. Children reported that the main significant barriers to a good adherence to the GFD were availability of gluten free foods, labeling of food and their cost [29]. Roma et al. reported the answers of 73 children questioned about the main causes of non-compliance and the most frequent reasons were: poor palatability (32%), dining outside home (17%), poor availability of products (11%), and asymptomatic disease diagnosed by screening (11%) [30].The parental knowledge of CD is also important for a good diet adherence and a better disease awareness of their children. A recent study conducted in the UK among parents of CD children at the time of diagnosis reported that 98% of the parents understand that GFD is the treatment of CD and 95% know that the GFD is for life. However, specific dietary knowledge was lacking with only one-third of the parents correctly identifying all the gluten containing food. The internet was the most common source of information (70.6%) [31]. Higher levels of parenting stress has been observed in parents of CD children compared to parents of healthy children [32].Patients’ support groups have played a relevant role in the last decades, organizing special social events for patients and families and providing education to patients, doctors, dietitians and the food service industry.The most recent and unique intervention in this matter is a randomized trial showing the efficacy of an interactive online intervention to improve the QoL and the GFD adherence [33].However, beside the external help, Ford et al. reported that personal tools such as self-efficacy and illness perception are influential factors and play a role in the individuals’ psycho-education. A targeted therapeutic intervention might improve GFD adherence and enhance psychological well-being for CD patients [34].In conclusion, the published studies indicate that, although there has been a positive change over time, there is still the need for additional training and education about CD and GFD [35].The question is: Has the CD QoL improved now that at school, at work, at restaurant the words “gluten free diet” very often do not require any other information to be understood?A review of the literature over time tells us that QoL of a CD patient before/at diagnosis is strongly influenced by the presence of gastrointestinal or non-gastrointestinal symptoms. Differently, after the diagnosis, the QoL of a CD patient is mainly related to the difficulty of having a chronic condition and the limitations imposed by the GFD as well as the difficulties in maintaining a good diet compliance [36,37]. Poor dietary adherence has been often associated with a poor QoL [38,39] but it is difficult to identify which is the cause and which the effect.“When I am at a restaurant with my friends, I often eat food with gluten because I do not want to be different”Celiac patients’ bad dietary adherence might be caused by the difficulty to accept their condition and the limitations that it imposes. A study conducted in 2001 showed that adult celiac patients reported inability to communicate feelings and a tendency to conformist behavior leading to a lifestyle limited by a lifelong condition [40]. At that time, patients feared that social contexts such as restaurants or convivial gatherings are occasions in which they may be identified by others as disease-affected, resulting in reputational damage. There are contradictory results about the relationship between dietary compliance and QoL. A 2010 long-term longitudinal study suggested that subsequent deterioration in QoL was associated with a lack of dietary adherence [41]. Other studies reported no differences in QoL scores between patients with full adherence and patients with partial/non adherence to GFD [42,43]. In particular, Barratt et al. reported that the perceived difficulty of adhering to a GFD and not the correct adherence to that may be associated with a decline in QoL [42].“I do not want to go to my friends’ birthday parties because I do not want them to say that I cannot eat the cake because I am ill”Looking at adolescents, Wagner et al. [44] found that bad compliance to the GFD was the major cause of low general QoL. Non-compliant adolescents had more physical problems, a higher burden of illness, higher feelings of “ill-being” and more family problems than GFD compliant adolescents. Moreover, in these patients, a late CD diagnosis was associated to more problems at school and in social activities. A school-based cross-sectional screening study recently found that children with undetected CD reported comparable health-related QoL to CD children on a GFD and those without CD [45]. Finally, a prospective study on screening-detected CD children described that ten years after diagnosis, the QoL of the children with CD was similar to that of the reference population [46].A qualitative study, using a focus group interview, investigated QoL in children and adolescents with CD to identify patient concerns in living with CD and on a GFD [47]. Two main categories were identified: “having CD” (composed by the diagnosis-process, self-perception, awareness of CD, social and emotional impact of CD, and thoughts about the future) and “coping with CD” (coping with food and coping with social situations). Several ways to alleviate stress were identified in the coping group: comparing CD to other chronic diseases, believing they were healthier than others as they did not eat as much unhealthy food, accepted their diagnosis and came to the conclusion that “there is nothing to be done about it” [47]. Living with CD is perceived as requiring several sacrifices which can influence the QoL in children on a GFD [48].“I was so ill before diagnosis, I daily had diarrhea, abdominal pain, headache… now I am a new person, I love the GFD”The GFD induces an improvement in QoL in symptomatic patients, while a similar effect has not been clearly reported in patients with subclinical/asymptomatic CD. A 2011 Finnish study [49] reported no change of the QoL in screen-detected patients on GFD and another Finnish study [50] in the same year described the QoL improved in symptomatic and in screen-detected symptomatic patients but not in screen-detected asymptomatic patients. Recently, Kurppa et al. [51] have described, using a randomized study, that asymptomatic CD patients benefited from the GFD for anxiety and better health, but not for social function when compared to similar CD patients following a gluten-containing diet. Finally, Paarlahti et al. [52] reported that a long duration of symptoms before diagnosis, psychiatric, neurologic or gastrointestinal co-morbidities and persistent symptoms were predictors of a reduced QoL.Apart from the symptoms commonly attributed to CD, other less explored complaints are known to improve on GFD and may have a positive effect of the QoL of patients. For example, sexual activity, which is hardly investigated dealing with a celiac patient, has been shown to be poor, unrelated to gastrointestinal symptoms, and to improve after one year of GFD [53].Another problem that is likely underestimated in celiac patients is the presence of eating disorders that are more frequent in untreated celiac women, dependent on the presence of gastrointestinal symptoms, and also improve on GFD [54]. Moreover, Zingone et al. [55] reported a reduced quality of sleep in CD patients both at diagnosis and on GFD compared with controls. A direct correlation between the quality of sleep and mood disorders and QoL was observed.In conclusion, the review of published papers reveals a noticeable increased knowledge of CD among doctors and the wider society. Despite this, the literature search indicates that celiac patients still present some vulnerability in the adaptation to a chronic condition, in particular in their social life. Planned intervention at diagnosis and follow-up to improve the patients’ adaptation to the condition and its limitations [56] and as a consequence their QoL has been suggested.It should be noted, however, that large controlled follow-up studies on the effect on QoL of any intervention, such as psychological support and repeated dietetic consultation, are still lacking.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Celiac disease is a permanent genetically determined intolerance to gluten that generally presents with gastrointestinal symptoms in young children and extraintestinal manifestations (endocrinological, dermatological, neurological, etc.) later. Furthermore, many studies demonstrate the close association between celiac and endocrine diseases, including growth and pubertal disorders, type I diabetes mellitus and autoimmune thyroid diseases, probably due to the presence of a common genetic predisposition. Follow-up for celiac children after the start of gluten-free diet is mandatory to avoid complications such as growth hormone deficiency. The present review deals with the problem of the diagnosis of endocrine-associated diseases in celiac children and gives suggestions for correct management and follow-up of these patients.Celiac disease (CD) is a systemic immune-mediated disorder triggered by dietary gluten in genetically susceptible subjects [1]. It is the most clinically relevant condition in which genetic predisposition and environmental factors interact to trigger an autoimmune response, and is the only human autoimmune condition in which the causative antigen is known.Treatment of CD consists of a gluten-free diet (GFD) which determines remission of intestinal damage, and protects against the risk of developing autoimmune diseases associated with CD and “normalizes” the mortality risk related to the disease itself [2,3].Classically, children with CD present with typical malabsorption signs and symptoms such as failure to thrive, diarrhea, abdominal distension, muscle wasting, poor appetite and irritability. Recent evidence points to the occurrence of extraintestinal manifestations (Table 1), such as iron and folic acid deficiency with or without anemia, dermatitis herpetiformis, delayed puberty, short stature, enamel defects, and recurrent aphthous stomatitis, before the diagnosis of celiac disease or during its management.Extraintestinal manifestations of CD.Osteopenia/osteoporosisArthritisEnamel defectsShort statureDelayed pubertyInfertilityEpilepsy with occipital calcificationsDepressionAtaxiaMyelopathyIron deficiency anemia refractory to oral iron supplementationHypertransaminasemiaDermatitis herpetiformisPediatricians frequently encounter the problem of short stature or stunted growth rate in CD subjects. In fact, the prevalence of CD in patients evaluated for short stature varies between 2% and 10% [4,5,6,7].Indeed, during the evaluation of a short child the first step is the exclusion of CD, which may be responsible for growth failure [8]. After the start of a GFD, catch-up growth is generally observed, and the celiac child usually returns to his/her normal growth curve for weight and height within 1–2 years. However, an endocrinological investigation including an evaluation of GH secretion should be performed in CD children who show no catch-up growth after at least one year of a strict GFD, and after seronegativity for anti-tissue transglutaminase and/or anti-endomysial antibodies have been confirmed. In subjects with CD and growth hormone deficiency (GHD), substitutive therapy with GH might be promptly started at standard doses, in order to obtain complete catch-up growth. The long-term effects of GH therapy in children following a strict GFD are similar to those observed in children with idiopathic GHD [9].Finally, the existence of a close relationship between CD and autoimmune diseases, such as thyroid disorders and diabetes mellitus type I, is suggested by the fact that CD is an autoimmune disorder. The pathogenetic mechanism is still not completely known and only partly linked to the increase of intestinal permeability.The present review contains some suggestions for the correct diagnostic work-up for CD in subjects who, even in the absence of gastrointestinal symptoms, show extraintestinal symptoms such as short stature and/or delayed puberty. The management of short stature and other endocrine related conditions in celiac children and adolescents is discussed.In a child with a stature below the 3rd percentile (Figure 1, Panel A) or with growth deceleration (Figure 1, Panel B), a diagnostic work-up should be started to identify any pathological cause [10].If a pre-pubertal child shows a stature close to the 3rd percentile, but grows along the same percentile, only an annual growth follow-up is indicated (Figure 1, Panel C).If, instead, a child grows on a percentile lower than his/her genetic target or when the first pubertal signs (appearance of the breast development in the female and increased testicular volume in the male) are already evident, the diagnostic work-up should be started immediately, without waiting for a growth rate decrease [10].First of all, it is necessary to evaluate whether the short stature is clinically suggestive of a normal variant of the growth pattern (e.g., familial short stature or constitutional delay of growth and puberty) or of a specific pathology (e.g., achondroplasia, hormonal dysfunction). In the differential diagnosis, to discern between familial short stature and/or constitutional delay of growth and puberty and endocrine conditions, a bone age evaluation is useful, since it indicates the subject’s growth potential.Therefore, the next step is the exclusion of other possible conditions responsible for growth failure including kidney or liver disease, skeletal disorders, subclinical hypothyroidism, intestinal malabsorption such as Crohn’s disease and CD. It is accepted practice to exclude celiac disease before evaluating GH secretion as well, in a short child in whom GHD is suspected. In fact, pathological GH responses to pharmacological tests have not been confirmed following the institution of the GFD [11,12]. Insulin-like growth factor I (IGF-I), which is considered the peripheral GH mediator, is low in patients with insufficient GH secretion, but is not a discriminating factor in the evaluation of GH secretion, since its level is influenced also by the nutritional status of the subject.Furthermore, in a patient with delayed appearance of pubertal signs (such as absence of the mammary gland over 13 years of age in female subjects and >4 mL testicular volume in male subjects over 15 years of age), it is necessary to exclude CD. In fact, after resolution of a pathological condition which may have determined growth deceleration, spontaneous catch-up growth is generally observed.Also in CD, after the start of a GFD, a significant catch-up growth is generally observed. Therefore, the celiac child usually returns to his/her normal growth curve for weight and height within 1–2 years (Figure 2).Therefore, a careful auxological follow-up is necessary to verify growth and weight catch-up, in addition to the annual evaluation of serological negativity. In fact, to confirm dietary compliance the annual monitoring of anti-tTG-IgA is recommended, in consideration of its high sensitivity and specificity. If after 1–2 years of a GFD the subject does not show clear catch-up growth, in the presence of seronegativity for specific celiac antibodies, the evaluation of GH secretion in response to at least two pharmacological stimuli is mandatory. In fact, it has been observed that 0.23% of children with short stature show an association between CD and GHD [13].Example of patients with height below the 3rd percentile (A), with height, initially above the 50th percentile, that decreases to a lower percentile (B) and with height within the 3rd percentile that does not decrease to a lower percentile (C).Example of a deceleration of growth rate in a CD child and catch-up growth after the introduction of a GFD.In the presence of a normal GH response to at least one pharmacological stimulus and IGF-I values within the normal range for sex and age, the auxological follow-up should be repeated every year. In the case of a pathological response (i.e., GH peak < 8 ng/mL to both stimulus tests) and in the presence of a negative specific serology, once the basal levels of FT4, TSH, ACTH and cortisol have been verified, substitutive therapy with recombinant human GH should be started as in patients with idiopathic GHD [14]. Nuclear magnetic resonance imaging of the brain is required to exclude any morphological abnormality of the hypothalamic-pituitary region. In the rare cases of GHD associated with a deficiency in one or more pituitary hormones (TSH, ACTH, LH, FSH, ADH), correct hormonal secretion should be restored by substitutive therapy with the missing hormones before starting GH treatment. A deficit in pituitary gonadotropins, LH and FSH, may be assessed only during the pubertal period when in normal children a pubertal gonadotropins increase.Celiac patients with GHD should be treated with the same GH dosage as patients with idiopathic GHD (0.23–0.25 mg/kg/week s.c.), administered in the evening before sleeping to mimic the physiological night-time elevation of the hormone. In the case of associated hormonal deficiencies, the doses of levothyroxine, hydrocortisone, estradiol or testosterone enantate according to gender and desmopressin are the same ones used in GHD patients. In celiac patients with GHD, the response to substitutive treatment is similar to that of subjects with idiopathic GHD [9,12,13]. Both height and growth velocity significantly improved during the therapy, confirming that the absence of catch-up growth after a GFD was not due to malnutrition, but to low GH secretion. The growth rate increases, especially during the first year of GH therapy, and then decreases, although it always remains above pre-treatment values [13]. Moreover, we have previously shown that CD children treated for associated GHD reach normal final height [9] (Figure 3).Height Standard Deviation Score (SDS) in CD-GHD patients at CD diagnosis, at GHD diagnosis, after 1 year of GH replacement therapy and at final height. Results are expressed as mean ± SEM (Standard Error of Mean) [9].Finally, compliance to GFD is very important in order to obtain a good response to GH therapy. Clinical results suggest, in fact, that CD patients with GHD do not respond to hormonal substitutive therapy if they do not observe a strict GFD [9].The existence of a close association between CD and endocrine autoimmune diseases such as autoimmune thyroiditis, type 1 diabetes mellitus (T1DM) and pubertal disorders has been widely demonstrated. A common genetic predisposition, such as HLA-DQ2 or DQ8 haplotypes, seems to partially explain the association between CD and autoimmune diseases. The development of multiple autoimmunity may be due to shared epitopes between an environmental agent and common antigens present in several endocrine tissues. In CD, the ingestion of gliadin causes an immune response against the enzyme tissue transglutaminase, which could share epitopes with other molecules present especially in endocrine glands [15,16].Furthermore, in CD patients the increased risk of developing other autoimmune diseases seems to be related to the duration of gluten exposure [17].The thyroid is frequently affected in CD patients, with highly variable percentages of hypo- and hyperthyroidism [18]. The frequent association of CD with autoimmune thyroid disorders is based on shared immunopathological mechanisms linked to the haplotype HLA-B8 and -DR3, which are more frequent in these patients compared with the general population. The prevalence of association between thyroiditis and CD varies from 4% to 14% and the most frequent association is between CD and Hashimoto thyroiditis [19]. Furthermore, early markers of thyroid autoimmune involvement, such as anti-thyroid peroxidase antibodies (anti-TPO) or thyroid echographic alterations with euthyroidism, have more frequently been observed in CD patients compared with the healthy population [20]. On the contrary, some authors reported that the presence of antithyroid antibodies in children with celiac disease has a low predictive value for the development of thyroid hypofunction during a three year-surveillance period [21].However, relying on our personal experience, in CD subjects, we suggest periodically monitoring thyroid function (FT4 and TSH) and autoantibodies (anti-thyroid thyroglobulin antibodies (anti-TG) and anti-TPO, in the case of a TSH increase.In the presence of anti-thyroid antibodies, if Hashimoto thyroiditis is suspected, a thyroid echography should be performed to evaluate the structure of the thyroid parenchyma. In the rare case of reduced FT4 and increased TSH, low-dosage treatment with levothyroxine (12.5–25 µg per day, to be taken orally at least 30 min before breakfast) should be started, and increased to the full dosage in 1–2 months, with biannual monitoring of FT4 and TSH, without considering anti-TPO and anti-TG levels. On the other hand, in some patients with autoimmune thyroiditis and CD following a GFD, the dose of replacement therapy can be progressively reduced and, in a few cases, the diet could normalize a subclinical hypothyroidism [19].Another widely documented association is that between CD and T1DM. In fact, CD is observed at a higher frequency in T1DM patients with prevalence rates up to 16.4% compared to 1% prevalence in the general population [22]. Genetic studies report a higher frequency of HLA-B8, -DR3 and -DQW2 both in patients with T1DM and in those with CD in comparison with the general population. Furthermore, alterations in β cells and enterocytes seem to be due to the same factors, such as proinflammatory cytokines (e.g., interferon-γ and TNF-α). In most cases CD is diagnosed months or years after the onset of T1DM, although at the onset of T1DM a temporary rise in antigliadin/antitransglutaminase antibodies is described in 3%–4% patients. The probability of developing CD increases with the duration of diabetes [23]. Predictive parameters of the risk of developing T1DM include the presence of anti-insulin antibodies (IA2), anti-glutammic acid decarboxylase antibodies (GAD), anti-zinc transporter 8 antibodies (ZnT8). At the onset of T1DM, and annually thereafter, it is common practice to evaluate CD serology and, in the case of abnormal anti-tTG levels, a duodenal-jejunal biopsy is required to detect any intestinal damage suggestive of CD [24]. In cases of potential CD, when a positive serological test does not correspond with any histological intestinal mucosal alterations (Marsh stage 0 or 1), an annual auxo-endocrinological and serological follow-up are recommended.In some CD patients, hypogonadism has been described with a delay in the onset of puberty and retarded menarche in untreated CD girls. Similarly, boys show tissue resistance to androgens characterized by reduced serum levels of dihydrotestosterone and increased levels of luteinizing hormone. Probably, a selective malabsorption of micronutrients (zinc, iron, folic acid, vitamins) important for the metabolism, for the functionality of sex hormone receptors and for autoimmune processes may have a role in the pathogenetic mechanisms of hypogonadism [25].In CD patients, an autoimmune hypophysitis resulting in hypopituitarism and impaired growth could be found. In fact, an autoimmune syndrome may hamper the growth response to a GFD. Some authors have detected anti-pituitary and anti-hypothalamus autoantibodies in CD and GHD children without catch-up growth after GFD, suggesting the onset of autoimmune hypophysitis involving somatotropic cells [26,27]. Therefore, the paediatric endocrinologist could also consider testing for anti-pituitary and anti-hypothalamus antibodies in all patients with CD-associated GHD.In conclusion, taking into account the high incidence of endocrine disorders in CD, and that an early diagnosis and treatment of CD might protect against the development of autoimmune conditions, annual serological screening for CD is recommended in all patients with T1DM or other autoimmune endocrine diseases [2].Children with CD should be monitored regularly to verify a normal growth and pubertal development, appearance of symptoms, and adherence to GFD. Evaluating adherence to GFD should be based on a combination of personal and dietary history and serology.GH secretion should be evaluated in CD children who show no catch-up growth after at least one year of a strict GFD, and after seronegativity for anti-tissue transglutaminase and/or anti-endomysial antibodies have been confirmed. In subjects with CD and GHD, substitutive therapy with GH should be administered at standard doses and should be started promptly, in order to achieve complete catch-up growth. The long-term effects of GH therapy in children who follow a strict GFD are similar to those observed in children with idiopathic GHD.Furthermore, CD patients should be monitored annually also for the occurrence of other autoimmune endocrine diseases, in particular thyroiditis.The authors are grateful to Susan West for English revision of the paper.M.B. and C.M. completely wrote the manuscript. A.V. revised the manuscript.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Since the first evidence demonstrating the dramatically high incidence of H. pylori infection and the subsequent medical challenges it incurs, health management of H. pylori infection has been a high priority for health authorities worldwide. Despite a decreasing rate of infection in western countries, prevalence of H. pylori infection in developing and in some industrial countries is still very high. Whereas treatment and vaccination against H. pylori is a contemporary issue in medical communities, selective treatment and prior high-throughput screening of the subject population is a major concern of health organizations. So far, diagnostic tests are either elaborative and require relatively advanced medical care infrastructure or they do not fulfill the criteria recommended by the Maastricht IV/Florence consensus report. In this review, in light of recent scientific studies, we highlight current and possible future approaches for the diagnosis of H. pylori. We point out that novel non-invasive tests may not only cover the requirements of gold standard methods in H. pylori detection but also offer the potential for risk stratification of infection in a high throughput manner.Helicobacter pylori is a gram negative microaerophilic fastidious bacterium which over centuries has successfully infected around 50 percent of human individuals throughout the world. Very often, infection occurs in childhood and persists lifelong if not treated. This human pathogen is known to induce several gastric disorders, but may also be associated with extragastric diseases like anaemia, dyspepsia, and some immunological disorders [1,2,3]. Almost all infected subjects develop chronic gastritis, and a considerable percentage of patients further develop ulcer disease or gastric cancer. Direct and indirect evidence suggests that the eradication of H. pylori or vaccination against it may decrease the risk of ulcer disease and gastric cancer. On the other hand, this approach is questioned by several scientists arguing that the co-evolution of H. pylori with the human population might imply beneficial effects of this bacterium for its host. Indeed, H. pylori infection has been shown to protect from childhood diarrhoea and asthma [4,5]. Here, the chronic infection leads to a constant stimulation of the immune system, the consequences of which might be more than local inflammation; it might lead to a persistent status of activation and induce a shift in immune regulation which could influence the course of many other diseases including asthma [6]. Therefore, prior to any treatment of H. pylori infection especially in larger populations and in asymptomatic individuals, exact diagnosis of the infection and characterization of the bacterium in the context of pathogenesis and virulence is necessary [7,8].Soon after the initial discovery of H. pylori by Marshall and Warren in 1983 and the subsequent confirmation of its causal role in gastric diseases, means of correct diagnosis as well as the clinical management of infected patients became a challenge to medicine and related biological sciences. Despite numerous academic research projects and commercial approaches, a standard method uniformly applicable, especially for indigent risk populations, is still missing. This, together with the high infection incidence worldwide on the one hand and the increasing need for individualized treatment of this infection on the other, demonstrates the emerging need of highly competent diagnostic methods for H. pylori [9,10]. While these methods should fulfill the common standards of clinical diagnostics like accuracy, sensitivity, and specificity, they should also be applicable in developing countries (areas) where hygiene standards and medical support are low. Certainly, the costs, time, necessary equipment and human resources as well as the availability of point of care and high throughput application are important issues which must be considered in the development of such a method. Currently, there are increasing numbers of attempts for development and several routinely employed methods for the diagnosis of H. pylori. According to the guideline of the Maastricht IV/Florence consensus report for the management of H. pylori infection, while the majority of older but still gold standard methods belong to the invasive approaches, non-invasive methods are more desirable and could cover the requirements for the more suitable tests as described above more conveniently [11]. In this review we attempt to categorize these tests, briefly describe their advantages and disadvantages and finally provide some hints regarding a future oriented standard test.The Rapid Urease Test (RUT) is a popular invasive diagnostic H. pylori test that is relatively quick, cheap and simple to perform. It detects the presence of urease in or on the gastric mucosa. Best results for RUT are obtained if biopsies are taken from both the antrum and corpus. The biopsy used for RUT can also be used for other tests such as for molecular-based tests of microbial susceptibility or for host factors [12]. False positive results are rarely observed. However, treatment with proton pump inhibitors or antimicrobial agents, which prevent the growth of urease producing H. pylori prior to the RUT, may cause false negative results. Moreover, the sensitivity of the test in patients with peptic ulcer disease with bleeding as well as in patients with intestinal metaplasia is significantly lower [12,13]. Therefore in these cases a negative RUT result should be complemented by a second method.The presence of typical spiral motile bacteria accompanied by inflammatory reaction in the histo-pathological sections of stomach was the first described method used for the diagnosis of the H. pylori [14]. Along with routinely applied stainings like Giemsa, hematoxylin, and eosin, there are some more specific staining procedures which facilitate the diagnosis of H. pylori infection. However, the accuracy of the histo-pathological diagnosis of H. pylori always depends on the number and the location of collected biopsy materials. While H. pylori can be detected in even a single biopsy taken from the correct site, to achieve a higher sensitivity, multiple biopsies are recommended. Moreover, the possible presence of other bacterial species with a similar morphology to H. pylori in the stomach [15,16] can be another source of error which negatively affects the accuracy of the test. In addition, treatment with proton pump inhibitors (PPI) or antibiotics prior to sampling may transform the shape of H. pylori to a coccoid form. Although molecular biology techniques like in situ hybridization [15] or immunological methods [17] are helpful solutions for the pitfalls mentioned above, the long assay time of between three hours and three days and the requirement of a relatively complex infrastructure for these methods compromise routine application.Although it should be stated that H. pylori culture is not a routine procedure in initial diagnosis, in many bacteriology laboratories H. pylori isolation via the culture of biopsy samples is a routine second line approach [18,19]. Because of the demanding character of this bacterium, this method remains challenging. This technique, although highly specific, is not as sensitive as other tests like histology and the rapid urease test. As well as for purposes of scientific research, cultured live H. pylori is used for diagnostic approaches and for the detection of antibiotic resistance if treatment failure is suspected [20]. H. pylori requires a microaerophilic atmosphere (5% to 10% oxygen, 5% to 12% carbon dioxide and 80%–90% nitrogen with humidity) and a complex culture media. The most commonly used media contains Brucella, Columbia Wilkins-Chalgren, brain-heart infusion or trypticase agar bases, supplemented with sheep or horse blood [21]. Because isolation of this microaerophilic organism from gastric biopsy specimens takes a long time, up to 5–7 days, to overcome the problem of growth of other competitors that exist in the sample, the culture media is supplemented with specific antibiotics. Although H. pylori could be cultured from stool specimens [22,23,24] due to the presence and growth of numerous other bacteria and especially microorganisms phenotypically similar to H. pylori, colonies have to be further characterized by other methods. Moreover, it is possible that the bacterium goes into a viable form that cannot be cultured (coccoid form) which leads to false negative results [25].PCR based detection of H. pylori could be categorized under invasive as well as non-invasive methods. Molecular diagnostics have dramatically changed the clinical management of many infectious diseases in the past decades. PCR currently remains the best developed molecular technique as it provides a wide range of clinical applications, including specific or broad-spectrum pathogen detection, evaluation of emerging novel infections, surveillance, early detection of bio threat agents, and antimicrobial resistance profiling [26]. While PCR could be applied for the detection of H. pylori in biopsies, this technique is more qualified for its use in samples taken from the oral cavity or from stool. PCR-based techniques, if applied as a non-invasive approach (i.e., from stool samples) tend to be more cost effective than other traditional methods. In addition to the improved specifications of this technology like high sensitivity and specificity, simplicity, and automated procedures, there are several other advantages to be considered. Practically, regardless of genome size, any genomic material could be used as a template sample for PCR, which allows sampling from multiple origins [27]. The high efficiency of this method also achieves fast results. Since antibiotic resistance is currently the major challenge in microbiology, it has to be pointed out that the fast acquirement of results of not only the diagnosis of H. pylori but also of its susceptibility to the right antibiotics is extremely important. There are a couple of PCR methods like multiplex PCR which have been employed and recommended as an alternative to culture for resistance testing to antibiotics like clarithromycin [28,29,30,31]. However, a limitation lays in the fact that PCR cannot detect metronidazole resistance. Here, bacterial culture would still be necessary to cover this pitfall especially if a resistance to antibiotics is suspected or again in the case of second line therapies.The achievement of the criteria mentioned above with its advantages is possible through different PCR approaches. In addition to the conventional PCR methods, a new approach like the colorimetric detection of H. pylori DNA using isothermal helicase-dependent amplification is a valuable tool. The rapid application of the test is complemented by reasonably high (up to 95%) sensitivity and specificity of the assay [32].Despite the numerous advantages of PCR-based techniques, there are still some challenges regarding its appropriate application in diagnostics. While correct sampling and preparation of samples prior to assay is a common issue in diagnostic assay, sensitivity and specificity of PCR-based assay are strongly dependent on the design of the method. For instance, due to the extraordinary variability of H. pylori genome, the selection of the target gene and PCR primer pairs dramatically influence specificity and sensitivity of the test [33]. As the whole genome of most known pathogens including many H. pylori strains has been successfully sequenced in the last decade, it is very important to design and select the PCR primers based on a comprehensive bioinformatics analysis of relevant genomes. Here, it is mandatory to design primers according to genomic sequences that are highly conserved in all H. pylori strains.The urease breath test (UBT) is one of the most common non-invasive tests used. This non-invasive test, available in different versions, has been evaluated in different studies, showing high sensitivity, specificity and accuracy [34,35]. The test is able to detect the infection indirectly by measuring the existence of bacterial urease produced by H. pylori in the stomach. There are different types of this test comprising 13C- or 14C-isotope labelled urea. If H. pylori is present, the urease hydrolyses the labelled urea and the exhaled isotope containing ammonia can be detected applying the samples to a measuring device. This test is recommended by the Maastricht IV/Florence Consensus Report as a valuable diagnostic tool for the detection of infection and for therapy control [11].It has been shown that UBT can distinguish an ongoing from a past infection; hence, it is able to detect the eradication progress after treatment [36]. A recent study was conducted comparing C13-UBT and a monoclonal H. pylori stool antigen test in infants and toddlers in South America [37]. The study suggests that both tests are reliable for H. pylori diagnosis in very young patients because of the high concordance between test results. In comparison, a study performed in Turkey evaluating C14-UBT in elderly patients also showed good diagnostic parameters with a sensitivity and specificity of 91.4% and 93.8%, respectively, compared to histopathology [38].In recent years, different application protocols and detection devices were developed. A recently published study describes a residual gas analyser-based mass spectrometry approach with possible point-of-care application [39]. A further study has evaluated a low dose capsule-based UBT approach compared to conventional UBT and invasive tests [40]. This approach seems to be superior to the conventional UBT in terms of sensitivity and specificity, resulting in 100% sensitivity and specificity but depending on the time of sample collection.One of the limitations is the presence of other urease producing bacteria in the stomach, e.g. H. heilmannii, which might lead to false positive results. Moreover, acute bleeding and co-medication can lead to false negative test results [41]. For reasons of required fasting prior to taking the test, it might be onerous for the patient. In addition, because of the detection devices needed, costs tend to be higher compared to alternative tests used [42]. Furthermore, according to different protocols available, the accuracy of UBT test results depends on the amount of urea applied, the point in time in which samples are taken and the set point of the cut off value [43].Fecal antigen tests detect antigens in stool samples. ELISA formats comprising monoclonal antibodies against H. pylori proteins showed improved results compared to polyclonal approaches [44]. The current guideline evaluates the use of the stool antigen test as equivalent to the UBT if a validated laboratory-based monoclonal antibody is used [11]. Korkmaz et al. compared the diagnostic accuracy of five different stool antigen tests in adult dyspeptic patients comparing monoclonal enzyme immunoassay tests (EIA) and rapid immunochromatographic assays (ICA). The sensitivity and specificity of the tests analysed had a high variation between 48.9%–92.2% and 88.9%–94.4%, respectively, depending on the test format. They conclude that EIA tests are more accurate compared to the currently available ICA-based test, that are fast and easy to use but provide less reliable results [45]. A recent meta-analysis conducted by Zhou and colleagues analysed forty-five studies, including 5931 patients and evaluated the test performance of a H. pylori stool antigen test in children. The average sensitivity and specificity was 92.1% and 94.1%, respectively [46]. Furthermore, the available stool antigen tests have been shown to be able to distinguish infected from treated patients [47], enabling the confirmation of treatment. Degradation of antigens in the intestine and consequent disintegration of epitopes might lead to false negative results. Moreover, the process of sample handling could be fastidious for patients. False negative results may occur when the bacterial load is low, due to proton-pump inhibitors or the recent use of antibiotics or bismuth [41,48].Immune responses against H. pylori are utilized to detect infection by analysing patients’ blood or serum for IgG and IgA antibodies. Serology is the only test which is not affected by those local changes in the stomach that could lead to a low bacterial load and to false negative results [11]. According to guidelines proposed by the Maastricht conference, only IgG detection is considered and the favoured method is ELISA. Currently, different formats of serological tests are available, including simple ELISAs that use whole lysates or recombinant produced H. pylori proteins as antigens. More recently, immuno-blots [49], luminex-based bead assays [50] and line assays [51] were developed; these allow a more specific evaluation of the infecting H. pylori strain in terms of bacterial virulence factors and host immune responses towards the human pathogen. Moreover, they show improved sensitivity and specificity [51,52,53] because additional and highly purified antigens are included. These non-invasive tests are easy and cheap to perform. The potential for developing a rapid diagnostic test makes serology an interesting option for testing populations in areas with little or no access to medical facilities. Using an automated approach, large cohorts could be tested within a short time, allowing population based studies. Good diagnostic parameters in terms of robustness, sensitivity and specificity are given as shown in different studies [51,52]. These studies show that improved serological test formats lead to increased sensitivity of over 95% and specificity ranging from 85%–96%, making serology a preferable alternative to other non-invasive tests. However, there are also contradictory studies with low sensitivity and specificity values for the serological test [54], probably due to the test format applied or the antigens used.Risk stratification might be an interesting issue, detecting combined antibody responses to different virulence factors. Promising studies have analysed different H. pylori antigens, showing significant correlations between positive immune responses and clinical outcomes like chronic atrophic gastritis, intestinal metaplasia, dysplasia, and gastric cancer [55,56,57]. In particular, there have been great improvements by applying single defined recombinant H. pylori antigens; the well-characterised oncogenic protein CagA that has been shown to correlate with severe gastric disorders and VacA that are in clinical use [58,59]. The immune responses towards VacA could be even more specific by differentiating between m1 and m2 variants of VacA [60]. More recently, antigens like NapA, GroEL, HyuA have been shown to have the potential to be used as biomarkers to predict infected individuals having a higher risk of developing premalignant changes that are a hallmark of gastric cancer development [61]. The combination and evaluation of different biomarkers adapted to the region where they are used might be the future method for ruling out patients at higher risk of developing severe diseases.Antibody responses towards antigens are sustained for a long period after eradication therapy, as shown in different studies [62,63,64,65]. These studies argue that a confirmation of treatment success by serology is not applicable for this reason.However, while some immune responses to certain antigens persist for a long period of time (especially CagA), for some antigens the antibody titre decreases within a short period, also depending on the Ig-class tested [66]. This phenomenon could also be utilized as clinical readout to confirm treatment success by analysing the decline of antibody responses, as shown in different studies [67]. Wang et al. conclude from their study that it would be the reasonable and even perhaps preferred method of monitoring H. pylori infections [68].Despite numerous publications on rapid diagnostic testing (RDT) methodologies for infectious diseases, such testing has become neither commonplace nor an integral component of services offered by clinical microbiology laboratories [69]. However, because of the emerging need for rapid diagnosis and treatment of virulent strains of different viral and bacterial infections, discussions regarding routine application of RDT are increasing within current medical circles. Debate on the value of RDT has broadened and continues to increasingly encompass new infections.Results of RDT testing become available within a couple of minutes to a few hours. A clinical specimen is processed in a few steps (preferably in a single step) at the site where it is collected (point of care). The quality and value of the RDT is determined by its sensitivity and specificity, the time required for results, and its cost and availability. According to the guidelines set forth by the Maastricht conference, it appears that those antigens with either high or low molecular weight are more specific [11].There are currently many H. pylori RDT kits commercially available. However, how far these tests fit in with standard clinical practice is still undetermined.As generally in microbiology laboratories, H. pylori RDT kits could also be performed based on the following four approaches: (A) testing of H. pylori specific antigens; (B) molecular detection of the specific H. pylori nucleic acid sequence; (C) rapid biochemical reaction test and (D) serologic detection of H. pylori specific antibodies.Regardless of which approach is applied, these tests have to be validated based on gold standards. In addition to specificity and sensitivity, the value of the overall agreement with the gold standard (biopsy or culture) plays a crucial role in the evaluation of the kit. Although the majority of the approved RDTs for H. pylori reach a non-ideal sensitivity and specificity (in general < 90%), there are promising findings that lead to significant improvement of the assays. For instance, in the process of testing of H. pylori specific antigens, H. pylori specific antibodies are required. While antibodies produced by immunization with whole H. pylori lysate or a mixture of bacterial compartments are more prone to be sensitive, they reduce the specificity of the test. On the other hand, antibodies which are produced against single molecules are very specific, although they gain more false negative results. The same argument is also valid in other sero-immunology based approaches that aim at detecting H. pylori specific antibodies. While utilization of single conserved molecule of H. pylori leads to reasonable specificity, this assay may lead to higher false negative results compared to the application of a mixture of antigens, which in turn may cause higher false positive results.As mentioned above, new promising studies have described new H. pylori antigens which have been successfully tested in preliminary assays. For example, FliD, a hook flagellar protein of H. pylori, is a highly conserved molecule in all H. pylori species. While this molecule has no or only low homology to other bacterial species which prevents cross reactivity, it has shown a strong antigenicity in animal studies. Assays which employ this protein as antigen in the diagnosis of anti H. pylori antibodies have shown high sensitivity and specificity [70].Serologic tests should therefore include antigens for sensitivity, specificity testing, and antigens that identify infections by more pathogenic H. pylori strains, which might provide the basis for decisions about further treatment.Continuous developments in both invasive and non-invasive based methods for detection of H. pylori infection will greatly contribute to further improvement of the health management of H. pylori associated disorders. Although this mysterious bacterium has been unanimously declared a human pathogen, only a minority of infected individuals develop an associated disease, which seems mainly attributed to the armament of virulence factors of H. pylori. Therefore, individual identification of virulence factors of the bacterium for risk stratification is discussed for risk assessment in combination with histopathological evaluation of gastritis. There are common diagnostic methods for detection of the infection and verification of the virulence factors. However, while gold standard methods are still mainly derived from biopsy-based methods, the high prevalence of the infection especially in areas with low medical support suggests the urgent need for introducing non-invasive and preferably high throughput applicable procedures. The selection of such a test depends on the sensitivity, specificity, availability, complexity, costs, and rapidity of results [71]. Unfortunately, none of the currently used methods cover these criteria perfectly. Although biopsy-based methods have a very high specificity, only a moderate sensitivity is observed in these assays mainly due to factors that are difficult to control such as the sampling site. Even in spite of correct sampling, there are other factors such as PPI treatment prior to sampling that possibly affect the growth or presence of detectable curved bacteria. PCR-based tests have slight advantages compared to other tests [71,72]. PCR, as a highly sensitive and specific method, is applicable not only in the detection of infection with H. pylori but also in the monitoring of treatment and therapy efficiency. Our experience showed that with the proper design of PCR, a single copy of genomic DNA is sufficient to obtain a positive signal indicating the presence of H. pylori in the sample. Moreover, DNA samples isolated from different sources such as gastric biopsy, samples from the oral cavity, and stool specimens could be subjected to PCR assay. Accordingly, precluding the possible chances of contamination, PCR could be used as a gold standard. The main disadvantage of PCR in our opinion is the level of diagnostic facilities and implementation of this method in regions with poor medical support. Since other biopsy-based methods generally have disadvantages such as the requirement of endoscopic facilities or additional systems such as RUT, they are not altogether suitable as gold standards. Recently, most efforts to introduce an adequate gold standard are focused on non-invasive methods. In addition to PCR, serological methods aiming at the detection of H. pylori specific antigens in different specimens have been significantly improved. Bioinformatics analysis greatly helps the selection of the optimal antigen applied in serological assays. Not only could the antigenicity of the antigen be predicted in these analyses, more importantly the homology of the antigen to other relevant microorganisms can be comprehensively analysed. Through the choice of a suitable antigen and the application of advanced methods in nanotechnology in assay design, new serological tests have recently reached relatively ideal performance [70]. Although the report of the Maastricht conference emphasizes that antibodies against H. pylori and especially against its most specific antigen CagA, remain elevated for long periods, for months or even years, there is evidence that while CagA is a dominant virulence marker, it is only present in around 70% of H. pylori strains depending on their geographic origin [51,73,74]. Here, recent studies identified other antigens in all H. pylori strains which possess not only high immunogenicity but also elicit specific antibodies which will fade away after a relatively short time after eradication. Advanced and simplified specific antibody detection techniques like line assay and rapid diagnostic tests have greatly improved the application of serological tests for the detection of H. pylori. These approaches cover most of the criteria mentioned for H. pylori diagnostic assays. Not only are they applicable in high-through-put studies of large cohorts but they are also highly cost effective, bedside applicable, simple for analysis and intelligible for medical personnel. Less so, but still in an equally acceptable relevant context, specific antigen tracing tests are currently being developed. In particular, noticeable progression has been made in the area of stool tests. Indeed, when a study focuses particularly on children of a high prevalence area, antigen-tracing systems like the stool antigen test are advantageous. Finally, there is apparent evidence suggesting that not every strain of H. pylori is harmful and should be treated [8,75,76,77]. In addition, there are accumulating data on the beneficial aspects of infection with H. pylori for its human host [75]. Therefore, conduction of risk stratification according to the epidemiological data is highly recommended. The feasibility of such assessments is now exclusively conceivable through serological assays, which can indicate the infection with pathogenic strains of H. pylori.In conclusion, we feel that current available diagnostic systems cannot meet the requirements posed by such a widespread and prevalent infection as H. pylori, especially when more than simple detection of positivity is required (i.e. risk stratification, antibiotic resistance). We thus suggest a collaborative effort from experts and health organizations across the world to strive for the development, validation, and introduction of such multi-tasking diagnostic tools which could not only become a new recommended gold standard for the screening of large infected populations but would also assist in guiding the treatment strategies for each infected individual.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).The gluten-free diet has long been considered the standard treatment for celiac disease. However, a significant number of patients continue to experience persistent symptoms despite following a gluten-free diet. Inadvertent gluten ingestion, fermentable carbohydrates, cross-contamination, and social or financial burdens present obstacles to maintaining a gluten-free diet. Proper diet education and follow-up by an expert Registered Dietitian (RD) is essential to ensure adequate nutrition on the gluten-free diet. Patients may experience unintended weight gain or elevated cholesterol levels after initiating the gluten-free diet due to adequate absorption and healing of the intestines. This review deals with the evolving gluten-free diet, optimal recommendations while considering the overall health of patients, and multi-factorial aspects of the permanent lifestyle change.Celiac disease is a chronic, systemic, immune-mediated disorder triggered by exposure to gluten in genetically predisposed individuals. [1]. Over the years, overt gastrointestinal symptoms that almost invariably previously accompanied a diagnosis of celiac disease have become less frequent and obvious, posing additional challenges to clinicians. An Italian study found the clinical presentation drastically shifted over the 15 year interval of the study [2]. The majority, 66%, were found to have non-classical symptoms like bloating, osteoporosis and anemia. Only 34% had classical symptoms of diarrheaand malabsorption [2].Medical treatment for celiac disease consists of life-long avoidance of gluten-containing foods. Gluten is used to describe specific amino acids in wheat (gliadin), rye (hordein), barley (secalin), and derivatives of these grains. Strict adherence to the gluten-free diet often minimizes bloating, diarrhea, weight loss, fatigue, and begins the healing process. Some patients feel improvements within days of starting the diet, although it may take up to a year for complete intestinal healing. This may be due to the degree of intestinal damage, or intermittent, unintended gluten ingestion.Wheat gluten was first isolated in 1745 [3]. Since then, there have been constant advances in the production and usage of wheat gluten. Gluten, which means glue in Latin, provides a viscoelastic network [4] in food. These proteins produce the wide variety of textures, including crispy, crunchy, flaky and fluffy, in baked products.Harvesting and consumption of cereals increased gradually over time, especially between World War I and World War II. In 1941, the Nutrition Society was formed in Britain to promote advances in nutrition and to arrange a rationing system for an increasing population. Its main objective was to increase global wheat production [5]. This shift correlates with an increase in the population of celiac disease: It was estimated that in 1950, 1 in 8000 in the United Kingdom had celiac disease [6]. Today, approximately 1% of the population in most Western countries has celiac disease [7].Dicke initiated experiments of wheat-free diets in 1934–1936, and in 1950, published his thesis that found that gluten caused anorexia, increased bowel movements and steatorrhea. Dicke’s thesis provided the framework for the development of the gluten-free diet [8].The duodenum is the uppermost section of the small intestine. It is lined with villi, small finger-like protrusions, which increase the surface area over which nutrients are absorbed. The villi themselves have microvilli on their tips, known as the brush border of the small intestine, which is responsible for breaking down proteins and a variety of sugars including lactose [9].If an individual with celiac disease consumes gluten, the immune system responds by damaging the small intestine, and particularly the villi. At diagnosis of celiac disease, the villi may be partially shortened or completely flattened. Without villi, the ability to properly digest and absorb nutrients diminishes considerably.Patients with celiac disease typically have malabsorption issues leading up to the time of diagnosis. Therefore, it is important to check for deficiencies upon confirmation of the diagnosis. There is a pressing need for international guidelines for vitamin and mineral supplementation in celiac disease. The most common nutrient deficiencies in patients with newly diagnosed celiac disease are iron, Vitamin B12, folate, calcium, Vitamin D, and zinc. The risk of nutrient deficiency is related to the degree of malabsorption, inflammation, or intestinal damage upon diagnosis [10]. If deficiencies exist, vitamin or mineral supplements are required to aid in proper nourishment and repletion. A multivitamin with minerals initiated at diagnosis can promote proper healing and compensate for the lack of fortification in gluten-free food products [11].The gluten-free diet can be balanced and healthy with the inclusion of foods that are naturally gluten-free before processing. However, many patients are drawn to the convenience of processed gluten-free foods that are high in fat, sugar, and sodium without vitamin and mineral enrichment or fortification. Patients should be steered toward nutrient-dense foods, which are naturally good sources of these vitamins and minerals, to provide a balanced and adequate intake. These include fruits, vegetables, beans, nuts, seeds, fish, lean meat, poultry and dairy.Iron-deficiency anemia has been detected in 46% of subclinical celiac disease cases, with a higher prevalence in adults than children [12]. Iron deficiency anemia in celiac disease is primarily due to malabsorption, as the duodenum, where villous atrophy is mostly located, is the main site of iron absorption. Both ferritin and CBC should be checked upon diagnosis [13]. Good sources of iron include beef, turkey, liver, egg yolks, sardines, and oysters. Treatment consists of a strict gluten-free diet and iron supplementation until iron stores normalize. This can take some time, typically shorter for children, but up to a year for hemoglobin and two years for iron stores to stabilize in adults [12].Vitamin B12 deficiency is reported in untreated celiac disease patients at rates of 8% to 41%. It correlates with extensive disease, as most absorption takes place in the ileum [12]. The gluten-free diet, along with a multivitamin, is often sufficient for repletion of vitamin B12, although additional supplementation may be necessary. Dietary sources of vitamin B12 include shellfish, liver, dairy, beef, eggs. Vegans are at risk due to lack of B12 in the diet considering avoidance of all animal products. Elevated levels of serum methylmalonic acid (MMA) may enhance the diagnostic accuracy if vitamin B12 levels are in the lower range of normal or if there is an additional folate deficiency [14].Studies have shown that 35%–49% of newly diagnosed celiac patients have folate deficiency [13]. Folate levels should be checked at diagnosis and annual follow-up visits. Unlike their gluten-containing counterparts, gluten-free foods are not fortified with folate. Women of childbearing age must be especially mindful of adequate folate levels and intake due to the importance of folate to the developing fetus.The connection between bone health and celiac disease has been well documented [15]. One review suggests 75% of untreated adult patients with celiac disease suffer from a loss of bone mass [16]. Bone mineral density is related to inflammation from active disease as well as poor absorption. Calcium and vitamin D are absorbed in the duodenum. Clinicians should check 25-hydroxy-vitamin-D and in adults obtain a bone mineral density test at diagnosis, to determine baseline bone health and need for supplementation. The bone mineral density test should be repeated within a year if the patient is found to have osteopenia or osteoporosis [15]. Bone density increases over time on the gluten-free diet, and especially rapidly in children; as the risk is reduced with good dietary adherence and reduction in intestinal villous atrophy [17]. Ideally, between the diet and supplements, adult patients with celiac disease should have a calcium intake of at least 1000 mg per day [18]. Many patients experience lactose intolerance which leads to gas and bloating that may overlap symptoms of celiac disease. Lactose intolerance may occur if villi are damaged, as lactase enzymes are in the brush border at the tips of the villi; but often improves with intestinal healing. Patients who suffer from gas, bloating, and diarrhea at diagnosis should be advised to avoid lactose to reduce symptoms [9]. Often after several weeks to months on a gluten-free diet, lactose containing products can be reintroduced without overt symptoms of intolerance.The medical treatment for celiac disease is a life-long avoidance of gluten-containing foods. Gluten is found in wheat (gliadin), rye (hordein), barley (secalin), and derivatives of these grains. Oats are not included in this group, but must be regarded with caution, due to risks of cross-contamination. The first step in managing the gluten-free diet is to understand which foods contain wheat, rye, and barley so they can be eliminated from the diet, and intestinal healing can begin. Fresh foods, without any processing or additives, from fruit, vegetables, dairy products, fish, and meat, meat alternative food groups are all naturally gluten-free. The diet is complicated and confusing, with misinformation traveling over the Internet and bewildering patients. The gluten-free food market continues to grow: some estimates state that gluten-free sales will reach $15.6 billion in 2016 [19].Oats are a significant source of vitamins, minerals, and heart healthy soluble fiber. Thompson and colleagues studied inherently gluten-free grains, and found gluten in 32% of samples not labelled gluten-free [20]. This prompted recommendations to only consume oats labelled as gluten-free, as the manufacturers follow a purity protocol to reduce the risk of cross contamination. The purity protocol involves frequent sampling of gluten-free oats (in the field, during shipment, processing and in storage facilities) to reduce the risk of cross-contamination [20].The FDA set a gluten limit of less than 20 parts per million (ppm) in foods that carry the label gluten-free [21]. This is similar to guidelines set by the Codex Alimentarius [22] and the European Union (EU) [23]. The EU guidelines consider 20 ppm to be gluten-free and <100 ppm gluten as “very low gluten” [23]. Since August 2013, a food labeled “gluten-free” that fails to meet the requirements of the regulation will be subject to regulatory action by the FDA.According to a study by Catassi, 50 mg of gluten introduced daily induced villous damage over three months [24]. Researchers found a significant decrease in the villous height to crypt depth (vh/cd) ratio in the group taking 50 mg gluten daily. No significant change was found in the vh/cd ratio in the group taking 10 mg gluten daily for three months. This study provided evidence for avoidance of minute amounts of gluten, with the cut-off of 10 mg gluten daily [24,25]. One would need to eat more than a pound of food (500 g) tested at 20 ppm of gluten to achieve 10 mg gluten in a day.Gluten exposure from cross-contamination may lead to ongoing disease activity, and is of concern. Considering risk of cross-contamination, it is recommended to avoid grains and flours from bulk bins, and to purchase flours and grains labeled gluten-free [11]. Condiment containers that allow use of spoons or other utensils may be at risk of cross-contamination in shared kitchens. Deli slicers, cutting boards, toasters, and colanders are risks of cross-contamination in shared kitchens.A gluten-free diet has become easier to follow with the explosion of gluten-free products and increasing options available when eating out. A major concern is eating out; as the establishment may have best intentions in mind but there is often cross-contamination. Despite a gluten-free menu, many restaurants do not follow strict guidelines to avoid cross-contamination. If the wait staff of a restaurant does not communicate closely with the kitchen; this poses risks as the gluten-free order may not be high priority, or overlooked. Fortunately, chefs have become more aware of the gluten-free diet and concerns regarding cross-contamination over the past decade [26].Proper gluten-free diet education and counseling by an expert RD is necessary to aid in adherence to the gluten-free diet. After the initial visit, the patient should have access to a RD to answer questions as he or she learns to navigate the gluten-free diet. Such access would ideally include follow-up visits, phone calls, as well as analysis of food logs to ensure compliance and understanding of the diet. Not all insurance carriers cover visits with a RD. It is advisable for patients to check insurance coverage before the visit to avoid billing issues.Misinformation online can lead to anxiety, confusion, and overwhelm the patient. The expert RD should review the diet, food logs, and lifestyle with a “fine tooth comb” to identify potential sources of gluten exposure. Important areas to focus on include cross-contamination in the home, frequency and locations of eating out, and products purchased that may be cross-contaminated. The RD can direct patients to reputable information sources. Ludvigsson has four steps to assess diet adherence: dietetic review, serum antibodies, clinical assessment of symptoms and follow-up biopsy [27].The R5 sandwich ELISA is a reliable test and used to test most foods worldwide. This test is unable to evaluate gluten content in foods with hydrolyzed proteins or fermented products. An example is the issue arising with gluten-removed beers. These beers are barley based, and are treated with enzymes that break the proteins at the amino acid proline. The R5 sandwich ELISA requires proline to be interpreted correctly. Therefore, no safe or accurate test is available to deem a gluten-removed beer safe for consumption [28]. Hopefully the future will bring advances with testing. It is advised to only consume gluten-free beers and avoid gluten-removed beer until an appropriate test is developed to ensure safety.Only a handful of studies have been done to assess gluten content in foods that are labelled gluten-free. In the past year, three studies had varying results. One study analyzing 275 U.S. gluten-free products found 1.1% containing ≥20ppm of gluten [29]. Another study found vastly different results with nearly 20% of foods labeled gluten-free contained ≥20ppm of gluten [30]. A study by Thompson found 5% of 158 gluten-free products with ≥20ppm of gluten [31]. A study of European foods analyzed from Italy, Spain, Germany, and Norway found 99.5% of the foods were under 20 ppm and 94% were under 5 ppm [32].A recent study found a four-fold increase in mercury blood levels in celiac patients following a gluten-free diet [33]. This study took into account amalgam fillings which contain mercury, as well as seafood intake; which did not correlate with blood and urine samples of mercury. Mercury is primarily absorbed in the duodenum. This outcome could be due to an altered response to mercury exposure, with a predisposition to accumulate it. Further studies are needed to clarify the concern of mercury in celiac disease and inspire guidelines for the surveillance of mercury in food.Rice is a main staple in the diet of many with celiac disease. Consumption of plain rice and convenience foods processed with rice has skyrocketed. These include rice crackers, rice milk, rice flours, rice noodles, which further increases the average intake. However, most varieties of rice have been shown to contain high levels of inorganic arsenic, a carcinogen [34]. This may pose concerns due to large volumes of rice consumed by those following a gluten-free diet. Large doses of arsenic are life threatening, but small doses may increase risk of heart disease, diabetes, and cancer [34]. In 2013, the Food and Drug Administration (FDA) conducted an analysis of 1300 rice-based foods to determine levels of arsenic. Varying levels of arsenic were found in the foods tested. The FDA plans therefore to conduct risk assessments as the analysis did not determine long-term risks, and it should also be noted that no “safe” levels of arsenic have been established by the FDA [35]. This furthers the importance of a diet with varied grains, an important consideration that could simply be overlooked in those who do not see a RD at diagnosis.Corn has long been a staple in the typical gluten-free diet. Similar to rice, corn is one of the predominant gluten-free options in convenience foods, including corn tortillas, chips, or other baked foods or snacks. Corn has been found to contain high levels of mycotoxins, specifically fumonisins [36]. In the initial study done in the Czech Republic in 1998, 88% of corn-based foods tested had high levels of mycotoxin fumonisins [37]. It was specifically high in extruded corn products, including polenta, but lower in corn flour or corn porridge [37]. A more recent study in Europe found 90% of gluten-free foods were contaminated with fumonisins, although the overall median of these values was below the European Union limit for adult consumption, which is 800 µg/kg [38]. Approximately 17.5% of the products contained fumonisins above the legal limit, with up to 3310 µg/kg [38]. The conditions corn sustains during the growth in fields, storage and processing allows for fumonisins to fester. This is yet another reason to support a varied diet, a restrictive diet could prove hazardous.A study in Spain found a significant decrease in lactobacillus and bifidobacterium, and fecal short chain fatty acids in celiac patients on a gluten-free diet for 2 years [39]. There was a reduction in the diversity of healthy bacteria in 5 of 11 treated celiac patients, although 6 of 11 had similar microbiota compared to healthy controls [39]. Another recent study found a decrease in lactobacillus in pediatric celiac disease patients following a gluten-free diet [40]. Further studies are warranted to determine if probiotics or prebiotics may be of benefit to increase beneficial bacteria in celiac disease.Gluten is versatile, as it provides a variety of functions in foods. When gluten is removed, the product is denser. Sugar and fat are often added to provide a similar mouthfeel, which can result in higher calories. A recent study in Spain analyzed the nutritional content of gluten-free and gluten-containing products, which revealed significant differences. Gluten-free products contained more fat, predominantly saturated fat, more sodium, and less fiber and protein than gluten-containing counterparts [41]. Recent utilization of bean and nut flours, which aid in the texture, composition and nutritivevalueof gluten-free foods, has made some gluten-free products quite similar to their gluten-containing counterparts [42] and may aid in reducing the effect of weight gain on a gluten-free diet.Shepherd et al. found those following a gluten-free diet had some nutritional inadequacies and excesses. Those following a gluten-free diet consumed inadequate fiber and folate, with higher fat and sugar content of gluten-free foods [43]. Further attention should be paid to the importance of fortification of gluten-free foods to mimic gluten-containing counterparts.It has been reported that between 39%–44% of patients are overweight or obese at diagnosis [44,45]. This reflects the general population with respect to overweight and obesity [45]. Approximately 81% patients with celiac disease gain weight on a gluten-free diet [44]. Metabolic syndrome is a cluster of risk factors for cardiovascular disease and type 2 diabetes, and encompasses abdominal obesity, high blood pressure, dyslipidemia and impaired glucose regulation [46]. The prevalence of metabolic syndrome is estimated to be approximately 20%–25% of the world’s population [47]. A recent study found nearly 30% of patients with normal weight at diagnosis shifted into metabolic syndrome after only one year on the gluten-free diet [48]. The researchers suggest an in-depth nutrition assessment and follow-up with a RD to help prevent weight gain and shifts in metabolism. A study at Columbia University found that education and counseling with a RD improved all categories of body mass index (BMI) [49]. In this study, 54% of overweight celiac patients and 47% of obese celiac patients lost weight after initiating a gluten-free diet [49]. Additionally, 66% patients who were underweight at diagnosis gained weight after a consult with a RD experienced in the gluten-free diet [49]. Cheng notes this further emphasizes the importance and impact of RDs in gluten-free diet adherence and quality of life. Expert diet counseling should encompass multi-factorial aspects of patient’s health, including obesity, high cholesterol, diabetes, and food allergies or intolerances. In 2012, Green stated, the “risk of weight gain needs to be addressed by the gastroenterologist, the primary care physician, and the registered dietitian” [50].In a study of more than 1000 patients over three years, 22% of patients that complied to the gluten-free diet gained weight, increasing their BMI >2 pts. At diagnosis, 20.5% were overweight and 11.5% obese [51]. Over three years on the gluten-free diet, 17% who started at normal weight crossed over into overweight or obesity. Fortunately, 19% who started out overweight shifted to normal weight [51]. In a study at the Cleveland Clinic, there was a two-fold increase in coronary artery disease in celiac disease versus non-celiac disease patients [52]. A study in Sweden found cardiovascular disease risk increased 60% after diagnosis of celiac disease [53]. This is of interest because systemic inflammation, as well as a healed small bowel, may result in elevated cholesterol levels.A study of over 1200 celiac patients found 23% considered as non-responsive celiac disease (NRCD) [54]. This study provided diet education to non-responders by a RD on a strict, gluten-free diet termed the “gluten-contamination elimination diet.” The diet emphasized avoidance of processed foods and minimized risk of cross contamination. After initiation of a strict gluten-contamination elimination diet, there were significant changes. Eleven of 14 subjects experienced resolution of symptoms, and were able to return to a normal gluten-free diet without issues. Five of six patients with Marsh 3 lesions on biopsy had resolution of symptoms and were no longer considered to have refractory celiac disease (RCD). Another recent study showed that in 90% of patients considered NRCD; the problem was due to continued gluten ingestion [55]. If a strict gluten-free diet does not resolve symptoms, it is important to consider a consultation with a RD to rule out obvious or inadvertent sources of gluten. If antibody levels are improving or normal but symptoms persist, consider other reasons for persistent symptoms. These considerations may include fermentable food molecules, small intestinal bacterial overgrowth, lactose or fructose intolerance, microscopic or collagenous colitis, irritable bowel syndrome (IBS), inflammatory bowel disease (Crohn’s disease or ulcerative colitis). There is often an overlap of IBS with celiac disease. A meta-analysis of over 3000 patients found that 40% of those with celiac disease also experienced IBS [56]. Paarlahti et al. found that 25% of patients suffered from persistent symptoms despite following a strict gluten-free diet. Approximately 78% suffered from depression [57]. Anxiety, stress and depression can affect the ability to follow a strict gluten-free diet. Verdu suggests an overlapping pathophysiology of celiac disease, IBS, gluten sensitivity and other food intolerances [58].Many patients experience gas, bloat, reflux, constipation and loose stools, despite initiation of the gluten-free diet. Some may have a heightened awareness of symptoms after initiating a gluten-free diet. Often, gluten-free sweets, granola bars, or other foods have added sugars or sugar alcohols to provide an acceptable product. Some are high in fermentable carbohydrates, which if malabsorbed could mimic symptoms of celiac disease. Fermentable dietary molecules (FODMAPs) are, “widespread in the diet and comprise monosaccharide (fructose), disaccharide (lactose), oligosaccharides (fructans and galactans), and polyols. Their ingestion increases delivery of readily fermentable substrate and water to the distal small intestine and proximal colon, which are likely to induce luminal distension and induction of functional gut symptoms” [59].In the area of gluten-free oats, there may be opportunity for oat cultivators to develop hybrid oats that are less likely to exacerbate the inflammatory response in celiac disease. A study out of Italy assessed duodenal biopsies to determine which cultivars of oats caused an inflammatory response similar to wheat. Gliadin-induced transglutaminase-2 (TG2) was measured on the epithelial lining to determine reactions. Three types of cultivars were reviewed: Nave, Potenza and Irina [60]. Nave cultivars were the only type of oat to significantly cause inflammatory response to TG2. This may help many to vary the gluten-free diet, especially those avoiding gluten-free oats due to overt symptoms.In the future, RDs can instruct celiac patients to enjoy and follow an adequate, balanced gluten-free diet [61]. Inclusion of education regarding weight management, heart healthy diet, low FODMAP, and consideration of IBS in celiac disease management is appropriate to avoid concomitant diseases or concerns in the future. Food companies and restaurants certainly have a role to play in the future to ensure that foods are properly tested to provide safe gluten-free products to consumers.Education about the gluten-free diet and its potential pitfalls are essential for successful adherence. Often, patients are not satisfied with their physician or RD visit when diagnosed. Unfortunately not enough RDs are specialized in the education of a strict gluten-free diet. This is a gap in the medical system, as coping strategies are not consistently included in medical or nutritional plans [62].In addition, the way patients and their families perceive the diagnosis of celiac disease affects compliance with diet. Anxiety, depression and fatigue are often linked with celiac disease before and after diagnosis [27]. Patients should have access to support groups; whether online or a local group to provide additional support. Adherence to the gluten-free diet with adequate symptom control improved health related quality of life [63]. Lifelong commitment of a strict gluten-free diet affects emotional and cognitive aspects, and interpersonal relationships. A recent study found a significant correlation between non-compliance and depression with a decrease in quality of life [64]. Barriers of compliance could minimize the therapeutic effect of the gluten-free diet. This could be likened to Type 2 Diabetes; as patients may not follow recommendations, despite education, including risks and complications. Not all are ready or willing to go gluten-free, especially those that may not have overt symptoms.Patients on a gluten-free diet perceive limitations on their social life including travel, eating out, and navigating food at parties. A study of approximately 1700 patients found celiac disease significantly affected quality of life, including economic, social and physiological aspects [65]. An economic burden falls on patients and families, as children and adolescents are not able to consume foods often provided at school and require additional steps to ensure gluten-free status of institutional food. Guandalini noted that “Effective communication between the physician and the patient is essential in minimizing the disease burden of celiac disease, particularly in young patients” [66].Motives to maintain a strict gluten-free diet vary significantly from patient to patient. A recent study found a significant correlation of following the gluten-free diet in those diagnosed with celiac disease with improvement of general physical symptoms and the feeling of hitting “rock bottom” with symptoms [61]. Patients aware of long-term health consequences maintained the diet better. “Fear was the motivation to start and stick with the diet.” It is not simple to understand the myriad of factors surrounding motivation [61]. This provides opportunity for clinicians, as well as basis for education at diagnosis to understand the disease and concerns of not remaining gluten-free.The gluten-free diet is the only treatment for celiac disease. There are many factors to ensure adherence to the diet. Proper education by an expert registered dietitian, support, and follow-up are necessary components to aid in permanent lifestyle change. Education on balanced and varied gluten-free diets is pertinent, as many fall into a holding pattern with gluten-free choices. The safety and consistency of gluten-free products has advanced, but more progress needs to be made to ensure validity of testing. The gluten-free diet has evolved, providing future opportunities for clinicians, consumers, restaurants and the food industry.The author would like to acknowledge Ronit Rose, Elizabeth Wall, RD and Stefano Guandalini, MD for their valuable time, assistance editing and reviewing the manuscript. The author declares no conflict of interest.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Recently, new therapeutics have been developed for hepatocellular carcinoma (HCC). However, the overall survival rate of HCC patients is still unsatisfactory; one of the reasons for this is the high frequency of recurrence after radical treatment. Consequently, to improve prognosis, it will be important to develop a novel anti-tumor agent that is especially effective against HCC recurrence. For clinical application, long-term safety, together with high anti-tumor efficacy, is desirable. Recent studies have proposed menahydroquinone-4 1,4-bis-N,N-dimethylglycinate hydrochloride (MKH-DMG), a prodrug of menahydroquinone-4 (MKH), as a promising candidate for HCC treatment including the inhibition of recurrence; MKH-DMG has been shown to achieve good selective accumulation of MKH in tumor cells, resulting in satisfactory inhibition of cell proliferation in des-γ-carboxyl prothrombin (DCP)-positive and DCP-negative HCC cell lines. In a spleen-liver metastasis mouse model, MKH-DMG has been demonstrated to have anti-proliferation and anti-metastatic effects in vivo. The characteristics of MKH-DMG as a novel anti-HCC agent are presented in this review article.Intracellular levels of vitamin K and its homologs are significantly lower in most hepatocellular carcinomas (HCCs) as compared with background non-tumor areas [1]; vitamin K-dependent carboxylation reactions are impaired in HCC cells. Vitamin K can inhibit the growth of HCC cells in a dose-dependent manner. However, vitamin K uptake is lower in HCC cells relative to normal hepatocytes in vitro [2]. These findings support the hypothesis that differences in the ability to absorb vitamin K lead to the individual sensitivity of HCC to vitamin K. Nowadays, the vitamin K2 (menaquinone) homolog menaquinone-4 (MK-4) is used for the treatment of osteoporosis, and its long-term safety has been confirmed [3,4,5,6]. There is a possibility that MK-4 can suppress the progression of HCC [7,8,9,10,11,12]. In some small-scale studies, MK-4 treatment has been reported to reduce the onset of HCC in patients with liver cirrhosis and HCC recurrence after curative surgical resection or radiofrequency ablation [13,14]. Indeed, MK-4 is expected to inhibit de novo carcinogenesis, HCC proliferation and HCC recurrence with long-term safety. However, no significant inhibiting effect was proven in a large-scale, double-blind and randomized control study [15]. A meta-analysis of randomized controlled trials, failed to confirm significantly better tumor recurrence-free survival at one year, and there was no beneficial effect on the overall survival [16].Because the anti-HCC effect of MK-4 may be dependent on the delivery of its metabolite, menahydroquinone-4 (MKH), it is hypothesized that effective delivery of MKH to HCC cells leads to inhibition of HCC proliferation, metastasis and recurrence. However, MKH itself has easily oxidizable characteristics and is unsuitable for clinical use. In our previous studies, menahydroquinone-4 1,4-bis-N,N-dimethylglycinate hydrochloride (MKH-DMG), the ester derivative of MKH, showed excellent MKH delivery potential regarding the liver without the reductive activation process of MK-4 to MKH [17,18]. To improve the prognosis of patients with HCC, an agent with high anti-tumor effectiveness and a good safety profile needs to be developed. Recent studies suggest that MKH-DMG has promising anti-HCC characteristics, such as intracellular MKH delivery and inhibition of tumor progression [19].The schema for the conversion process associated with the MKH delivery system in HCC cells is shown in Figure 1. Des-γ-carboxy prothrombin (DCP) also known as the protein induced by vitamin K absence-II (PIVKA-II), an abnormal prothrombin with incomplete carboxylation, is a HCC-specific tumor marker [20,21]. Additionally, DCP functions as a predictive factor for vascular invasion, metastasis and tumor progression; it is associated with the poor prognosis of HCC patients [22,23,24,25,26,27]. The vitamin K content in HCC cells has the ability to restrict DCP production [1,28,29,30]. MKH, a fully reduced form of MK-4, is a cofactor of γ-glutamyl carboxylase (GGCX) that converts glutamate residue into the γ-carboxyglutamate residue of vitamin K-dependent proteins such as prothrombin [31,32,33]. Therefore, ancillary to vitamin K-dependent carboxylation, MKH is stoichiometrically converted into menaquinone-4 epoxide (MKO). Utilization of MKH is restricted in HCC tissue. Because MKH availability regulates the rate of carboxylation [34], reduction of MKH availability in HCC cells may result in the increase in DCP.Schema of MKH delivery system in HCC cells. MKH-DMG, menahydroquinone-4 1,4-bis-N,N-dimethylglycinate hydrochloride; MK-4, menaquinone-4; MKH, menahydroquinone-4; MKO, menaquinone-4 epoxide; DCP, des-γ-carboxyl prothrombin; PIVKA-II, protein induced by vitamin K absence-II; VKORC1, vitamin K 2,3-epoxide reductase complex subunit 1; VKORC1L1, VKORC1 like-1; HCC, hepatocellular carcinoma.MKH-DMG, an MKH prodrug, is hydrolyzed into MKH by esterase after its uptake by cells. GGCX is required for its activity and depends upon MKH, which is generated mainly by vitamin K 2,3-epoxide reductase complex, subunit 1-like 1 (VKORC1L1). VKORC1L1 promotes the reduction of MK-4 to MKH and supports vitamin K hydroquinone-mediated intracellular antioxidation, which is critical for cell survival [35]. In our previous studies, MKH-DMG effectively supplied MKH to HCC cells without reductive activation [17,18].MKH-DMG has an inhibitory effect regarding cell proliferation in HCC cell lines in vitro. In our cell viability assay, MKH-DMG suppressed the proliferation of HCC cells in a time- and dose-dependent manner both in DCP-positive (PLC/PRF/5 and Hep3B) and DCP-negative (SK-Hep1) cell lines [19]. A rapid and intensive suppression effect was evident at 48 h after MKH-DMG administration at a concentration of 20 μM (Table 1). In contrast, the effect of MK-4 was clearly weaker and the suppression of cell proliferation was not distinct until 72 h at a concentration of ≥40 μM (Table 1). MKH-DMG showed significantly lower IC50 values (14–37 μM) and exhibited 4–18 times stronger proliferation-suppressing activity than MK-4. When cell injury was estimated by LDH release into the medium, no indication of injury was evident during MKH-DMG treatment. MK-4 is usually prescribed at a dose of 45 mg/day (three times per day) with a good long-term safety profile in the treatment of osteoporosis patients. At this dose, the maximum plasma concentration is 1 μM [36]; in a previous distribution study of oral MK-4 administration, the hepatic concentration was >10 times higher than the plasma concentration [37]. These results indicate that the IC50 values of MKH-DMG used in the treatment of HCC cell lines may be clinically reasonable levels, and that MKH-DMG is potentially a promising anti-HCC agent available for safe clinical use.Effects for HCC cells (Reference [19]).* MKH-DMG was dissolved in drinking water (40 μmol/L) and provided ad libitum.Inhibition of HCC proliferation, metastasis and recurrence by MKH-DMG and MK-4 are determined by their ability to deliver MKH to HCC cells. It is difficult to measure MKH levels accurately in HCC cells because of its highly oxidative characteristics regarding MK-4. However, MKO levels can be substituted for MKH levels (Figure 1). MKH delivery via MKH-DMG or MK-4 (both at a concentration of 25 μM) to HCC cell lines has been estimated by means of intracellular MKO and MK-4 levels [19]. MKH-DMG treatment induced a rapid and time-dependent increase in intracellular MKO and MK-4 levels (Table 1). In this case, MK-4 is an oxidation product of MKH (Figure 1). MKH-DMG is taken up by HCC cells and effectively converted into MKH. Conversely, MK-4 administration did not lead to an increase in MKO and MK-4 levels (Table 1), which supports the evidence that the MK-4 uptake rate was lower in HCC cells relative to normal hepatocytes [2]. When the AUC of the intracellular concentration versus time profile was determined, the AUC0-72 h values for MKH were 3.5–15 times higher after MKH-DMG treatment than those after MK-4 treatment, regardless of whether DCP-positive or DCP-negative cell lines were used. These results indicate that MKH-DMG administration is an effective method for the delivery of MKH to HCC cells.MKH-DMG as an MKH prodrug shows satisfactory cell-membrane permeability and is effectively hydrolyzed into MKH by esterase present in HCC cells, leading to the rapid and intensive inhibition for HCC proliferation. Perhaps the uptake process for MKH-DMG is different to that for MK-4. Investigation of MKH-DMG uptake including transporter system may be an important target for future research.In a recent investigation, DCP/PIVKA-II was reported to act as a growth and metastasis factor for HCC, and to exacerbate its prognosis [27]. Therefore, depression of DCP may be a prospective target for developing a novel treatment against DCP-positive HCC. This therapeutic strategy is applicable to MKH-DMG administration, which can achieve sufficiently high MKH delivery. The effect of MKH-DMG regarding the DCP level in a DCP-positive PLC/PRF/5 cell line was evaluated by measuring the DCP concentration in culture media at 72 h after treatment [19]. DCP levels had clearly decreased after MKH-DMG (10 μM) and MK-4 (10 μM) treatments (2.0 ± 0.0 mAU/mL, and 1.3 ± 0.6 mAU/mL, respectively) relative to untreated controls (43 ± 3.6 mAU/mL). However, as described above, cell proliferation was only suppressed after MKH-DMG treatment and not after MK-4 treatment. Because growth inhibition caused by MKH-DMG has also been demonstrated in DCP-negative HCC cells, DCP suppression may not be important for MKH-DMG and MKH regarding the inhibition of HCC growth. Other vitamin K-dependent proteins, with the exception of DCP, may be critical regarding the effect of MKH-DMG, and such vitamin K-dependent proteins should be explored.The underlying mechanism concerning the effect of MKH-DMG on HCC cell growth suppression should be investigated. Generally, cell-cycle arrest has been considered to be primarily associated with the anti-proliferative action of MK-4 [7,8,9,11,37,38]. Aberrant expression of NF-κB is linked to cyclin D1 [10], and to the onset and progression of HCC tumorigenesis [38]. Because both cyclin D1 and NF-κB are associated with cellular migration [39,40], downregulation of NF-κB and cyclin D1 by MKH-DMG treatment may contribute to the inhibition of HCC cell growth and invasion.In our analysis, it is suggested that G1/S arrest is one of the mechanisms involved in the inhibition of HCC cell proliferation induced by MKH-DMG [19]. Flow cytometric analysis of MKH-DMG-treated PLC/PRF/5 cells in vitro revealed an increase in G1 phase cells and a decrease in S phase cells. In Western blot analysis, the expression of cell cycle-related proteins (cyclin D1, cyclin D3 and CDK4) decreased and was completely suppressed after 48 h of MKH-DMG treatment in both DCP-positive (PLC/PRF/5 and Hep3B) and DCP-negative (SK-Hep-1) HCC cell lines. Conversely, similar treatment using MK-4 induced only a slight decrease in the levels of these proteins. Additionally, NF-κB was downregulated by MKH-DMG treatment in these HCC cell lines, which might have resulted from effective MKH delivery to the tumor cells using MKH-DMG.The clinical benefit of MKH-DMG treatment has been demonstrated using xenografted human HCC cells in vivo [19]. In a preceding pharmacokinetic study after oral administration of MKH-DMG, MKH-DMG was found to be delivered to and absorbed by hepatocytes in the same esterized form, and then converted to MKH in vivo. Using this MKH delivery system, the effects of MKH-DMG in combating hepatic metastasis and the proliferation of HCC cells (PLC/PRF/5) were examined in a mouse spleen-liver metastasis model (nu/nu mice). Macroscopic findings of liver tumors were estimated at 50 days after transplantation of PLC/PRF/5 cells in MKH-DMG treated and untreated groups. Increases in both liver weight and the percentage of cancer surface area were significantly suppressed in the MKH-DMG treatment group as compared with the untreated group. In the MKH-DMG treatment group, plasma DCP, which is produced by HCC cells, was not detected, although liver metastasis could not be suppressed completely. These findings mean that MKH is definitely delivered to the metastasized HCC cells and that DCP is suppressed by MKH; they suggest the possibility that MKH-DMG could function as an anti-HCC agent in humans. However, subsequent investigation is required to show whether or not a similar effect can be demonstrated after MKH-DMG treatment of human HCC.The effects of MKH-DMG and MK-4 regarding HCC cells are summarized in  Table 1. Reduced uptake of therapeutic agents by cancer cells is one of the important factors affecting resistance to chemotherapy. Consequently, the development of compounds with tumor-specific cell penetration properties is still a primary focus of cancer research. Effective supply of MKH, an active form of MK-4, is a promising approach concerning the suppression of HCC growth with low toxicity. MKH-DMG, a MKH prodrug in esterized form, can deliver substantial amounts of MKH to HCC cells, and can suppress HCC proliferation effectively both in vitro and in vivo. The anti-HCC effect of MKH-DMG may be the result of cell-cycle arrest with downregulation of NF-κB and cyclin D1 expression. MKH-DMG is a promising new candidate for suppression of the onset of HCC, its recurrence and metastasis without significant adverse effects.We wish to thank Naoki Magario for his assistance with the DCP assay. All authors contributed to the submitted work. Especially, ME and JT wrote and supervised the review; KM prepared the figures.The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Over the last decades, improvement of medical and surgical therapy has increased life expectancy in Marfan patients. Consequently, the number of such patients requiring secondary interventions on the descending thoracic aorta due to new or residual dissections, and distal aneurysm formation has substantially enlarged. Surgical and endovascular procedures represent two valuable options of treatment, both associated with advantages and drawbacks. The aim of the present manuscript was to review endovascular outcomes in Marfan syndrome and to assess the potential role of Thoracic Endovascular Aortic Repair (TEVAR) in this subset of patients.Marfan syndrome (MFS) is an autosomal dominant disorder of the connective tissue caused by mutation in the FNB1 gene on chromosome 15, with high penetrance and varying phenotypic expression, which involves the cardiovascular, ocular and muscolo-skeletal systems [1]. The incidence is of 1/10,000 patients with no known gender or ethnic predilection. The gene encodes fibrillin 1 glycoprotein, which is essential for the formation of elastic fibers found in connective tissue, provides a scaffold for elastin deposition in the extracellular matrix, fundamental to maintain the integrity of the vessel wall and serves as a regulator of transforming growth factor beta (TGF-β) signaling [2]. The Ghent criteria, first reported in 1996 and reviewed in 2010 by Loeys et al., are currently utilized to diagnose the Syndrome [3]. Patients with MFS are prone to developing vascular complications such as aortic dilatation, dissection and rupture. The life expectancy of these individuals has increased tremendously, by 30 years, over the past 30 years [4]. This is due to medical management with β-blocker and AT1 antagonists therapy [5,6,7] but especially to improved surgical results of early prophylactic aortic interventions on the proximal thoracic aorta [8]. If in the early 1970s, the mean life expectancy for these patients was 32 ± 16 years, between 1972 and 1995 the median (50%) cumulative probability of survival was 48 to 72 years [8,9].In 1999, in a survey involving 10 major Marfan centers, Gott and colleagues [10] showed excellent surgical outcomes reporting a 30-day mortality rate of 1.5% for patients undergoing elective aortic root replacement. However, because of the progressive nature of these diseases, even after successful aortic root replacement, the number of MFS patients requiring secondary interventions on the descending thoracic aorta (DTA) or thoraco-abdominal aorta due to new or residual dissections, rupture and distal aneurysm formation has substantially increased [11].Open repair of the descending aorta in these patients is highly challenging and excellent surgical expertise is important to minimize catastrophic complications such a stroke, paraplegia or paraparesis, postoperative hematomas and death [12]. Higher complication rates have been reported by low-volume centers with fewer than four cases/year, where in-hospital postoperative mortality can be as high as 27%, compared to less than 10% recorded in some high-volume centers [13,14,15].Thoracic Endovascular Aortic Repair (TEVAR) has emerged as valid alternative to open surgery in the past decades gaining wide acceptance in the treatment of acute and chronic type B aortic dissection, considering the unsatisfactory results of open repair. However, the role of TEVAR in MFS remains a frequently debated issue.To date, only small series of MFS patients treated with endovascular approach have been published. Baseline and follow up results of these studies are summarized in Table 1 and Table 2.Ince [16] and coll. reported a series of six consecutive MFS patients treated with TEVAR. Indications for intervention were chronic type B dissections after previous aortic root repair in five patients and solitary type B dissection in one patient. Talent stent grafts were successfully deployed in all cases, with no 30-day mortality or 1-year procedure-related mortality. Complete aortic remodeling was observed in two cases. At a mean follow up of 51 months three patients required open conversion at 12, 22 and 43 months. One patient with a known dilated aortic root suddenly died after 24 months, and another was reported under surveillance for retrograde false lumen flow without aortic diameter increase.Thoracic Endovascular Aortic Repair (TEVAR) in Marfan Syndrome: Preoperative status and early outcomes.NA: Not avaible.TEVAR in Marfan Syndrome: Follow up.NA: Not avaible.Nordon [17] and coll. reported on seven MFS patients undergoing TEVAR for aneurismal chronic dissection after previous aortic root repair. Technical success was 100%, one patients (14%) died due to pneumonia and irreversible cardiac failure. No perioperative adverse neurologic events occurred. At a median follow of 16 months, two patients developed endoleaks successfully managed within the first year by additional stent graft deployment. However, at imaging follow up, all patients demonstrated continued thoracic aorta dilatation with an alarming average of 7 mm per year.Geisbusch [18] and colleagues treated 167 patients with TEVAR between 1997 and 2007. Of them, eight patients presented with a diagnosis of connective tissue disease (six Marfan and two Ehler-Danlos). The pathologies included thoracic and thoracoabdominal aortic lesions (one degenerative aneurysm and seven chronic expanding aortic dissections). Technical success was 88% (one primary type one endoleak). There were no operative deaths. At a median follow up of 31 months, seven (88%) were alive. Endoleaks were observed in three patients after endovascular treatment of residual chronic type A dissection. Half of patients showed progression of disease with de novo aneurysms or aortic expansion. No patients needed early or late conversion to open procedures, but three (38%) underwent endovascular re-interventions.Botta [19] and colleagues investigated the feasibility and outcomes of TEVAR of the descending aorta in 12 MFS patients already submitted to open aortic root/arch surgery. Stent graft procedures were performed urgently in five patients and electively in seven. Neither in-hospital death nor adverse neurologic events occurred. At a median follow up of 31 months, one patient underwent open surgical conversion for persistent type 1 endoleak about 3 months after endovascular repair. Two patients showed extension of the dissection at 1 and 24 months after the procedure. No late death or aortic rupture was observed.Marcheix [20] and coll. analyzed short and mid-term outcomes of 15 MFS patients with chronic aortic dissection from the 457 enrolled in the European Talent registry. Eleven patients had previously aortic root surgery. Thirteen patients with progressive descending aortic dilatation were treated electively. Two patients were treated urgently due to impending aneurismal rupture. Technical success was achieved in all cases, and no conversion to open surgery was required during the procedure. No deaths or paraplegia occurred. One patient suffered a transient hemispheric stroke. Primary endoleaks were detected in five patients (type 1, n = 4; type 2, n = 1). The primary type 2 endoleak spontaneously healed. Of the remaining four patients with type 1 primary endoleak, one successfully underwent secondary conversion to open surgery one month after TEVAR, one died after endovascular reintervention, and two died of rupture of the descending aorta or of the abdominal aorta (within post-operative day 335) before scheduled reinterventions. After a mean follow up time 2.1 years, 12 patients were alive. Of them, five successfully underwent secondary conversion to open repair and seven showed different degrees of false lumen thrombosis.Waterman [21] and coll. examined the outcomes in 16 patients with MFS undergoing TEVAR between 2000 and 2010. Fifteen patients had previous surgery of the ascending aorta or arch. Chronic dissection and/or aneurysmal dilation of the descending aorta were the indications for elective intervention in 13 patients. Two acute dissection/malperfusion cases and one anastomotic disruption early after an open surgery led to emergency intervention in three patients. Six patients (38%) had successful therapy and required no subsequent interventions over a median follow up of 6.8 months. Seven (44%) patients experienced primary treatment failure defined as type 1 endoleak, persistent false lumen flow in the stented aorta leading to aneurysmal degeneration, and/or need of subsequent open or endovascular interventions. Five of these patients required open conversion. Five patients died during follow-up. Of them, two died perioperatively: the patient who underwent emergent TEVAR for anastomotic disruption, and a patient who required multi-visceral revascularization in conjunction with a second TEVAR. Two patients died following discharge: one because of a respiratory failure at 3 months, and the other because of a cardiac arrest after 4 months. The remaining patient died more than 6 years after EVAR from advanced age (84 years).Eid-Lidt and coll [22] evaluated mid-term follow-up in 10 Marfan patients with acute aortic syndrome complicating a chronic type B dissection (four contained rupture, one malperfusion syndrome and five acute aortic expansion). Five patients had previous surgical procedure. Technical endovascular success was achieved in all patients. In-hospital mortality was 10% (one patient died of aortic rupture 5 days after TEVAR during an open surgical repair). There were no cases of paraplegia or paraparesis. One patient had a transient ischemic attack 2 days after TEVAR without recurrence and one patient had vascular access complications. At a mean follow-up of 59.6 months, the cumulated mortality was of 20% and late mortality 11.1%. The rate of secondary endoleak was 44.4% and late reintervention of 33.3%. Survival freedom from cardiovascular death at 8 years was 80.0% and positive remodeling was documented in 37.5% of patients.Despite the small number of patients and the heterogeneity of treated diseases (acute and chronic dissection and aneurism), common finding of these studies are the feasibility of endovascular technique in MFS patients with percentage of success nearly to 100% with low morbidity and mortality rates. However, mid term follow up data reveal a high reintervention rate due to primary and secondary endoleaks and reduced rate of positive aortic remodeling. Despite major improvement in interventional materials and techniques, the increase in circumferential stress of the vessel wall created by the radial force of the stent graft might be deleterious in this clinical setting where a particularly fragile aortic wall is usually encountered. Landing a stent graft in the native aorta in patients with connective tissue disease is subjected to early and late complication. In a series reported by Dong et al. [23] the most common complication in MFS patients was retrograde aortic dissection (rTAD). This was confirmed in an analysis from the European registry on endovascular aortic repair complications, where the presence of a connective tissue disorder accounted for 8.4% of the underlying disease [24]. Consequently, it is prudent to consider TEVAR in MFS patients only with devices without proximal bare springs, if logistically feasible. Also, in a report of 650 patients treated for type B aortic dissection with endovascular approach, 22 events of stent graft–induced new entry (SINE) tears occurred. The mortality of the new tears was substantial, with nearly 30% of patients dying from the event. The incidence of SINE in the patients with MFS was 33%, which was significantly higher than the 3% among non-Marfan patients [25]. In general, patients with genetically triggered aortic diseases are excluded by the criteria currently approved by the US Food and Drug Administration for using commercially available thoracic endovascular devices to treat aneurysms in the descending thoracic aorta. In addition, the expert consensus document for endovascular therapy in patients with descending thoracic disease does not recommend stent grafts unless conventional repair poses a prohibitive risk [26,27]. More recent in a position statement [28] TEVAR is not recommended in patients with connective tissue disease except as a bail-out procedure or bridge to definitive open surgical therapy, or as a procedure following prior aortic repair when both landing zones lie within previously sited prosthetic grafts. Finally, last European guidelines on diagnosis and treatment of aortic diseases say that, in case of Marfan disease, surgery should be preferred over TEVAR (class IIa, level of evidence C), except in emergency situation in order to get initial stabilization as a bridge to definitive surgical therapy [29]. In summary, TEVAR could be an option in patients with absolute contraindication to open thoracic aorta surgery or, in selected patients, when the graft can be placed within a proximal and distal prosthetic graft landing zone. Certainly, endovascular treatment remains important in the emergency conditions, such as complicated type B acute aortic dissections and ruptured true or false aneurysms for which surgical therapy is associated with sobering outcomes and rapid control of the disease is essential to increase patients’ survival. Finally, close clinical and imaging surveillance is mandatory to detect early stent graft complications and/or disease progression of the native aorta.In MFS patients, the management of the aortic pathology is complex and often requires remedial procedures. Open surgery remains the mainstay of the treatment but endovascular therapy can provide a useful adjunct or bridge to open surgical treatment in anatomically suitable patients. However, failure of endovascular therapy is common, and its use should be judicious with close follow-up to avoid delay if open surgical repair is required.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Metabolomics is the study of low molecular weight molecules or metabolites produced within cells and biological systems. It involves technologies such as mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) that can measure hundreds of thousands of unique chemical entities (UCEs). The metabolome provides one of the most accurate reflections of cellular activity at the functional level and can be leveraged to discern mechanistic information during normal and disease states. The advantages of metabolomics over other “omics” include its high sensitivity and ability to enable the analysis of relatively few metabolites compared with the number of genes and messenger RNAs (mRNAs). In clinical samples, metabolites are more stable than proteins or RNA. In fact, metabolomic profiling in basic, epidemiologic, clinical, and translational studies has revealed potential new biomarkers of disease and therapeutic outcome and has led to a novel mechanistic understanding of pathogenesis. These potential biomarkers include novel metabolites associated with cancer initiation, regression, and recurrence. Unlike genomics or even proteomics, however, the degree of metabolite complexity and heterogeneity within biological systems presents unique challenges that require specialized skills and resources to overcome. This article discusses epidemiologic studies of altered metabolite profiles in several cancers as well as challenges in the field and potential approaches to overcoming them.Metabolomics, the study of metabolites produced in the body, has the potential to be useful in identifying novel diagnostic biomarkers and understanding cancer etiology. Metabolomics is considered most closely related to a patient’s phenotype [1,2]. The metabolome is the complete set of small-molecule metabolites that are found in a biological sample, and the human metabolome contains approximately 2500 metabolites [3]. Like the transcriptome, epigenome, and proteome, the metabolome is dynamic and changes over time. Metabolomics is the quantitative assessment of endogenous metabolites within a biologic system. Knowing a metabolome may allow early cancer detection and diagnosis or it may be a predictive and pharmacodynamic marker of drug effects. Metabolic and molecular imaging technologies, such as positron emission tomography (PET) and MRI, enable noninvasive metabolic marker detection. The HIF-1 pathway affects response to radiotherapy by HIF-1 protection of vasculature after irradiation and the regulation of glycolysis and the pentose phosphate pathway, thus increasing tumor antioxidant capacity. [18 F]-fluorodeoxyglucose-PET images can be used to quantitatively determine glucose metabolic rate and pharmacokinetic rate constants in tissue volumes, which is useful for the radiotherapy pharmacokinetic analysis conducted to determine the rate constants of the fluorodeoxyglucose metabolism in 41 patients (104 lesions). The highest glucose metabolic rate tumor regions had high cellular uptake and phosphorylation rate constants with relatively low blood volume. In regions with less metabolic activity, the blood volume fraction was higher and cellular uptake and phosphorylation rate constants were lower. Thus, the tumor glucose phosphorylation rate was not dependent on nutrient transport [4,5].Metabolomics may prove useful in following the effects of pathophysiological stimuli, as metabolomic profiling reflects changes that occur during disease development, progression, and response to therapy. Metabolomic biomarkers have been used in cancer surveillance [4]. Technologies such as mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) can be used to analyze samples that are isolated from the biofluids and tissues of cancer cases and controls. Compared to genetic and proteomic approaches, metabolomics generates more complex data. Koo et al. proposed the construction of metabolomic association networks that use high-dimensional MS data [5,6]. Tissue-specific metabolites have been identified in different cancer samples [1]. It is important to note that metabolomic markers themselves are not considered to be tumor-specific, but their altered correlations (representing different pathways) lead to concentrations and patterns of metabolomic intermediates and end products that are specific to either patients or healthy individuals. Both targeted and untargeted (or non-targeted) approaches are applied in metabolomics and epidemiology. The untargeted approach applies when no prior knowledge is available about the biomarkers to be identified; the methods applied are used to detect all possible metabolites. The targeted approach applies when a small number of suspected metabolites need to be quantified so that cases and controls can be distinguished. When using metabolomics, large sample sizes are needed for successful epidemiologic studies with minimum variability [7]. Cohort consortia with thousands of samples are an excellent resource for such studies. Furthermore, metabolomics is a high-throughput technique that is cost-efficient, fast, and adaptable for epidemiologic studies.This section describes examples of selected tumor types for which metabolomic and epidemiologic studies have been conducted. The information provided could be useful in stratifying patients for a variety of tumor types based on their metabolomic profiling.Bladder cancer is the fifth most common cancer in the world and has a high rate of recurrence. The surveillance protocol for this cancer involves urine cytology and cystoscopy every three months for two years, every six months for the next two years, and then yearly thereafter [8,9]. Promising markers for bladder cancer include bladder tumor antigen, nuclear matrix proteins, minichromosome maintenance protein, cystatin B, profiling B, and a number of glycosylated proteins. In one epidemiologic study, bladder cancer cases and controls could be distinguished based on liquid chromatography (LC)-MS metabolomic profiling [10]. In this study, carnitine transferase and pyruvate dehydrogenase levels were much higher in cases compared to controls. Zhang et al. reported on the use of urinary modified nucleosides as biomarkers for monitoring urothelial bladder cancer [11].Breast cancer accounts for the largest number of newly diagnosed cases in female cancer patients, and biomarkers are needed that can detect this cancer early and identify responders among patients undergoing treatment. In the United States, the number of breast cancer cases is projected to increase each year, which poses a burden for both the health care system and the economy. Tang et al. identified metabolites associated with breast cancer heterogeneity [12]. Gas chromatography (GC)-MS and LC-MS analysis of tumor samples characterized under The Cancer Genome Atlas (TCGA) project of the National Cancer Institute (NCI), National Institutes of Health (NIH), revealed that 18% of metabolites were present at higher levels in ER− breast cancer samples compared to ER+ samples. The main metabolites were glycolytic and glycogenolytic intermediates. Higher levels of glutathione in ER− breast cancer suggested the ability to handle oxidative stress and toxins better than ER+ breast cancer. In another study, the level of patient frailty was determined based on metabolomic profiling [13]. Some of the metabolites found to be altered in frail patients were amino acids, including serine, tryptophan, hydroxyproline, histidine, and its derivative 3-methylhistidine, cystine, and beta aminoisobutyric acid. A prediction model intended to discriminate between breast cancer patients who might respond to treatment from those who would not respond was proposed based on metabolomic profiling [14]. Another model was proposed based on lipidomics, in which lysophosphatidylcholine and sphingomyelin levels predicted cancer occurrence [15]. Such models might be useful in personalized and precision medicine.Although mammography has been successful in screening high-risk breast cancer patients, about 20% of cases remain undetected by this technology. It is hoped that metabolomic profiling combined with other detection technologies may improve the sensitivity and specificity of cancer detection and diagnosis. Toward this end, the role of chemokine receptor CXCR4 in breast cancer aggressiveness was demonstrated by Vermeer et al. [16].Colorectal cancer is one of the most prevalent and deadly cancers worldwide. Microbiome involvement, especially bacteria, has been proposed in the development of this cancer. Johnson et al. used colon microbiome metabolomics information to identify metabolites that can discriminate between healthy individuals and colon cancer patients [2]. Microbiota collected from the colon was analyzed for metabolites, and upregulation of polyamine and diacetylspermine metabolites was observed. Other investigators proposed using valine as a biomarker for detecting colon cancer in patients with polyps [17]. Holst et al. observed branched glycan elevation in colon cancer patient samples [18] and hypothesized that the interaction of proteins with colon cancer glycans contributes to carcinogenesis. Metabolomic profiling was shown to be useful in validating transcriptomic results of colon cancer samples when transcriptomic expression was functionally evaluated by metabolomic profiling [19]. Other investigators found that a combination of NMR, chemomatrix analysis of colon cancer patient serum, and comparison with controls indicated higher levels of pyridoxine, orotidine, and taurocholic acid in colon cancer samples [20]. The major metabolomic cycles involved include bile acid biosynthesis, vitamin B6 metabolism, methane metabolism, and glutathione metabolism.Although more prevalent in Asian countries, gastric cancer incidence and prevalence are increasing in Western countries [4]. Metabolomic profiling has been proposed as an alternative screening method for gastric cancer because of the high cost of endoscopy [21]. 5-Hydroxyindoleacetic acid levels were found to be higher in gastric cancer patients compared to controls [22]. Urine metabolomics were applied in this study, and the authors suggested including this urine marker along with other gastric cancer markers to improve the sensitivity of diagnostic assays.Liver cancer is one of the most lethal malignancies. In the United States, the rate of liver cancer has increased by 7% during the past two decades [23]. Early detection biomarkers are needed that can be used to screen populations. The Kernel approach, a new metabolomic analysis approach, was used with liver cancer samples and reduced complications from missing values in epidemiologic studies [24]. Luo et al. analyzed 854 metabolite ion pairs in liver cancer samples and proposed a multiple reaction-monitoring ion pair finder that is useful in identifying new biomarkers [25]. Another study identified long-term elevated serum bile acids as potential risk factors for liver cancer [26]. Based on metabolomic profiling, Shao et al. identified butyrylcarnitine and hydantoin-5-propionic acid as biomarkers for diagnosing liver cancer [27]. Coen et al. used metabolomics profiling to evaluate the hepatotoxicity of different treatment agents [28]. Other investigators also have reported liver cancer-specific biomarkers [1]. Zheng et al. analyzed more than 100 serum samples from controls and liver cancer patients and identified tryptophan, glutamine, and 2-hydroxybutyric acid as early markers for liver cancer [29].Cigarette smoking is the major risk factor for lung cancer, although additional factors have been identified [30]. Chronic obstructive pulmonary disease (COPD) may increase the risk of lung cancer. Metabolomic approaches were applied to distinguishing COPD and lung cancer in serum samples collected from patients with one of these diseases (TNM stages I, II, III, and IV). Higher levels of acetate, citrate, and methanol were found in individuals with COPD compared to those with lung cancer; and N-glycosylated proteins, leucine, lysine, mannose, and choline levels were higher in those with lung cancer [30].Blood samples from patients with pancreatic cancer and cachexia were characterized by metabolomic approaches to identify contributing metabolites [31]. Two groups, one with and another without cachexia, were followed longitudinally. Serum levels of IL-6, tumor necrosis factor (TNF)-alpha, and leptons as well as loss of body weight were determined in both groups using GC-MS. Compared to the cachexia-free group, levels of these markers varied day to day and were higher in cachexia patients. Most patients with advanced stage pancreatic cancer develop cachexia with symptoms such as decreased dietary intake, anxiety, and depression.The incidence and prevalence of prostate cancer is very high in the United States and worldwide. Although prostate-specific antigen (PSA) is used for the diagnosis and prognosis of this cancer, the sensitivity and specificity of this antigen is low. It is difficult to make a clinical decision for treatment if PSA levels are lower than 0.4 microgram per mL. Metabolomic biomarkers with potential for use in diagnosing prostate cancer include sarcosine, proline, kynurenine, uracil, and glycerol-3-phosphate in urine [32]. These metabolites can be measured in longitudinally collected urine samples by LC-MS [33].Metabolomics currently involves a variety of challenges. For example, metabolomics data generally are complex and it has been observed that the data matrix frequently contains missing values, making quantitative analysis difficult. Peaks that are present in the chromatogram can be missed by investigators during peak picking. Zhan et al. proposed using the Kernel-based scoring approach to address missing data [24]. The Kernel method is available in the R statistical computing environment.Metabolomics requires the development of sophisticated and powerful statistical methodologies to make clinical observations easy to follow. These statistical approaches enable comparison of the abundance levels of a metabolite between cases and controls to assess their significance. Metabolomic association networks that use high-dimensional MS data have the potential to improve data analysis and use by investigators.Although the analytical platforms for metabolomic analysis are robust, sample pretreatment procedures differ among institutions such as clinics and hospitals. This contributes to systematically biased results. For example, when tumor tissues are collected, normal cells may be included in these samples and confound the analysis. Laser microdissection is a potential solution, but the procedure is highly technical, time-consuming, and expensive for epidemiologic studies. Fasting status shows different metabolomic patterns in serum versus urine collected from the same patient. Techniques that show minimum variability of metabolites in samples from patients under fasting or non-fasting conditions are needed. Intra-individual variations have been reported for nutritional status (food frequency) and physical exercise [34].In epidemiologic studies that seek to identify race- and ethnicity-specific metabolites, cohorts containing multiple ethnic groups are needed so that findings can be generalized to all populations. Additional challenges include: variability in metabolomic measurements and related implications; the need to create new techniques for analysis, computation, and interpretation; epidemiologists have limited training in metabolomics; the need to integrate metabolomic data with genomic, epigenomic, transcriptomic, and proteomic data; and the availability of highly purified standards.Opportunities include the fact that biofluids such as urine and blood can be collected noninvasively and are suitable for both epidemiologic and clinical studies. Applications of metabolomics in drug-resistant breast cancer cells were described recently [35], and similar approaches can be investigated in other cancers.Understanding the metabolic basis of cancer has the potential to provide the foundation for the development of novel approaches targeting tumor metabolism. Tumors characterized by aerobic glycolysis and/or glucose dependence could be more sensitive than other tumors to agents targeting the tumor vasculature and glucose transport. Tumors characterized by impaired TCA cycle function and/or respiration that is glutamine-dependent could be sensitive to agents targeting glutamine metabolism (such as glutaminase). Tumors that have impaired mitochondrial/electron transport function could be sensitive to agents that target the reductive carboxylation and fatty acid synthesis pathways. Malignancies that are characterized by IDH1, IDH2, FH, or succinate dehydrogenase mutations could affect TET2 function, resulting in hypermethylation phenotypes [36]. Such malignancies could be responsive to hypomethylating agents.Metabolites are the end products of biological regulatory and metabolomic processes, and they can be measured in biological fluids and tissues. Their levels can be regarded as the response of biological systems to genetic, lifestyle, and environmental changes. Molecular profiling based on metabolomic analysis may facilitate the stratification of patients with cancer into homogeneous biological groups to facilitate the clinical management of these patients. Similar to other omics biomarkers, metabolomic biomarkers should have high sensitivity and specificity. Because metabolites are closely related to a patient’s phenotype, their potential for use in disease diagnosis and prognosis is significant. Metabolomics has the potential to become a valuable tool for precision medicine.We thank Joanne Brodsky for her editorial assistance.MV contributed in developing the outline and preparing the first draft of the manuscript. HNB provided comments and finalized the manuscript.The authors declare no conflicts of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Metabolic syndrome is an ever-increasing health problem among the world’s population. It is a group of intertwined maladies that includes obesity, hypertriglyceridemia, hypertension, nonalcoholic fatty liver disease (NAFLD), and diabetes mellitus type II (T2D). There is a direct correlation between high triacylglycerol (triglyceride; TAG) level and severity of metabolic syndrome. Thus, controlling the synthesis of TAG will have a great impact on overall systemic lipid metabolism and thus metabolic syndrome progression. The Acyl-CoA: monoacylglycerolacyltransferase (MGAT) family has three members (MGAT1, -2, and -3) that catalyze the first step in TAG production, conversion of monoacylglycerol (MAG) to diacylglycerol (DAG). TAG is then directly synthesized from DAG by a Acyl-CoA: diacylglycerolacyltransferase (DGAT). The conversion of MAG → DAG → TAG is the major pathway for the production of TAG in the small intestine, and produces TAG to a lesser extent in the liver. Transgenic and pharmacological studies in mice have demonstrated the beneficial effects of MGAT inhibition as a therapy for treating several metabolic diseases, including obesity, insulin resistance, T2D, and NAFLD. In this review, the significance of several properties of MGAT physiology, including tissue expression pattern and its relationship to overall TAG metabolism, enzymatic biochemical properties and their effects on drug discovery, and finally what is the current knowledge about MGAT small molecule inhibitors and their efficacy will be discussed. Overall, this review highlights the therapeutic potential of inhibiting MGAT for lowering TAG synthesis and whether this avenue of drug discovery warrants further clinical investigation.TAG stores and transports fatty acids (FAs) as energy for fuel and provides substrates for lipid synthesis that maintain membrane phospholipid synthesis [1]. In mammals, adipose tissue stores TAG as a primary energy substrate to sustain animals during fasting. TAG also protects cells from lipotoxicity, provides natural ligands for nuclear hormone receptors, and is involved in cell signaling [2]. A hallmark of a variety of metabolic pathological conditions including obesity, insulin resistance, T2D, coronary heart disease, hypertriglyceridemia, and NAFLD is the excessive accumulation of TAG in human adipose and non-adipose tissue [3,4]. TAG-rich lipoproteins comprise a vast array of secreted particles from intestine and liver. Multiple clinical trials and models have established the relationship between elevated plasma TAG and increased risk of coronary and cerebrovascular ischemic events [5]. Mendelian randomization studies have also demonstrated a causal involvement of TAG-mediated pathways and conronary heat disease [6]. Limiting TAG production in humans provides a potential pharmacological intervention of these metabolic disorders.TAG synthesis and metabolism have been topics of study since the 1950s. The conserved pathway for TAG synthesis in all higher eukaryotes is the Kennedy or sn-glycerol-3-phosphate (G3P) pathway [7] (Figure 1). Here, G3P is acylated by glycerol-3-phosphate acyltransferase (GPAT) to produce lysophosphatidic acid (LPA). LPA is then acylated by acylglycerol-3-phosphate acyltransferase (AGPAT) to generate phosphatidic acid (PA), which is dephosphorylated by lipins/PA phosphatases (PAP) to generate DAG [8,9,10,11,12]. Finally, DAG is converted to TAG by DGAT. TAG can also be synthesized by a second distinct MAG-dependent pathway that is found exclusively in animals, playing a predominant role in dietary fat absorption in the small intestine [13,14]. Here, MAG is converted to DAG by MGAT enzymes (MGAT1/2/3), and like the G3P pathway, DAG is converted to TAG by DGAT. While the G3P pathway uses G3P as an initial acyl acceptor, the MAG pathway uses MAG. Interestingly, both pathways use acyl-CoA thioesters as acyl donors to produce DAG, and both converge at the final DGAT acylation step to produce TAG [15,16,17,18] (Figure 1). The endoplasmic reticulum (ER) is the main site for these reactions, but mitochondria can also synthesize TAG. Interestingly, lipid droplets can produce the TAG precursors LPA and PA [1,19] and it has been shown that dihydroxyacetone phosphate (DHAP) can be esterified to form the LPA precursor, 1-acyldihydroxyacetone-phosphate in all animal tissues and yeast [20,21].TAG is de novo synthesized in the liver and adipose tissue, while dietary TAG is broken down and re-synthesized in the small intestine. In the liver, TAG is used for very low density lipoprotein (VLDL) assembly. Newly formed VLDL is secreted into the circulatory system where it transports neutral lipids including TAG to peripheral tissues [1]. In the small intestine, dietary TAG is hydrolyzed by pancreatic lipase to FA and MAG that are re-absorbed in the intestinal lumen. Enterocytes then re-synthesize TAG and secrete it as ApoB-containing chylomicrons that deliver dietary fat to tissues [22]. Most tissues including liver and adipose use the G3P pathway for the synthesis of TAG. In contrast, the small intestine predominately relies on the MAG pathway [23,24,25,26].Schematic representation of the two triglyceride synthesis pathways using either MAG or G3P as the initial acyl acceptor. The G3P pathway is dominant in liver, whereas MGAT mediated MAG to TAG conversion is predominant in the intestine.As stated above, MGAT activity is essential for the biosynthesis of TAG in the small intestine. The fact that MGAT expression level is found to be elevated in humans with obesity, while hepatic MGAT expression is decreased in patients after Reux-en-Y gastric bypass (RYBG) surgery [27,28] indicates that MGAT itself is a major regulator of TAG homeostasis in response to diet. Below, the structural and functional aspects of the MGAT family of proteins (MGAT1, MGAT2 and MGAT3) will be discussed. Related acyltransferases (e.g., DGAT1) will be touched upon as to their molecular relationship with MGAT. Finally, the review will examine whether MGAT is a viable pharmacological target for the treatment of metabolic disorders.Monoacylglycerolacyltransferase (MGAT) is an ER bound enzyme [29,30,31].Three known MGAT genes, Mogat1, Mogat2 and Mogat3, were identified in the early 2000s based on their sequence homology with the DGAT2 gene [29,30,31,32]. In mammals, MGAT is involved in intestinal dietary fat absorption and TAG synthesis in suckling rat liver, as very little MGAT activity has been detected in adult rat liver microsomes [33]. Interestingly, MGAT genes share homology with DGAT2 but not DGAT1.MGAT1 was the first family member to be cloned from mouse liver in 2002 [31] based on sequence homology to DGAT2 [32]. Mouse MGAT1 is located on chromosome 1, while in humans it localizes to chromosome 2. MGAT1 contains a core amino acid sequence that shares homology to a domain found in phosphate acyltransferases, two putative N-linked glycosylation sites, and a possible tyrosine phosphorylation site. Interestingly, MGAT1 does have in vitro DGAT activity, as demonstrated using a radio-labeled DGAT activity assay. However, activity towards DAG is significantly less than DGAT2 [31]. Mouse MGAT1 expression is found to be highest in the stomach and kidney; it is also present at a lower level in white and brown adipose tissue, uterus, and liver. Importantly, MGAT1 expression is found to be absent in mouse small intestine.Human MGAT1 is also expressed in the stomach, uterus, kidney, adipose, and liver, but unlike the mouse, expression is seen in brain, lung, thymus, prostate, testes, colon, and notably, small intestine [31]. There are two identified splice variants of human MGAT1, the larger of the two is predominantly found in the thymus and testes [34]. Work from Hall et al., showed that the putative full-length MGAT1 transcript was not amplified in human liver, gall bladder, adipose, or intestine, which suggests the presence of multiple splicing variants [27].The function of MGAT1 outside the small intestine has yet to be elucidated. One possibility is that it plays a role in polyunsaturated FA preservation in tissues other than the small intestine, where fast turnover of TAG degradation and re-synthesis occurs [33]. For example, when FA is required as the major source of energy in suckling rat and chick embryo, despite the high rate of hepatic beta-oxidation, the long-chain polyunsaturated derivatives of FAs (e.g., C18:2n-6 and C18:3n-3) are selectively retained. The preference of MGAT activity for MAGs that contain C18:2 and C18:3 FA in the sn-2 position could lead to greater retention of essential FAs by preventing them from being used during β-oxidation [33]. In humans, the expression of MGATs is up-regulated in the livers of insulin-resistant patients who have nonalcoholic fatty liver disease (NAFLD) [27]. In the mouse, MGAT1 (the dominant isoform) expression and activity are increased in diet-induced obese (DIO) and ob/ob mice. This up regulation may be explained by increased expression of the hepatic peroxisome proliferator-activated receptor (PPAR)γ, which directly regulates MGAT1 promoter activity [35].Antisense oligonucleotide (ASO) knockdown of MGAT1 in mice fed a high trans FA, fructose, and cholesterol diet (HTF-C diet) prevented weight gain, improved glucose tolerance and hepatic insulin signaling, and decreased hepatic TAG content when compared to control ASO-treated mice fed the same diet (Table 1) [36]. Similar findings were observed for DIO and ob/ob MGAT1 ASO treated mice [37]. Interestingly, in spite of the metabolic beneficial effects due to knockdown of MGAT1, no reduction was seen in hepatocyte ballooning, macrophage infiltration, or stellate cell activation, nor did it attenuate the expression of inflammation markers [36]. Unexpectedly, the knockdown of MGAT1 increased total DAG content in both membrane and cytosolic compartments of hepatocytes [37]. The increased DAG accumulation was not due to compensatory increases in phosphatidic acid phosphatase or TAG hydrolase activities, but may have been due to decreased DGAT activity; as stated above MGAT1 does have DGAT activity [31]. Thus, although reducing intrahepatic lipid content will most likely protect the liver from the development of nonalcoholic steatohepatitis (NASH), inhibition of TAG synthesis by MGAT1 inhibition may not necessarily be beneficial, at least in mice. Important to this statement is the fact that MGAT1 protein expression is undetectable in humans. Thus, small molecule discovery targeting MGAT2 should not have to be concerned with potential cross-reactivity, selectivity issues, and toxicity associated with inhibiting MGAT1.The first mammalian MGAT2 was cloned and characterized in mice in 2003 [13], with cloning of the human MGAT2 coming shortly afterwards [30,38]. Human MGAT2 shares 81% identity with the mouse MGAT2 and 47% amino acid identity with DGAT2. MGAT2 and DGAT2 localize adjacent on chromosome 11q13.5, suggesting a possible duplication event. Human MGAT2 has a splice variant (hMGAT2V) that lacks MGAT2 activity.Human MGAT2 is primarily expressed in the small intestine and liver. It can also be detected in adipose and colon at a much lower expression level. hMGAT2V also shows expression in the small intestine, but what its molecular function is remains to be elucidated. MGAT2 is predominantly expressed in the small intestine and kidney in the mouse [13]. While MGAT2 is found in human liver, its activity is barely detectable in human liver microsomes. This finding was in line with reports that showed hepatic MGAT2 specific activity was very low in adult rat, chick, and guinea pig compared with neonatal rat, fetal guinea pig, and chick embryo [33,39,40,41]. Thus, hepatic TAG synthesis in adult mammals is thought to occur primarily via the sequential acylation of G3P. The lack of detectable MGAT activity in human liver may be due to the very high lipase activity in adult liver, which depletes the MAG substrate required for DAG synthesis by MGAT2 [42]. This is backed up by experiments using the lipase inhibitor, MAFP, which showed there was active MGAT2 activity in human liver microsomes [27]. There is still a debate whether this activity truly comes from MGAT2 or MGAT3, as MGAT2 protein cannot be detected in human liver microsomes [27]. Taken together, the majority of the present data indicates that MGAT2 activity is substantially lower in human liver than in the small intestine.Genetic Manipulation of MGAT1 and MGAT2 in Mouse models.n.c., no change; DIO, diet-induced obesity; HFD, high fat diet; n.d., not determined.Much information has been gleaned about MGAT2 function using transgenic mouse models (Table 1). Mogat2−/− mice are metabolically healthy, are resistant to high-fat diet induced obesity, have improved insulin sensitivity, and have decreased fat accumulation in the liver and adipose tissue [43]. Moreover, the deletion of MGAT2 increases the level of gut incretin glucagon-like peptide-1 (GLP-1), which has many beneficial effects in treating metabolic disorders including stimulating insulin secretion, reducing glucagon, slowing gastric emptying, and inducing satiety [45]. Interestingly, Mogat2−/− mice fed a high fat diet consume and absorb a similarly high percentage of dietary fat when compared to their wild-type litter mates on the same diet. The fat level in the feces of Mogat2−/− mice was not increased and vitamin A and E levels were normal, indicating MGAT2 is dispensable for fat absorption. Both whole body Mogat2−/− and intestine-specific deletion (Mogat2iko) mice exhibit significantly lower intestinal MGAT2 activity and have impaired intestinal TAG synthesis [44]. MGAT2 deficiency also slows gastric emptying and changes the kinetics of fat absorption.Mogat2−/− mice display an increase in energy expenditure when compared to their wild-type counterparts. Re-introduction of MGAT2 in the intestine of Mogat2−/− mice remediates the MAG absorption defect, thus allowing for the conversion of dietary MAG → DAG → TAG, and restores the rate to which dietary fat enters the circulation [46]. Mogat2iko mice also show an energy expenditure rate that is somewhere in between Mogat2−/− and wild-type mice [46]. Data from Nelson et al., indicated that the change in energy expenditure in Mogat2−/− mice was unrelated to high-fat diet consumption, as when these mice were fed a normal chow diet increased energy expenditure remained intact [47]. In contrast, data from Mul et al., showed that Mogat2−/− mice only increased their energy expenditure when fed a high-fat diet but not with normal chow [28]. Interestingly, Mogat2−/−mice have a preference for eating carbohydrate over fat, which correlates with an increase in locomotive activity, suggesting that shifting macronutrient preference away from fat may act as a protective mechanism against high-fat diet induced obesity [28]. Thus, targeting MGAT2 for inhibition may have beneficial effects on obesity.Adult-onset MGAT2 deficiency in mice brought on by expressing a tamoxifen-inducible Cre recombinase demonstrated that MGAT2 was involved in regulating energy balance. Somatic loss also protected mice against diet-induced weight gain, hepatic steatosis, and glucose intolerance. Just as important is the fact that inactivation of MGAT2 in diet-induced obese mice reduced their body weight and improved glucose tolerance [48]. This observation indicates that MGAT2 deficiency during early development is not required for the protective effects MGAT2 inactivation gives. However, germ line MGAT2 knockout mice do suffer from malnutrition and gain less weight during the suckling postnatal period. Moreover, the reduction in weight gain in MGAT2-inactivated adult mice is less than germ line MGAT2 knockouts. With that said, these results do indicate that targeting MGAT2 for small molecule discovery will be efficacious, and that adult patients with various metabolic disorders will respond to treatment.The accumulation of specific lipid intermediates, including DAG, acyl-CoA, and ceramide is thought to drive the progression of NAFLD in humans [49,50]. Although in the liver, most DAG and TAG are synthesized through the sequential acylation of G3P. MGAT2 may provide an alternative pathway for the generation of DAG. Unlike in mice, human MGAT2 mRNA expression can be detected in liver, as well as in small intestine [27,34]. While MGAT2 mRNA is highly expressed in pooled human liver RNA, MGAT2 protein expression is not detectable in human liver lysates among 200 pooled donor samples. In contrast, pooled human intestine samples have robust MGAT2 protein expression. This may suggest that liver MGAT2 expression may be under tight promoter regulation, or that the stability of MGAT2 protein is highly regulated due to the detrimental consequences of liver TAG accumulation [27]. Thus, the question of why the MGAT2 pathway is found in human liver still needs to be investigated. As stated above, and importantly, MGAT2 expression is up regulated in patients with obesity and NAFLD, and down regulated following gastric bypass surgery. This suggests that MGAT2 may have a positive role in lipid-related liver disease progression. As there are very few therapeutics out there to treat NAFLD, targeting of MGAT2 for inhibition may not just help in lowering TAGs, but may also potentially reduce or eliminate the progression of NAFLD, which ultimately progresses to NASH, and later cirrhosis.Among the three MGAT isozymes identified to date, the MGAT3 gene, Mogat3, is thought to only exist in higher mammals and not in rodents, one publication has documented its expression and function in rats (discussed below); murine Mogat3 is a pseudogene. Human MGAT3 was cloned in 2003 and it has been shown that over-expressing MGAT3 in baculo virus gives robust MGAT enzyme activity. Like MGAT2, MGAT3 appears to have substrate specificity for the acylation of 2n-monoacylgycerol over other stereoisomers [51]. MGAT3 was initially thought to be strictly expressed in the human intestine, however, it was shown later on that MGAT3 was expressed in human liver [27]. The sequence of MGAT3 is more homologous to DGAT2 than to MGAT1 or MGAT2.Thus, MGAT3 exhibits significantly higher DGAT activity than MGAT1 and MGAT2 when MAGs or DAGs were used as substrates, suggesting another gene duplication event giving rise toMGAT3 from DGAT2 [29].Besides being expressed in human intestine, MGAT3 expression has been seen in pooled human liver RNA and protein in cell lysates. MGAT3 expression level increases in patients with NAFLD and its level decreases after gastric bypass surgery-induced weight loss. The MGAT activity present in livers of obese human subjects correlated with Mogat3 gene expression, not MGAT2 [27]. This suggests MGAT3 might play a more important role than MGAT2 in obesity related hepatic insulin resistance and NAFLD progression in humans.As the function of murine MGAT3 has yet to be determined, its true in vivo physiological importance in TAG biosynthesis and absorption in mammal remains a mystery. While MGAT3 was reported to be functional in the rat [14], and its expression pattern in this rodent was shown to be similar to human MGAT3, knockout studies in rodents are lacking. Until then, the functional relationship between MGAT2 and MGAT3 will remain unanswered. For instance, can MGAT3 compensate for MGAT2 function when MGAT2 is inhibited? What is the substrate specificity of MGAT3, and how does that affect lipid metabolism? What is the role of MGAT3 in NAFLD progression? There are a number of excellent hypotheses that can be tested once transgenic animals are created.As discussed above, MGAT isoforms share sequence homology and similar substrate specificities with the DGAT family member, DGAT2. Two DGAT isozymes have been identified in mammals: DGAT1 and DGAT2 [32,52]. The DGATs do not share similar tissue expression patterns (DGAT1 is mainly expressed in adipose and small intestine; DGAT2 is expressed in liver). Moreover, DGAT1 does not share sequence homology with DGAT2, again suggesting duplicate functions between DGAT2 and MGATs. DGAT2 knock mice are lipopenic and die shortly after birth [53] due to profound reductions in substrates for energy metabolism and an impaired permeability barrier in the skin. Interestingly, DGAT1 expression was unable to compensate for the loss of DGAT2, indicating a fundamental role for DGAT2 in TAG biosynthesis, much more so than DGAT1. DGAT1 knockout mice were found to be viable and were protected from high-fat diet induced obesity and insulin resistance. For information concerning the biochemical function and physiological significance of each DGAT, the reader is referred to an excellent review of this material [54].Pharmacological interventions using DGAT1inhibitors have been developed as a therapy for obesity and T2D. These inhibitors have been tested in rodent and dog models and have been shown to delay gastric emptying and lower fat absorption, decrease postprandial plasma TAG, increase GLP-1 level, stimulate FA oxidation, reduce liver TAG, and improve insulin sensitivity while causing weight loss [55,56,57,58,59,60,61]. In human clinical trials, similar results were seen: a reduction in postprandial plasma TAG, an increase GLP-1 level, normalized insulin sensitivity, and weight loss. However, the beneficial effects of these inhibitors are often counterbalanced by intolerable gastrointestinal side effects including nausea, diarrhea, and vomiting. These adverse symptoms are thought to be the result of on target side effects; as unlike mice, humans appear to solely rely on DGAT1 activity for TAG synthesis in the small intestine, while DGAT2 can compensate for the loss of function of DGAT1 in DGAT1 deficient (Dgat1-/-) mice. The inhibition of human intestinal DGAT enzyme blocks TAG synthesis completely and has led to severe fat malabsorption, as observed in patients with a null mutation in the DGAT1 gene [62,63]. Thus, all gastrointestinal side effects were thought to be due to the severe malnutrition and buildup of DGAT substrates. For all these reasons, the use of DGAT1 inhibitors as anti-diabetes and/or anti-obesity agents remains uncertain. Currently, there are ongoing clinical studies using DGAT1 inhibitors (Novartis) to treat patients with familiar chylomicronemia [64].The biosynthetic pathways leading to TAG synthesis are still attractive targets for pharmacological intervention even in light of the side effects associated with DGAT inhibition. One possible problem that was not foreseen in inhibiting DGAT was that both pathways converge on the DGAT step for TAG synthesis. A more attractive target may be MGAT2. The thought is that MGAT2 inhibition in the small intestines is unlikely to have DGAT-like side effects and cause severe deficiency in TAG synthesis, as the alternative G3P pathway is active in human intestine. Because of the similar expression and function of MGAT2 in human and mice, MGAT2-knockout mice have been very useful in helping in the discovery of small molecule MGAT2 inhibitors aimed at treating several lipid-related maladies, such as obesity, hypertriglyceridemia, and T2D. Another advantage of targeting MGAT2 is that drug discovery is still in the infancy stage, as there are only 10 MGAT2 inhibitor patents published. Examples of small molecule MGAT2 inhibitors are listed in Table 2.MGAT2 inhibitors and structures.The first patent describing MGAT2 inhibitors was published in 2010 (WO2010/095767, Banyu Pharmaceutical Co. Ltd., Tokyo, Japan), where pyrimidine-4(3H)-one derivatives were identified. The inhibitory activity (IC50 = 56 nM) of these compounds was examined in a radioactive-labeling assay with membrane fractions prepared from human MGAT2-expressing yeast. Bicyclic pyrimidine derivatives (WO2012/091010, Dainippon Sumitomo Pharma, Japan) and N-containing heterocyclic derivatives (WO2012/124744, Taisho Pharmaceutical Co. Ltd., Tokyo, Japan) were next identified. Both of these chemotypes showed improved inhibitory effects against MGAT2 in a radio-labeling enzyme assay using the membrane fractions prepared from human MGAT2-expressing Sf-9 cells; IC50 = 4.1 nM and IC50 = 2 nM, respectively. A non-radioactive fluorescence assay was later developed for the N-containing heterocyclic derivatives using a 7-diethylamino-3-(4-malimidophenyl)-4-methylcoumarin (CPM) fluorescent substrate assay that detected the release of CoASH from MGAT2 using a membrane fraction prepared from human MGAT2-expressing Sf-9 cells.Aryl dihydropyridinones and piperidinones derivatives (WO2013/082345, WO2014193884 Bristol-Myers Squibb Co., New York, NY, USA) inhibited MGAT2 with an IC50 = 14 nM in a radioactive-labeling enzyme reaction assay using human MGAT2-expressing Sf-9 cell membrane fraction. Using a stable radioactive-labeled substrate and high-resolution LC/MS with human MGAT2-expressing STC-1 cell based assay, these chemotypes demonstrated IC50 = 4 nM and selectivity over MGAT3 (>1000-fold), an acyl-CoA wax alcohol acyltransferase 2 (>1000-fold), and DGAT1 (>1000-fold) [65]. Phenyl methanesulfonamide derivatives that were developed by Eli Lilly (WO2013112323) had excellent MGAT2 inhibitory effects (IC50 ~12 nM) in a radioactive-labeling enzyme reaction assay using a human MGAT2-expressing Sf-9 cell membrane fraction. In ahumanMGAT2-expressing Caco2 cell based LC-MS assay, the chemotype inhibited MGAT2 with IC50 = 17.7 nM. The benzyl sulfonamide derivatives (WO2014074365, Eli Lilly, USA) showed an improved MGAT2 inhibitory effect over the phenyl methanesulfonamide derivatives with IC50 = 2.28 nM in a radioactive-labeling enzyme reaction assay using a human MGAT2-expressing Sf-9 cell membrane fraction. In human MGAT2-expressing Caco2 cell based LC-MS assay, the chemotype inhibited MGAT2 with IC50 = 3.8 nM. In a dog oil bolus model, the compounds were dosed at 10mg/kg and reduced TAG circulation by more than 70%.Morpholynyl derivatives (US20150005305, Eli Lilly, USA) showed MGAT2 inhibitory activity with an IC50 = 12 nM in a radioactive-labeling enzyme reaction assay using a human MGAT2-expressing Sf-9 cell membrane fraction. In human MGAT2-expressing Caco2 cell based LC-MS assay, the chemotype inhibited MGAT2 with an IC50 = 16 nM. Finally, in a dog oil bolus model where compounds were dosed at 30mg/kg and 75mg/kg, TAG circulation was reduced by 43% and 64%, respectively.The singleton 3-ethyl-3-methyl-2,5-dioxo-N-phenyl-2,3,4,5-tetra hydro-1H-1,4-benzodiazepine-7-sulphonamide 1 [66] showed MGAT2 inhibitory activity using a Rapidfire LCMS® assay, giving an IC50 = 1.6 nM.The singleton had > 10,000-fold selectivity over the acyl-CoA/wax alcohol acyltransferases, AWAT1/AWAT2, and DGAT1. It had variable selectivity over MGAT3 and no selectivity towards MGAT1, and achieved a high bio-availability, while reducing circulating TAG after corn oil oral gavage by 68% in mice.N-phenylindoline-5-sulfonamide derivatives [67] achieved an IC50 = 3.4 nM towards MGAT2 and >30,000-fold selectivity over MGAT3, DGAT1, DGAT2, and ACAT1. They are highly bio-available and effectively suppress post-olive-oil-loaded TG excursion after oral gavage in C57BL/6J mice.JTP-103237, a 7-(4,6-Di-tert-butyl-pyrimidin-2-yl)-3-(4-tri-fluoromethoxy-phenyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine derivative, potently inhibited MGAT2 activity in a rat intestinal S9 fraction and membrane fractions of MGAT2-expressing COS-7/Sf9 cells using radioactive-labeling assay [68]. JTP-103237 has an IC50 = 19 nM which is >300-fold selective over MGAT3 and >1000-fold selective over DGAT2. Plasma PYY level, but not GLP-1 was increased after administrating JTP-103237 in rats. As PYY is a satiety factor, this may explain the decreased cumulative food intake in rats fed a 35% fat diet and JTP-103237 when compared to control group. JTP-103237 not only prevented high-fat diet induced metabolic syndromes, it also caused a significant decrease in body weight, plasma glucose levels, and food intake in obese BDF1 mice fed with high-fat diet. Interestingly, the decreased glucose level seen and decreased food intake brought about by JTP-103237 is more significant than the marketed anti-obesity drug orlistat, a pancreatic lipase inhibitor. In addition, JTP-103237 also reduced fat weight and hepatic TAG content.Excessive TAG synthesis in the intestine due to dietary fat absorption followed by increased accumulation of TAG in the liver and adipose plays an integral role in the progression of metabolic disorders including obesity, insulin resistance, T2D, and fatty liver disease (Figure 2). The mode of action of MGAT2 inhibitors in the treatment of metabolic disorders. In mouse liver, inhibition of MGAT1 has shown to improve insulin sensitivity as well as hepatic steatosis. In human liver, MGAT2/MGAT3 expression is correlated with progression of NAFLD, in the small intestine, MGAT2 inhibition results in changes in TAG absorption and synthesis, as well as incretin secretion. These actions contribute to the weight loss, improvement of insulin sensitivity and hypertriglyceridemia, and prevention of NAFLD progression.Blocking dietary TAG absorption and resynthesis in the intestinal lumen has been seen as a viable pathway in which to design small molecules targeting obesity and metabolic syndrome; inhibitors to the intestinal lipase (e.g., orlistat) are currently one of the few medicines available on the market, and inhibitors to DGAT1 are currently in clinical trials. However, both types of inhibitors exhibit unwanted gastrointestinal side effects [63,69]. Inactivation of MGAT2 by genetic manipulation and pharmacological intervention supports the idea that targeting the MAG pathway as a therapeutic for metabolic syndrome is a viable option for inhibiting intestinal TAG synthesis. It has already been shown that inhibiting intestinal MGATs, especially MGAT2, results in dynamic changes in TAG and cholesterol absorption, which leads to the changes in systemic energy balance and gut incretin release. It is also possible that inhibition of the MGAT2 isozyme in the liver will improve steatosis by attenuating fat accumulation and insulin resistance. In adipose, MGAT2 inhibition may reduce fat biosynthesis and improve glucose uptake. Based on all the data accumulated thus far, the combined effects of MGAT2 inactivation in various tissues will have beneficial effects in reducing body weight, improving insulin resistance, decreasing hyperlipidemia, and attenuating hepatic steatosis.We are grateful to Martin Adelson and Eli Mordechai for their discussion and input. This work was supported by the Genesis Biotechnology Group. We also appreciate the many discussions with our colleagues at the Institute for Metabolic Disorders. We thank Pedro Martins for graphic design in Figure 2.The authors declare no conflict of interest.nonalcoholic fatty liver disease;triacylglycerol;acyl-coA: monoacylglycerolacyltransferase 2;monoacylglycerol;diacylglycerol;fatty acid;sn-glycerol-3-phosphate;acyl-coA: diacylglycerolacyltransferase;glycerol-3-phosphate acyltransferase;lysophosphatidic acid;acylglycerol-3-phosphate acyltransferase;phosphatidic acid;lipins/PA phsphohydrolases;endoplasmic reticulum;dihydroxyacetone phosphate;very low density lipoprotein;glucagon-like peptide-1;Reux-en-Y gastric bypass;diet-induced obese;peroxisome proliferator-activated receptor;antisense oligonucleotides;high trans FAs, fructose and cholesterol diet;nonalcoholic steatohepatitis.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Cancer is a leading cause of death in the United States. Angiogenesis inhibitors have been introduced for the treatment of cancer. Based on the fact that many anticancer agents have been developed from botanical sources, there is a significant untapped resource to be found in natural products. American ginseng is a commonly used herbal medicine in the U.S., which possesses antioxidant properties. After oral ingestion, natural ginseng saponins are biotransformed to their metabolites by the enteric microbiome before being absorbed. The major metabolites, ginsenoside Rg3 and compound K, showed significant potent anticancer activity compared to that of their parent ginsenosides Rb1, Rc, and Rd. In this review, the molecular mechanisms of ginseng metabolites on cancer chemoprevention, especially apoptosis and angiogenic inhibition, are discussed. Ginseng gut microbiome metabolites showed significant anti-angiogenic effects on pulmonary, gastric and ovarian cancers. This review suggests that in addition to the chemopreventive effects of ginseng compounds, as angiogenic inhibitors, ginsenoside metabolites could be used in combination with other cancer chemotherapeutic agents in cancer management.Cancer is a leading cause of human death in the United States [1]. The clinical management of cancer invariably involves diverse conventional modalities, including surgery, radiation, and chemotherapy [1,2]. Because commonly used chemotherapeutic agents usually affect processes that occur in all rapidly dividing cells, many normal cells throughout the body that are undergoing active growth and cell division can also be damaged. As a result, chemotherapeutic agents induce significant side effects [3]. To reduce the adverse effects caused by chemotherapeutic agents, in recent years, targeted therapies were developed to interact with specific molecules that are part of the pathways [4].Tumor growth and progression depend on angiogenesis, a process of new blood vessel formation from a preexisting vascular endothelium. The newly formed blood vessels provide nutrients and oxygen to the tumor, increasing its growth. Inhibiting angiogenesis has been proposed as a potential cancer treatment strategy [5]. Therefore, angiogenesis is considered as a powerful target to suppress tumor growth and metastasis [6,7].Complementary and alternative medicine (CAM), which covers a wide spectrum of ancient to new-age approaches that purport to expand options for preventing and treating diseases, is gaining more attention for cancer management [8,9,10]. The emergence of CAM represents a natural experiment of huge dimensions, as millions of Americans have begun self-medicating with natural products [11,12]. Natural products have been valuable sources of new therapeutic candidate compounds [13,14,15]; an analysis of the number of chemotherapeutic agents and their sources indicates that nearly 80% of approved drugs are derived from natural compounds [16]. With the advent of high throughput screening technologies, natural products are likely to provide many of the lead structures for the construction of novel compounds with enhanced anticancer properties.Natural products are most often administered orally so that they are exposed to the trillions of microbial organisms that live in the gut. Previous reports revealed that compound biotransformation resulted from reactions carried out by the enteric microbiome [17]. These studies involved monitoring compound metabolism during the ex vivo fecal incubation with a given compound [18], showing that orally ingested natural products can be biotransformed to their metabolites by microbiota in the gut. However, information regarding the role of the enteric microbiome in botanical bioactivity is still limited and this situation obstructs the evaluation of natural products with anticancer potential.Because ginseng is a very commonly used antioxidant natural product in the U.S, in this review, using American ginseng as an example, we have summarized recent research progress on the anticancer activities of ginseng parent compounds and their intestinal microbiome metabolites, focusing on their angiogenesis inhibitory potentials. Crucial information on ginseng’s interactions with the enteric microbiome was obtained and the contribution of intestinal microbiota to ginseng’s anticancer activity is discussed. Molecular mechanisms involved in the ginseng metabolites’ actions, including those targeted on angiogenesis are discussed.Ginseng is the name of a group of botanicals in the genus Panax of the Araliaceae family. Three species in the genus are commonly used as herbal remedies in oriental countries, i.e., Asian ginseng (Panax ginseng), notoginseng (Panax notoginseng), and American ginseng (Panax quinquefolius).Asian ginseng is distributed in Eastern Asia, including northeastern China, Korea, and the far east of Russia, while commercially available Asian ginseng is cultivated in China and Korea [19,20]. Like Asian ginseng, notoginseng is a Chinese herbal medicine that has a long history of use in Asian countries. Notoginseng is distributed in southwestern China, Burma, and Nepal, and this herb is cultivated commercially in southwestern China [21,22]. The root is the most commonly used plant part of Asian ginseng and notoginseng.American ginseng is an obligate shade perennial plant native to eastern North America. The commonly used part of the plant is the root, which is harvested after several years of cultivation. Although both the U.S. and Canada have cultivated American ginseng, the largest growing area is in Wisconsin, USA [23,24]. In recent years, American ginseng has also been grown in northeastern China on a small scale, however, studies on the chemical profiles and pharmacological effects of Chinese produced American ginseng are limited [25].It is generally believed that the active compounds in Asian ginseng, notoginseng and American ginseng are triterpene glycosides or dammarane saponins, commonly referred to as ginseng saponins (ginsenosides and notoginsenosides). These ginseng saponins are the major active ingredients in the herb, and their levels can be used to develop quality controls for these herbs [26,27,28]. There are over 50 different known ginseng saponins, and they are characterized by a four trans-ring rigid steroid aglycone skeleton and attached sugar moieties [29]. Based on the aglycone skeleton, ginseng saponins can be divided into the protopanaxadiol group and protopanaxatriol group, except for ginsenoside Ro, which is derived from the oleanolic acid group (Figure 1).Although all three ginseng plants have both protopanaxadiol group (PPD group) and protopanaxatriol group (PPT group) saponins, their constituent compositions vary. As we know, ginsenosides Rb1, Rb2, and Rd belong to the PPD group, while ginsenosides Re, Rg1, and notoginsenoside R1 belong to the PPT group. The major saponins in Asian ginseng are ginsenosides Rb1, Rb2, and Rg1; those in notoginseng are ginsenosides Rb1, Rd, Rg1 and notoginsenoside R1; and the major saponins in American ginseng are ginsenosides Rb1, Rd, and Re.American ginseng has a high saponin content, specifically having abundant PPD group saponins [30]. Previous observations suggested that the PPD group ginsenosides, especially those with lesser sugar moieties, showed more potent anticancer activity [31,32]. The higher PPD group saponin content in American ginseng supplied sufficient resources for further chemical or biochemical transformation to produce active anticancer ginsenosides, which will be discussed later.Several beneficial effects of American ginseng, such as cardioprotection and adjuvant cancer therapy, have been reported [33,34]. These pharmacologic activities are, to a significant extent, considered to be due to the antioxidant properties of this botanical. Our group observed anti-diabetic and anti-obesity activities of American ginseng extract [35,36,37]. These effects are likely related to the anti-oxidant property of the ginseng compounds. In a clinical trial, ginseng extract prevented acute oxidant injury following cardiac reperfusion [38]. Our group has demonstrated that acute oxidant stress induced cardiomyocyte injury was protected by the treatment of American ginseng [39]. For cancer chemotherapy studies, we observed that American ginseng possesses the potential for treating chemotherapeutic agent-induced side effects, which is partly mediated by antioxidant mechanisms [40,41].Chemical structures of ginseng saponins in American ginseng. Rg3, F2, and CK are gut microbiome metabolites of PPD group saponins.Bacteria are an important component of the human body. The microbiome is a community of living microorganisms assembled in particular ecological niches of a host that contain trillions of bacterial cells, 10 times more cells than the number of cells constituting the body [42]. A considerable portion (approximately 70%) of this microbial cosmos is localized in the gut, while the colon is the site where the gut microbiota reaches its highest concentration [43].Like many other herbal medicines, the route of administration of American ginseng is nearly always oral. After oral ingestion, ginseng saponins are metabolized extensively by intestinal microbiota. Although investigations on the comprehensive metabolic profile of American ginseng are very limited because of the chemical complexity and limitation of analytical methods, we recently systemically evaluated the biotransformation and metabolic profiles of American ginseng extract by human intestinal microflora. Using a highly sensitive and selective liquid chromatography/quadrupole time-of-flight mass spectrometry method, 25 metabolites were detected, of which 15 metabolites were derived from original protopanaxadiol saponins, including the three highest ones. This indicated that the PPD-type ginsenosides generated comprehensive biotransformation and were more easily metabolized than other ginsenosides under the same conditions. Several major metabolic pathways can be observed for PPD-type ginseng saponins by human intestinal microbiota such as deglycosylation and dehydration. One way is to selectively eliminate the C-3 sugar moieties to produce F2 and then compound K (CK) [44,45]. CK can be further converted to 3-oxo-compound K and monooxygenated compound K. Another way is to selectively eliminate the C-20 sugar chain to produce ginsenoside Rg3 [46]. It is interesting to note that Rg3 can be further transformed to Rk1 and Rg5 by intestinal microflora via dehydration (Figure 2) [46].Metabolic pathways of PPD group ginseng saponins converted by the intestinal microbiome.The three most abundant metabolites are ginsenoside Rg3, ginsenoside F2, and CK, which are transformed from ginsenosides Rb1, Rb2, Rb3, Rc, and Rd (Figure 1). The main metabolic pathways of ginseng saponins are deglycosylation reactions by intestinal microflora through the stepwise cleavage of sugar moieties. Subsequent dehydration reactions also occur. Compared to those of the PPT group, PPD group triterpenoids are easy to metabolize.We recently observed that CK showed significant antiproliferative effects on human colorectal cancer cells, while its parent compound Rb1 did not show any effects at the same concentrations [47]. Thus, ginseng’s bioactivity appears to be highly dependent on metabolism by enteric microbiota. Several microbial species including Prevotelki oris appear to have this capability [44,45,48]. However, information on the role of enteric microbiota in ginseng’s cancer chemoprevention is still not clear. Nevertheless, available evidence has suggested that the intestinal microbiota may play an important role in mediating the metabolism and bioactivity of American ginseng.Early ginseng anticancer evaluations largely focused on the herb’s parent compounds, i.e., the ginsenosides Rb1, Rd, and Re. The antiproliferative effects of these ginsenosides on different cancer cell lines were evaluated. Unfortunately, in several in vitro screenings, even in very high concentrations, these parent compounds did not show obvious antiproliferative activities [31,49].Orally administered ginsenosides are poorly absorbed, and some appear to require bacterial metabolism to be absorbed and biologically active [50]. After ginseng ingestion, both Rg3 and CK are major metabolites reaching the systemic circulation [29,46,51]. CK possesses very significant anticancer activities compared to its parent compound Rb1 [52]. The IC50 of CK for the inhibition of colon cancer cell proliferation was 30–50 µM, suggesting that its antiproliferative effect is greater than that of Rg3 (IC50 100–150 µM), the compound derived from Rb1 via steaming treatment [47,53,54].Although ginsenoside Rg3 showed moderate antiproliferative effects, Rg3 was approved as a new anti-cancer drug in China [55]. Rg3 has been shown to inhibit NF-κB signaling [56] and enhances the susceptibility of prostate cancer cells to docetaxel and other chemotherapeutics [57]. We observed that the inhibitory effect of Rg3 on colon cancer cells is in part mediated by inhibiting β-catenin/Tcf transcriptional activity and suppressing PNCA expression in colon tumors, thus inducing cancer cell apoptosis [58].CK induced apoptosis in several tumor cell lines by regulating various signaling pathways, such as the activation of caspase-8 [59] and AMP-activated protein kinase (AMPK) [60,61], suppression of nuclear factor-kappa B (NF-кB) pathways [62] and Janus activated kinase 1 (JAK1)-signal transducer and activator of transcription 3 (STAT3) signaling [63]. A recent study indicated that CK increased generation of reactive oxygen species and activation of c-Jun NH2-terminal kinase signaling pathway, which in turn stimulated autophagy and apoptosis in human colon cancer cells [64]. Our data showed that multiple pathways, including p53/p21, PI3K/Akt, and transforming growth factor beta (TGF-β), were involved in CK induced cancer cell death [65].Anti-angiogenic compounds have emerged as potential agents to be used alone or in combination with other chemotherapeutic drugs for the treatment of cancer. Early studies have revealed that red ginseng extract inhibited tumor metastasis, and active compound ginsenoside Rg3 exhibited a significant decrease in the number of blood vessels oriented toward the tumor mass [66]. As shown in Figure 2, Rg3 is also a major intestinal metabolite from parent PPD group ginsenosides. Rg3 was found to inhibit the proliferation of human umbilical vein endothelial cells.The vascular endothelial growth factor (VEGF) is an important growth factor that promotes angiogenesis. Overexpression of VEGF results in increased angiogenesis, whereas its suppression results in the inhibition of angiogenesis in both normal and pathological conditions. Increased VEGF expression is associated with solid tumor growth, and its inhibition has been shown to be effective in the control of several cancers [67]. Rg3 suppressed the capillary tube formation of the cells in the presence or absence of VEGF. The VEGF-induced cell chemoinvasions were significantly attenuated by Rg3 [68].Rg3 also showed anti-angiogenic effects on pulmonary, gastric, and ovarian cancers [69,70,71]. As a potential angiogenic inhibitor, Rg3 processed synergistic effects when it was used in combination with other cancer chemotherapeutic agents, such as gemcitabine, cyclophosphamide, and capecitabine [72,73,74]. The combination use of chemotherapeutic agent plus angiogenesis inhibitor could be a promising strategy in cancer management.As a major ginsenoside metabolite in the gut, CK showed significant anticancer activity. In early studies, in addition to inducing cancer cell apoptosis, CK also showed antiplatelet and anti-angiogenic activities and caused the suppression of primary tumor growth in a spontaneous metastasis model [45]. MMPs (matrix metalloproteinases), especially MMP-9, involved in extracellular matrix degradation, are crucial for the endothelial cell migration, organization, and angiogenesis [75]. It has been reported that MMP-9 expression levels are significantly higher in tumors than in normal tissue. The upregulation of MMP-9 is implicated in the process of invasion and metastasis of malignancies [76]. CK significantly inhibited the secretion and protein expression of MMP-9. The inhibitory effect of compound K on MMP-9 expression was correlated with decreased MMP-9 mRNA levels and the suppression of MMP-9 promoter activity [77].Using human umbilical vein endothelial cells (HUVECs), the molecular mechanisms of the anti-angiogenic effects of CK were further evaluated. CK significantly inhibited the migration and tube formation of HUVECs at non-cytotoxic concentrations, reduced the secreted level of VEGF and increased the secreted level of pigment epithelium-derived factor (PEDF) in HUVECs. The anti-angiogenic activity of CK was found to be a result of the inhibition of p38 MAPK and AKT in the HUVECs [78]. Anti-angiogenic effects of CK were also validated by a recently published study [79]. Therefore, the anti-angiogenic effects of CK might have therapeutic potential for controlling the growth and invasiveness of different cancers.Anti-angiogenic compounds have emerged as potential agents to be evaluated for the treatment of cancer. Many anticancer agents have been developed from botanical sources. American ginseng is a commonly used antioxidant herbal medicine in the United States. Ginsenoside Rg3 and CK are major ginseng metabolites biotransformed by the enteric microbiome. Compared to their parent compounds, these metabolites exert much better anticancer activities with multiple molecular mechanisms. Interestingly, both Rg3 and CK also showed significant anti-angiogenic effects on pulmonary, gastric, and ovarian cancers. Therefore, in addition to the chemopreventive effects of ginseng compounds, as angiogenic inhibitors, ginsenoside metabolites could be used in combination with other cancer chemotherapeutic agents in cancer management.This work was supported in part by the NIH/NCCAM grants AT004418, and AT005362.C.-Z.W. and C.-S.Y. wrote the manuscript. Y.C. and S.A. edited the manuscript. All authors read and approved the final manuscript.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).A mosaic pattern of lung attenuation on chest computed tomography (CT) may be due to various etiologies. There is limited published data on CT results when used to evaluate pulmonary hypertension (PH). We retrospectively studied the frequency of mosaic pattern in patients with PH and the cause of the PH by diagnostic group, as well as the correlation between the mosaic pattern and the following: demographics, severity of the PH, main pulmonary artery (PA) size, PA/aorta (PA/Ao) ratio, pulmonary function tests (PFT), and ventilation perfusion scan results. Overall, 18% of the cohort had CT mosaic pattern (34/189). Mosaic pattern was present in 17/113 (15%) in Group 1 pulmonary arterial hypertension, 5/13 (28%) in Group 2 pulmonary venous hypertension and 8/50 (16%) in Group 3 PH. Conversely, Group 4 chronic thromboembolic PH was more prevalent in 4/8 (50%). Main PA size, PA/Ao ratio, and segmental perfusion defect were positively associated with mosaic pattern. In contrast, factors such as age, gender, body mass index, functional class, hemodynamic data, and PFT values were not associated with mosaic pattern. Mosaic pattern is not specific as an isolated finding for distinguishing the subtype of PH.A mosaic pattern of lung attenuation on chest computed tomographic (CT) scan is defined by the Fleischner Society glossary as a patchwork of regions of differing attenuation seen on CT of the lungs [1]. It is characterized by heterogeneous lung attenuation with well-defined borders corresponding to the secondary pulmonary lobules as shown in Figure 1 [1,2]. This pattern is nonspecific and may be due to various etiologies, but the main pathologies are pulmonary vascular, small airway, and infiltrative lung disease [2,3,4,5,6].Chest computed tomographic (CT) scan demonstrates mosaic pattern of lung attenuation where there is a patchwork of regions of differing attenuation with well-defined borders (black arrows).The mosaic pattern related to pulmonary hypertension (PH) consists of relative hypoattenuation and hyperattenuation from adjacent areas with disparate perfusion [3,6,7]. The chest CT findings in patients with PH may also demonstrate enlargement of the main pulmonary artery (PA) with peripheral tapering [8,9,10,11]. The PA enlargement may be assessed by cross-sectional diameter or comparison to the diameter of the aorta (Ao), referred to as the PA/Ao ratio.Chest CT scan is often utilized as part of the evaluation to define coexistent conditions related to PH [12]. Little is known regarding the frequency of the mosaic pattern in the PH population and factors predisposing to the presence of this pattern. We retrospectively examined the frequency of mosaic pattern among patients with PH and by cause of the PH (i.e., group). We also sought to determine whether the presence of mosaic pattern attenuation on chest CT in patients with PH correlated with the severity of PH based on functional class and hemodynamic data from right heart catheterization (RHC), PA dimensions, pulmonary function test, and ventilation/perfusion lung scan.The study was approved by the Mayo Clinic Institutional Review Board. We retrospectively reviewed the patients referred to our PH Center from January 1992 to December 2006. Data was collected, including age, sex, body mass index, PH diagnosis group, modified New York Heart Association functional class, forced expiratory volume in 1 second/forced vital capacity ratio, total lung capacity, residual volume, ventilation/perfusion lung scan results, and hemodynamic data if the patients had RHC, including right atrial pressure (RAP), mean pulmonary arterial pressure (MPAP), pulmonary vascular resistance (PVR), cardiac output (CO). Patients who had either complete pulmonary function test or spirometry to review were defined to have obstructive airway disease if they had forced expiratory volume in 1 s/forced vital capacity ratio <70%, restrictive lung disease when total lung capacity <80%, and air trapping when residual volume >120%.Of the 306 patients who were referred for PH evaluation, 117 patients without RHC were excluded. Chest CT scans on 189 patients had been personally reviewed by the one of the authors (CDB) who had evaluated the patients in the PH Center and noted whether a mosaic pattern was present in the physician notes in the medical record [1]. Those 189 patients served as the study cohort. Another author (KU) independently reviewed the imaging. For the correlation between PA dimensions and mosaic pattern evaluation, the original study was not available to provide sufficient cuts to determine the correlation of the PA diameter and mosaic pattern; therefore, there were only 162 in the secondary analysis. The main PA was measured at the widest short axis diameter in centimeters at the level of PA bifurcation on the axial section. The adjacent aorta was also measured and calculated for PA/Ao ratio.All patients had undergone a clinical evaluation for PH. PH was defined as MPAP ≥ 25 mm Hg at rest by RHC. The patients with a PH diagnosis were clinically classified into five groups based on the 2013 World Symposium on PH [13]. Group 2 patients had PH related to left heart disease defined by a MPAP ≥ 25 mm Hg in association with a pulmonary capillary wedge pressure >15 and transpulmonary gradient <12 mm Hg [14].Demographic data were described using descriptive statistics. Categorical data were described by frequency and percentage. Continuous data were presented as mean ± SD. Comparison between groups were assessed by chi-squaretests for categorical variables and student t tests for continuous variables. Mann-Whitney U-test was used if they were not normally distributed. p values < 0.05 were considered significant. All data were analyzed using SPSS version 16.0, Inc., Chicago, IL, USA.We reviewed 189 patients with RHC who underwent a PH evaluation. The baseline demographic data of the patients are summarized in Table 1. Most patients were non-obese women. Most had functional class III–IV symptoms. More than half of the patients had Group 1 pulmonary arterial hypertension (PAH) with the second largest number of patients in Group 3 PH. All 189 patients were included for the presence or absence of mosaic pattern of lung attenuation [1] with a minority positive for this pattern. Overall, approximately 18% of the cohort had CT mosaic pattern as shown in Table 2. Mosaic pattern was seen in 17 of the 113 (15%) Group 1 PAH patients, 5 of the 18 (28%) Group 2 PVH, 8 of the 50 (16%) Group 3 PH and 4 of the 8 (50%) Group 4 chronic thromboembolic PH (CTEPH). Patients with Group 4 CTEPH were more likely to have mosaic pattern compared with other groups (50% vs. 17%, Fisher’s Exact test = 0.036 with the value of Phi at 0.175).Chest CT scans in 162/189 (86%) were available for measurement of the diameter of the main PA and Ao. Overall, the mean diameter of the main PA was larger than normal [15,16] (3.45 ± 0.59 cm) with a normal Ao diameter (3.06 ± 0.42 cm) and resulting PA/Ao ratio of 1.14 ± 0.26. Patients with PH and mosaic pattern had significantly larger main PA size and PA/Ao ratio compared to the patients with PH and no mosaic pattern (Table 2). In contrast, factors such as age, gender, body mass index, functional class, hemodynamic data, and pulmonary function test results (available in 175 of 189 or 93%) were not associated with mosaic pattern. Comparison of the ventilation lung scans (available in 69 of 189 or 37%) /perfusion lung scans (available in 133 of 189 or 70%) demonstrated more segmental perfusion defects in the patients with mosaic pattern. Of note, all of those patients had Group 4 CTEPH. There was no statistical difference in delayed ventilation washout between patients with mosaic pattern and those without. Intraobserver variability in vascular dimension measurements showed high agreement with mean main PA difference at 0.3 mm (SD = 0.07, correlation at 0.98) and mean Ao difference at 0.5 mm. (SD = 0.05, correlation 0.97).Pulmonary Hypertension Demographics.BMI—body mass index; PH—pulmonary hypertension; WHO—World Health Organization.Summary statistics of demographic and clinical variables by mosaic pattern of lung attenuation on chest CT scan status.BMI—body mass index; PH—pulmonary hypertension; PA/Ao—pulmonary artery diameter/aorta diameter; RAP—right atrial pressure; MPAP—mean pulmonary arterial pressure; PVR—pulmonary vascular resistance; CO—cardiac output; FEV1/FVC—forced expiratory volume in 1 second/forced vital capacity; TLC—total lung capacity; RV—residual volume.All patients had RHC. The mean RAP was 11 ± 6 and mean MPAP was 46 ± 14 mmHg, respectively. The mean CO was 4.9 ± 2.1 L/min with a calculated PVR of 615 ± 471 dyne s·cm−5.In this single-center retrospective review, a mosaic pattern was observed on chest CT in approximately 1/5 patients evaluated for PH. The presence of mosaic attenuation in the setting of PH has been well described and is often attributed to Group 4 CTEPH but also has been reported in many PH conditions including idiopathic PAH and congenital heart disease [2,5,8,9,11,17]. The finding is explained by increased arterial vessel caliber in geographic areas of increased attenuation, or hyperemia, compared with decreased vessels in areas of low attenuation, or oligemia [8,9,10,11].The purpose of our study was to examine the prevalence of the mosaic pattern in the various diagnostic groups of PH as defined during the 5th World Symposium on PH [13]. Our results suggest that a mosaic pattern is not specific enough to distinguish PH diagnostic groups because the pattern can be present in all PH diagnostic groups, although perhaps more prevalently in Group 4 CTEPH. The distribution across diagnostic groups did not appear to be influenced by demographics or PH severity as defined by functional class or hemodynamics (Table 2). Additionally, we could not determine the screening ability of mosaic pattern in PH due to the lack of a control cohort.Published literature lacks the comparison among the PH diagnostic groups present in this study. For example, Sherrick et al. [5] retrospectively reviewed the frequency of mosaic pattern in 64 patients divided into three groups: PH due to lung disease (21 patients), cardiac disease (17 patients), and vascular disease (23 patients). Mosaic pattern was seen in 1/21 (5%) patients with PH due to lung disease, 2/17 (12%) with PH due to cardiac disease, and 17/23 (74%) with PH due to vascular disease. The latter group included pulmonary emboli, pulmonary veno-occlusive disease, lymphangitis metastasis and idiopathic PAH (four patients, two of which had mosaic pattern). By comparison, the present study demonstrated a lower frequency of mosaic pattern in Group 1 PAH; however, it had a different spectrum of subtype classification of PH and a larger cohort, with more confirmed diagnoses. Although we report a higher prevalence of mosaic pattern in Group 4 CTEPH, which is consistent with previous studies [8,11,17,18], the number of patients with CTEPH in our cohort is small. The time frame of the study predated the availability of pulmonary thromboendarterectomy at Mayo Clinic, Jacksonville, Florida; therefore, patients were typically referred to medical centers with that capability. A selection bias is highly likely as those patients often bypassed evaluation in the PH Center and were directed to centers offering surgical intervention.In addition to examining the prevalence of mosaic attenuation in PH by diagnosis grouping, we sought to determine any correlation with the size of the main PA on chest CT, presence of obstructive or restrictive lung disease on pulmonary function tests, and corresponding abnormalities on ventilation/perfusion lung scanning. In particular, we were interested in correlative findings that might better elucidate mechanisms of the disparate attenuation in the lung parenchyma. For example, air trapping is a well-known mechanism of mosaic pattern on chest CT [2,3,4,5,6]; however, it did not seem to play a prominent role in this PH cohort (Table 2) as evidenced by the lack of prominence in Group 3 PH. Specifically, there was a similar prevalence of obstructive disease by pulmonary function test and air trapping by ventilation/perfusion lung scan.Overall, the PH cohort in this study had larger than normal PA size and increased PA/Ao ratios. Normative values were based on two studies. The largest population cohort in which PA dimensions were assessed by CT, comes from the Framingham Heart Study, that reported a 90th percentile sex-specific cutoff value for main PA of 29 mm for men and 27 mm for women without cardiopulmonary risk factors [16]. The second study examined main PA diameters in 112 adult patients with documented normal MPAP by echocardiogram [15]. The mean PA diameter was 27 ± 2.8 mm in men and 25.9 ± 3 mm in women, with an upper limit of normal for the main PA considered to be 32.6 mm [15]. Both the mean values for both the PA size and PA/Ao were elevated in our study population. Furthermore, patients with PH and mosaic pattern had significantly larger main PA size and PA/Ao ratio compared to the PH patients without mosaic pattern (Table 2). As a mosaic pattern is felt to represent disparate pulmonary blood volume in adjacent lung segments, the areas with relative oligemia are felt to have more severe pulmonary vasculopathy [3,6,7]. One might hypothesize that the mosaic pattern may be indicative of more severe pulmonary vascular remodeling with increased vasoconstriction and PA narrowing. In addition, a reduction in PA compliance may produce more pronounced regional alterations in pulmonary arterial blood flow. The resulting increased PVR and larger upstream pressures might dilate the main PA; however, the lack of a correlation between mosaic pattern and hemodynamic data in this cohort argues against this hypothesis. More recent attention to ventriculoarterial coupling mechanisms with methods to access pulmonary impedance and PA distensibility [19] may offer opportunities for future research to understand the link between pulmonary vasculopathy and the upstream impact on the right ventricle and main PA. Nonetheless, the correlation of mosaic pattern with PA size and perfusion abnormalities suggests a link between heterogeneous small vessel disease and proximal PA dilation that may distinguish a clinical PH phenotype independent of diagnostic group and hemodynamic profile.Our study had a number of limitations. Not every subject had pulmonary function tests, ventilation/perfusion lung scan, and CT scans available for second review.Because the study was retrospective, the imaging protocol for the chest CT scan was variable depending on the clinical indication. Inspiratory and expiratory views were not performed routinely to evaluate the degree of air trapping. Lack of lung histology data also limited the actual diagnosis especially for Group 1 PH subtypes or Group 1' pulmonary veno-occlusive disease, but all patients were thoroughly evaluated and reviewed to ensure appropriate classification into the proper PH diagnostic group. The small sample size of each subgroup limits firm conclusions. The effect of PAH therapy and outcome were not evaluated in our study.A mosaic pulmonary parenchymal pattern was observed on chest CT in approximately 1/5 patients evaluated for PH in our cohort. Analysis by the PH diagnostic group, revealed an increased prevalence in Group 4 CTEPH compared to the other groups. Regardless, mosaic attenuation is not specific as an isolated finding for distinguishing the subtype of PH. The correlation between mosaic pattern and main PA diameter, PA/Ao ratio, and segmental perfusion defect suggests a possible link between heterogeneous small vessel disease and proximal PA dilatation independent of PH diagnostic group or severity, a possibility that requires further study.No specific funding was received for this study.Kamonpun Ussavarungsi contributed to the collection, analysis, and interpretation of data, drafting and critical revision of the article, and generation of the tables. Augustine S. Lee contributed to the conception and design of the study, analysis and interpretation of the data, and critical revision of the article. Charles D. Burger contributed to the conception and design of the study, collection of the data, and critical revision of the article. All authors approved the final draft of the article.Augustine S. Lee has ongoing grant funding from Intermune, Roche, Gilead, and the NIH; however, none of these are relevant to this study. The authors declare no conflict of interest.

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+Current Address: Department of General Surgery, First Hospital of Hebei Medical University, Shijiazhuang 050031, China.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Hepatitis B virus (HBV) infection, a cause of hepatocellular carcinoma (HCC), remains a serious global health concern. HCC development and human hepatocarcinogenesis are associated with hepatic inflammation caused by host interferons and cytokines. This article focused on the association between the HBV core protein, which is one of the HBV-encoding proteins, and cytokine production. The HBV core protein induced the production of interferons and cytokines in human hepatoma cells and in a mouse model. These factors may be responsible for persistent HBV infection and hepatocarcinogenesis. Inhibitors of programmed death (PD)-1 and HBV core and therapeutic vaccines including HBV core might be useful for the treatment of patients with chronic HBV infection. Inhibitors of HBV core, which is important for hepatic inflammation, could be helpful in preventing the progression of liver diseases in HBV-infected patients.Hepatitis B virus (HBV) infection can lead to acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) and is a global health concern [1]. Although the HBV vaccine and drugs, such as peginterferon and nucleos(t)ide analogues (NUCs), are effective against HBV infection, further investigation is needed to eradicate HBV [2,3].HBV is a hepatotropic virus, belonging to the Hepadnaviridae family, with a circular, partially-double-stranded DNA of approximately 3.2 kb in length [4]. On the shorter plus stranded DNA, at least four genes (surface (S), core (C), X and polymerase (P)) are encoded, and these genes partially overlap [4]. The Pre-S gene, which consists of the pre-S1 and pre-S2 genes, is located upstream of the S gene. The S gene encodes an envelope protein, HBV surface antigen (HBsAg), which comprises 226 amino acids. The function of the HBx protein in the HBV life cycle remains unclear [5]. It is reported that HBx is associated with hepatocarcinogenesis [6]. HBV polymerase (P) protein is important for HBV replication. HBV polymerase encodes the RNA- and DNA-dependent DNA polymerase [5]. The Pre-C region is located upstream of the C gene. HBV pre-C and C regions encode both HBV core genes (183 codons) and HBeAg (149 codons) [4,7].HBV core protein self-assembles to form the viral capsid [8]. The functions of HBV core protein during the life cycle of HBV or in HBV infection of human liver are not well understood [8]. Liver cirrhosis is the strongest risk factor for the development of HBV-related HCC [9]. The production of inflammatory cytokines, such as TNF-α and TGF-β, is linked to hepatic fibrosis and hepatocarcinogenesis [9]. In this article, we focus on the association between HBV core protein and cytokine production.HBV replicates in human hepatocytes. We examined whether HBV core protein enhances 84 interferon- and cytokine-related gene expression levels in the human hepatoma cell line HepG2 with stable expression of HBV core (HepG2-HBV core) compared to a HepG2 control by a real-time RT-PCR-based array (toll-like receptor signaling pathway PCR array, Qiagen, Hilden, Germany) [7,9]. After transfection of the plasmids and three weeks of G418 selection, to avoid monoclonal selection, all cells were collected, and these cells, HepG2-HBV core and HepG2 control, were established for further analysis. Out of 84 genes, only eight (9.5%) genes [nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100) (NFKB2), tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A), ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1), myeloid differentiation primary response 88 (MYD88), prostaglandin-endoperoxide synthase 2 (PTGS2), toll-like receptor adaptor molecule 1 (TICAM1), ECSIT signaling integrator (ECSIT) and interleukin-1 receptor-associated kinase 1 (IRAK1)] were downregulated by at least 1.2-fold in the HepG2-HBV core compared to the HepG2 control, and four of them (PTGS2, TICAM1, ECSIT and IRAK1) were downregulated by greater than 10-fold or more. Out of 84 interferon- and cytokine-related genes, 76 (90.5%) were upregulated by 1.2-fold or greater in the HepG2-HBV core cells compared to the HepG2 control. Fifty-eight genes were upregulated 50-fold or more in HepG2-HBV core cells (Table 1). Our results showed that HBV core protein seems involved in the induction of several interferon- and cytokine-related genes.Interferon- and cytokine-related genes upregulated by at least 50-fold in HepG2-HBV core cells analyzed using real-time RT-PCR.TNF-α and the interferon system are important for HBV clearance [10,11,12,13,14,15]. Tzeng et al. [15] reported that HBV core is critical for inducing TNF-α to clear HBV and for TNF inhibition, which eliminates HBV core-induced viral clearance effects in mice. We also found that IL6 protein production was much higher in cell culture medium of HepG2-HBV core than in the HepG2 control [7,9]. Serum IL6 levels are higher in patients infected with HBV than those without HBV (Figure 1). Of interest, the IL6 level of HBeAg-positive patients is less than that of HBeAg-negative patients, although these patients had normal ALT levels. We also reported that the inflammatory cytokines, such as IL6, were downregulated in HBeAg-positive HepG2, which stably expresses HBeAg, compared to HBeAg-negative HepG2 cells [7,9].Serum IL-6 levels in patients with normal ALT levels. IL-6 levels were determined by enzyme-linked immunosorbent assay (ELISA; KOMABIOTECH, Seoul, Korea) following the manufacturer’s protocol. Non-B, non-C (NBNC), patients without HBV or HCV infection (males, n = 15; mean age, 57 ± 14 years); HBe antigen (HBeAg)(+), patients with HBeAg-positive asymptomatic carriers (males, n = 6; mean age, 34 ± 15 years); HBeAg(−), patients with HBeAg-negative asymptomatic carriers (males, n = 7; mean age, 51 ± 14 years).Lin et al. [16] found that C57BL/6 mice injected with mutant HBV DNA did not express the HBV core gene and that approximately 90% of the established HBsAg persisted after six months. Conversely, C57BL/6 mice administered a single intravenous hydrodynamic injection of the HBV DNA without mutation exhibited HBV replication, which resulted in approximately 20%–30% of these mice carrying HBV for more than six months. Lin et al. [16] reported that the C-terminal domain of HBV core is necessary for HBV clearance. HBV core protein may influence HBV persistence by evoking innate immunity through the binding of the C-terminal domain of HBV core to membrane heparin sulfate on the surfaces of immune cells, which stimulates proinflammatory cytokine production [16].Thus, the HBV core protein seems to play an important role in persistent HBV infection. Therefore, targeting therapies against HBV core also look important as one of the new treatment options against HBV infection. Treatment with monoclonal antibodies against the immunologic receptor programmed death (PD)-1, a 55-kDa transmembrane protein, reversed the impairment of HBV core-specific interferon-γ T-cell response in C57BL/6 mice with HBV persistence [17]. Tzeng et al. [17] found that knock-out HBcAg, but not HBeAg, led to HBV persistent infection in mice and reported that the immune response triggered in mice by HBcAg176~185 during exposure to HBV is important in the determination of HBV persistent infection.Although it has been reported that PD-1 is associated with apoptosis [18,19], inhibition of TNF-α led to higher maintained serum HBV viral load with an increased number of intrahepatic PD-1(high)CD127(low)-exhausted T-cells, which resulted in the reduction of HBV clearance in the mouse model [20]. Treatment with monoclonal antibodies of PD-1 might be useful for patients with HBV reactivation, especially those treated with immune-suppressing and anti-cancer drugs, as reactivation is now becoming a global issue [21,22,23,24,25].NVR 3–778, a candidate HBV core inhibitor, disrupts the HBV lifecycle by inducing the assembly of defective viral capsids, but it is also a potent inhibitor of HBV replication in both cell culture models and a humanized albumin enhancer/promoter urokinase plasminogen activator (UPA)/severe combined immunodeficiency (SCID) mouse model of HBV infection [26,27]. Treatment with NVR 3–778 for six weeks resulted in the suppression of HBV DNA replication. The efficacy of NVR 3–778 was superior to peginterferon and was similar to the nucleoside analog, entecavir, in a humanized UPA/SCID mouse model [27]. The phase 1a clinical trial of NVR 3–778 was completed in New Zealand in 2014, and the phase 1b clinical trial of NVR 3–778 is currently enrolling patients [26].Previously, a single CTL epitope (HBV core 18–27: FLPSDFFPSV) vaccine CY-1899 was used to treat patients with chronic hepatitis B [28]. Although CY-1899 initiated CTL activity, low-level CTL activity does not seem to be associated with HBV clearance [28]. Epitopes of both HBsAg and HBV core were loaded on dendritic cells, and epitope-pulsed dendritic cells were used in chronic hepatitis B patients. The results of the epitope study demonstrated the potent antiviral effects of dendritic cell vaccines that contain HBsAg and HBV core epitopes [29]. A phase III clinical trial of an HBsAg/HBV core-based therapeutic vaccine, administered through the nasal and subcutaneous routes, is currently underway in chronic hepatitis B patients [29].Hepatic inflammation induced by interferons and cytokines is associated with hepatocarcinogenesis [30]. The HBV core protein could produce a response through interferon and inflammatory cytokines to induce hepatic inflammation and, possibly, hepatocarcinogenesis, although HBe antigen (HBeAg) inhibits cytokine production and prevents persistent HBV infection [31,32]. Thus, inhibitors of HBV core may be useful for preventing the progression of liver diseases in patients infected with HBV.This study was supported by grants from the Ministry of Health, Labour and Welfare, Japan.T.K. wrote the manuscript. All authors discussed, edited and approved the final version.Tatsuo Kanda reports receiving grant support from Merk Sharp and Dohme (MSD) and lecture fees from Chugai Pharmaceutical, MSD, Tanabe-Mitsubishi, Ajinomoto, Bristol-Myers Squibb, Daiichi-Sankyo, Janssen Pharmaceutical and GlaxoSmithKline. Osamu Yokosuka reports receiving grant support from Chugai Pharmaceutical, Bayer, MSD, Daiichi-Sankyo, Tanabe-Mitsubishi, Bristol-Myers Squibb, Taiho Pharmaceutical and Gilead Sciences. The other authors declare that there is no conflict of interest regarding the publication of this paper.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Hepatocellular carcinoma (HCC), the second leading cause of cancer deaths worldwide, is difficult to treat and highly lethal. Since HCC is predominantly diagnosed in patients with cirrhosis, treatment planning must consider both the severity of liver disease and tumor burden. To minimize the impact to the patient while treating the tumor, techniques have been developed to target HCC. Anatomical targeting by surgical resection or locoregional therapies is generally reserved for patients with preserved liver function and minimal to moderate tumor burden. Patients with decompensated cirrhosis and small tumors are optimal candidates for liver transplantation, which offers the best chance of long-term survival. Yet, only 20%–30% of patients have disease amenable to anatomical targeting. For the majority of patients with advanced HCC, chemotherapy is used to target the tumor biology. Despite these treatment options, the five-year survival of patients in the United States with HCC is only 16%. In this review we provide a comprehensive overview of current approaches to target HCC. We also discuss emerging diagnostic and prognostic biomarkers, novel therapeutic targets identified by recent genomic profiling studies, and potential applications of immunotherapy in the treatment of HCC.Hepatocellular carcinoma (HCC) is the sixth most common cancer and the second leading cause of cancer deaths worldwide [1,2]. Agents causing chronic liver disease and eventually cirrhosis are risk factors for developing HCC [3]. These include infection with hepatitis B virus (HBV) and hepatitis C virus (HCV), alcoholic liver disease, and nonalcoholic fatty liver disease [4]. Other predisposing factors with lower prevalence are hereditary hemochromatosis, alpha1-antitrypsin deficiency, autoimmune hepatitis, and Wilson’s disease [4]. In the United States, the incidence of HCC has tripled over the past decade, but the five-year survival rate of 12% has not changed [4]. The poor prognosis associated with HCC is due to multiple factors, some of which include: (1) occurrence usually in the background of severe liver disease; (2) lack of effective therapeutics for advanced disease; and (3) aggressive and heterogeneous tumor biology [5,6,7]. Cures are possible if HCC is diagnosed early and treated with resection, liver transplantation, and/or ablation [8]. In this review we will discuss different approaches (Table 1) to targeting HCC including targeting the tumor anatomically and targeting the tumor biology. We will also review emerging biomarkers and molecular targets which promise to improve early detection and treatment of late-stage disease.Representative articles were selected from references found by searching Pubmed for the following key words: Targeting HCC anatomically section: “HCC”, “HCC and surgical resection”, “HCC and anatomical resection”, “liver transplantation and HCC”, “living-donor liver transplantation”, “percutaneous ethanol injection”, “radiofrequency ablation”, “microwave ablation”, “transarterial chemoembolization”, “transarterial radioembolization”, “cryoablation”, “irreversible electroporation”, “laser ablation”, and “high-intensity focused ultrasound;” Targeting HCC tumor biology section: “sorafenib and HCC”, and “kinase inhibitors and HCC”; Future directions section: “serum biomarkers and HCC”, “AFP and HCC”, “DKK1 and HCC”, “microRNAs and HCC”, “metabolomics and HCC”, “gene-expression signatures and HCC”, “immunotherapy and HCC”, “JX-594”, “CTLA-4 and HCC”, and “glypican-3”.The goal of surgical resection is to remove the tumor while preserving as much liver function as possible to prevent postoperative liver failure [9]. Surgical resection is one of the most effective treatments for patients with HCC, yet more than 70% of patients are ineligible [10]. Factors contributing to ineligibility include: extrahepatic metastasis, multiple and bilobar tumors, involvement of the main bile duct, or presence of tumor thrombus in the main portal vein and/or vena cava [11].The selection of appropriate candidates for resection not only involves the assessment of feasibility of complete tumor resection, but also the remnant liver function and a prediction of how much liver volume can be safely removed. Ideal candidates for resection have HCC without cirrhosis, usually in the context of HBV infection. However, such cases are rare and account for only 5% of patients in Western countries [8]. Most patients with cirrhosis have a high morbidity and mortality following anesthesia and surgery [12]. The use of Child-Pugh classification, model of end-stage liver disease (MELD), or clearance of indocyanine green to predict the risk of postoperative complications can be informative, but has not yet been proven to be reliable [13,14]. The presence of portal hypertension assessed by a hepatic venous pressure gradient ≥10 mmHg or suggested by the presence of a platelet count below 100,000/µL [3], splenomegaly or history of varices, has been reported to be a significant predictor of postoperative hepatic decompensation [8,14].In patients with normal liver function, it is usually considered safe to remove up to 70% of the total liver volume (TLV) [9]. However, the regenerative capacity of the liver is significantly decreased in patients with cirrhosis, and at least 50% of TLV should be preserved [9]. For cases in which the predicted remnant liver volume is below safety limits, preoperative portal vein embolization can stimulate liver hypertrophy to increase future remnant liver size [15].Operative considerations include the extent of resection (limited vs. anatomical) and method (open vs. laparoscopic). Since HCC is assumed to metastasize to other sites via the portal vein, Makuuchi et al. introduced the practice of “anatomical resection” or tumor removal with its portal tributaries by segmentectomy [16]. For tumors between 2 to 5 cm sizes, anatomical resections achieve significantly better disease-free and overall survival than limited resections [9,17,18]. For patients with tumors <2 cm size and poor liver function, limited resections are preferred since small tumors have a low risk of dissemination [9,11]. When considering open vs. laparoscopic resections, multiple studies have shown long-term oncologic outcomes are similar. However, laparoscopic resections are associated with reduced blood loss, postoperative complications, and length of hospital stay [19]. Despite these advantages, laparoscopic resections have numerous technical challenges and should be performed only by experienced surgeons [20].In a study of 6785 cirrhotic patients treated by liver resection, the Liver Cancer Study Group in Japan reported that short-term survival rates are good (one-, three-year 88%, 69%), but long-term survival rates are poor (five-, 10-year 53%, 28%) [21]. High rates of tumor recurrence (>80% five years after resection) contribute to poor long-term survival [22]. Preventing recurrence with neoadjuvant or adjuvant therapies has had limited success [8]. Randomized controlled trials (RCTs) using preoperative hepatic artery chemoembolization or adjuvant systemic chemotherapy have not improved overall survival [23,24,25,26]. In fact, Ono and colleagues reported that systemic chemotherapy actually correlated with a lower disease-free and overall survival [27]. Immunotherapy, internal radiation, and differentiation therapy (retinoids) may lengthen disease-free survival after resection, but these therapies require testing in large RCTs [8,23,28,29,30,31,32]. The oral multikinase inhibitor sorafenib has proven benefit in the treatment of non-resectable HCC. Whether sorafenib can reduce recurrence rates is under investigation in the Sorafenib as Adjuvant Treatment in Recurrence of Hepatocellular Carcinoma (STORM) trial [33].Current treatment approaches for patients with HCC.1: Emerging indication, but not widely used. Abbreviations: TLV, total liver volume; year, year; tx, treatment; pts, patients; w/, with; CR, complete response; PR, partial response; LDLT, living donor liver transplantation; ECD, expanded criteria donor.Liver transplantation (LT) is one of the most effective therapeutic options for patients with HCC as it removes both macroscopic and microscopic tumors and treats the underlying liver disease [11]. Before 1996, LT was reserved for patients with unresectable large or multifocal HCC. The results of such LTs were disappointing due to the high rate of recurrent disease in the new allograft and poor survival [82,83]. A landmark publication by Mazzaferro et al. established the Milan criteria by demonstrating that patients who have either one tumor <5 cm in diameter or 2–3 tumors each with a diameter of <3 cm have lower rates of disease recurrence [37]. Furthermore, patients transplanted within the Milan criteria have a five-year survival (70%–80%) similar to patients transplanted for non-HCC indications [37,39].Due to the scarcity of donor organs, selecting patients who will benefit most from LT has promoted strict adherence to the Milan criteria. In the United States, LT waitlist priority (currently starting MELD equal to 22 with additional points every three months) is only given to HCC patients within the Milan/T2 staging criteria [84,85,86,87]. However, multiple centers have reported acceptable outcomes when transplanting patients outside of the Milan criteria [48]. The University of California, San Francisco (UCSF) group transplanted patients with single tumors <6.5 cm or 2–3 tumors <4.5 cm with a total diameter <8 cm (UCSF criteria) and reported excellent survival [48]. At our institution we found similar five-year survival rates for patients transplanted within the Milan vs. UCSF criteria (79% vs. 64% p = 0.061) [49]. Nevertheless, the question still remains whether expanded criteria which results in slightly lower survival rates can justify the use of scarce donor organs [88]. Bruix and colleagues proposed transplantation for HCC patients should only be considered when patients’ five-year expected survival is at least 50% [89]. Yet, when comparing the survival benefit of patients transplanted outside the Milan criteria to the harm inflicted on other patients on the waiting list, Volk and colleagues proposed a five-year expected survival cutoff at 61% [90].While the time from listing to transplantation varies based on geographic location, many HCC patients experience tumor progression and drop out from the waiting list. The UCSF group showed the probability of dropout at six, 12, and 24 months to be 7.3%, 25.3%, and 43.6% [40]. To lower dropout rates, bridging treatments such as radiofrequency ablation, transarterial chemoembolization, or percutaneous ethanol injections are recommended, especially if waiting time is expected to exceed six months [91,92,93]. In fact, waiting more than six months on the transplant list drastically improved post-transplant survival as it seemed to select out the tumors with poor biology [94]. Surgical resection prior to transplantation is also an option. Belghiti et al. demonstrated that the resection of tumors within the Milan criteria did not increase transplantation surgery risk, nor reduce post-transplant survival [95].The most effective strategy to reduce waitlist dropout is to expand the donor pool. Use of marginal or extended criteria livers (non-heart beating donors, split livers, domino transplants from patients with amyloidosis, and advanced aged-donors) has expanded the donor pool but not enough to significantly reduce wait times [8]. Living donor liver transplantation (LDLT) is considered a feasible alternative to cadaveric liver transplantation since it eliminates the need to wait and patients with HCC are often suitable candidates for small allografts [7]. Survival rates for patients undergoing LDLT are similar to cadaveric donor transplant [50]. When following UCSF criteria, a multicenter study in Korea showed three-year survival for LDLT was 91% compared 88% for cadaveric donors [96]. Despite these favorable outcomes for the recipient, the risk to the living donor is not negligible. Postoperative complications arise in 20%–40% of donors and the risk of mortality is 0.3%–0.5% [50,97].HCC recurrence occurs in 8%–18% of patients after transplantation and is associated with a median survival of only nine months [38,49,98]. Identifying adjuvant therapies to prevent recurrence is an urgent need. Studies investigating systemic cytotoxic chemotherapy after transplantation have shown conflicting results, but the current consensus is that cytotoxic chemotherapy has failed to show major improvements in disease-free or overall survival [85,99,100,101]. Switching post-transplant immunosuppression from calcineurin inhibitors (CNI) to mTOR inhibitors may lower rates of recurrence [102]. CNIs can activate pro-tumorigenic pathways while mTOR inhibitors have both anti-proliferative and anti-angiogenic effects [103,104,105]. Encouraging results from multiple uncontrolled clinical studies have shown patients on sirolimus-based immunosuppressive protocols have a higher disease-free and overall survival compared to patients on CNI-based protocols [106,107,108,109]. However, a recent retrospective report comparing tumor recurrence and survival of >1000 patients transplanted for HCC found no differences between sirolimus users vs. non-users [110]. Perhaps the most promising adjuvant option is sorafenib [77,111]. At UCLA, in a case-controlled match study, Saab et al. showed sorafenib was well tolerated in transplant recipients and extended the disease-free survival (sorafenib 85.7% vs. control 57.1%) [47]. Based on these results, we started the POST trial, a phase II randomized, blinded, multicenter prospective study (NCT01624285) to determine if sorafenib is effective in preventing recurrence in high-risk HCC patients’ post-transplant.Since most patients within the Milan/UNOS criteria undergoing transplant will not have tumor recurrence, prognostic indicators are needed to determine who will benefit from active surveillance and adjuvant therapy. Tumor size and pathologic features including differentiation, presence of vascular invasion, and nuclear beta-catenin localization have been shown to be independent predictors of recurrence [41,102,112,113]. An evaluation of molecular signatures based on tumor expression of 20 metastasis-associated microRNAs [42], a comparison of microRNA expression between tumor and adjacent benign tissue [43], or the tumor expression of five genes (HN1, RAN, RAMP3, KRT19 and TAF9) [44] can also predict prognosis. Levels of the serum markers alpha-fetoprotein (AFP) and des-gamma carboxyprothrombin (DCP) correlate with tumor recurrence post-transplant [45,114]. Still, no single pathological or serum marker has proven >90% sensitivity and specificity in predicting recurrence, and multiple groups have proposed a combination of markers can better predict prognosis [46,115]. Recently, we developed a nomogram incorporating laboratory values (pre-transplant AFP, total cholesterol, neutrophil-to-lymphocyte ratio), pathologic features (nuclear grade, vascular invasion), radiographic tumor size, and response to downstaging therapy that showed excellent accuracy in predicting recurrence in 865 liver transplant recipients [46].Anatomical targeting of HCC by locoregional therapy is the next best treatment option for patients who are ineligible for surgical resection, liver transplant, or those who require downstaging or bridging therapy prior to transplantation. There is a growing list of locoregional approaches which can be divided into ablative and transarterial therapies. Local ablation involves killing tumor cells by chemical (ethanol, acetic acid) or thermal means (e.g., radiofrequency, microwave frequency, cryotherapy, laser) [56]. Transarterial therapies are characterized by arterial injection of therapeutic agents into the tumor followed by the occlusion of tumor blood supply [56].Percutaneous ethanol injection (PEI) is considered a safe, inexpensive, and effective ablative therapy for small HCC tumors [8,60,116]. Under ultrasound or CT guidance, absolute ethanol is injected into the tumor causing tumor coagulation necrosis [116]. Depending on the tumor size, injections are repeated weekly for six to eight weeks. Other agents, such as acetic acid, have been tried, but outcomes are not superior to ethanol injections [117,118]. In Child class A patients with tumors under 5 cm, PEI results in five-year survival rates up to 50% [51,52,116,119]. However, high rates of tumor recurrence (40% at two years for tumors >3 cm) and multiple treatment sessions required have limited the use of PEI in current clinical practice [120].Radiofrequency ablation (RFA) has replaced PEI at most centers due to superior efficacy and shorter treatment times. RFA is performed percutaneously by advancing an electrode into the tumor and delivering energy in the form of radiowaves. The energy induces the rapid vibration of ions in the tissue resulting in frictional heat causing thermal destruction (coagulative necrosis) of the tumor [60]. Five RCTs have compared the efficacy of RFA to PEI. All studies reported lower rates of tumor recurrence in RFA-treated patients. Three studies demonstrated RFA confers a survival benefit for patients with tumors >2 cm [53,54,55,121,122]. However, drawbacks of RFA include higher cost, increased incidence of adverse events, and limitations depending on tumor location. In a meta-analysis of four RCT complications such as peritoneal bleeding, tumor cell seeding, or intrahepatic abscesses were observed in 4.3% of RFA-treated patients vs. in 2.7% of PEI-treated patients [123]. Since the mechanism of RFA-induced necrosis is dependent on heat, the cooling effect of blood flow makes RFA less effective for highly vascular HCC tumors or tumors adjacent to blood vessels [60]. RFA is also contraindicated for subcapsular tumors and tumors in close proximity to the gallbladder. Clinical experience has shown these locations are associated with incomplete tumor ablation and risk of damage to adjacent structures [58,59,116].Similar to RFA, microwave ablation (MWA) destroys tumor tissue with heat. Instead of agitating ions within tissue, MWA uses high frequency electromagnetic radiation to heat intracellular water molecules resulting in coagulative tumor necrosis [56]. MWA is advantageous compared to RFA for several reasons including the ability to achieve higher intra-tumoral temperatures, ablate larger volumes, and it is not constrained by proximity to blood supply [56,116,124]. Still, there is no conclusive evidence showing MWA is more effective than RFA [57,125,126]. Several recent studies proposed that MWA may be an effective treatment for medium-to-large HCC tumors (>4 cm) [127,128,129,130]. Additional RCTs are needed to determine which patients are most likely to benefit from MWA.As HCC tumors grow (>2 cm) they become more vascularized and receive blood flow almost entirely from the hepatic artery [72]. Transarterial chemoembolization (TACE) takes advantage of this dependence on the hepatic artery and is the treatment of choice for tumors greater than 4 cm or multifocal HCCs [7]. Performed under angiography, TACE involves advancement of a catheter into the hepatic artery, injection of a chemotherapy emulsion (usually doxorubicin and lipiodol), followed by arterial embolization, most frequently with 1 mm gelfoam cubes [8]. The survival benefit of TACE has been debated due to mixed results in several RCTs [69,70,131,132]. However, a meta-analysis confirmed TACE, in comparison to conservative management, increased the two-year survival of patients with multifocal HCC, preserved liver function, and lack of extrahepatic spread and vascular invasion [133]. In contrast, TACE has failed to show survival benefit in patients with decompensated cirrhosis (Child-Pugh B/C) [134,135].While TACE can lengthen survival in select patients, ultimately 70%–80% will die due to tumor progression [70]. One way to potentially improve the efficacy of TACE is to increase the concentration and duration of the chemotherapy that reaches the tumor with drug-eluting beads (DEBs). In a European multi-center phase II prospective randomized trial (PRECISION V) the DEB-TACE group had fewer adverse events and a higher complete response rate (CR) and objective response rates (ORR) compared to the TACE control group (CR 27% vs. 22% and ORR 52% vs. 44%) [73]. Another promising approach is combining DEB-TACE with anti-angiogenic agents. Instead of undergoing necrosis, resistant tumors respond to TACE-induced hypoxia by increasing production of pro-angiogenesis factors such as VEGF [135,136]. Several trials have investigated the synergy of DEB-TACE and sorafenib, which inhibits VEGFR [74]. While initial efficacy data is promising, questions remain regarding the optimal dosing schedule (sequential, interrupted, or continuous) of sorafenib [75]. Results from two ongoing phase III RCTs (SPACE study and ECOG 1208), each with different dosing schedules, will provide insight to this question.HCC is a radiosensitive tumor; however, external beam radiotherapy is contraindicated for patients with cirrhosis due the risk of radiation-induced hepatitis [137]. To reduce the radiation of normal liver parenchyma and selectively target HCC, a type of brachytherapy involving the injection of yttrium-90 microspheres into the tumor-feeding vessels of the hepatic artery is used. Unlike TACE, transarterial radioembolization (TARE) maintains the patency of the hepatic artery and is suitable for patients with portal-vein thrombosis, a contraindication for TACE [66]. In a retrospective study by Sangro et al., TARE was associated with a median survival of 15.4–16.6 months in patients who were poor candidates for TACE (bilobar, bulky disease, multiple >5 tumors) [67,68]. Patients with portal vein thrombosis were also treated in the study, and while survival was poor, it was comparable to survival associated with standard-of-care chemotherapy [66,67,68]. Several matched patient cohort studies have demonstrated TARE in comparison to TACE results in increased OS, relative response rate, and greater effectiveness in downstaging prior to transplantation [138,139,140]. RCTs comparing TACE and TARE are ongoing and will hopefully provide consensus if and when TARE is superior to TACE.Although less commonly used, cryoablation, irreversible electroporation, laser ablation, and high-intensity focused ultrasound have important advantages to consider when optimizing treatment for HCC patients (Table 2). Cryoablation is performed percutaneously by advancing cryoprobes into the tumor and using either liquid nitrogen or argon gas to rapidly freeze tumor tissue [61]. Freezing to −35 °C induces the formation of ice crystals which damage cell membranes and organelles, leading to cell death [61]. Two recent analyses, one RCT and one prospective study, reported that the safety profile and outcomes of cyroablation were similar to RFA and MWA in the treatment of HCCs <2 cm [141,142]. However, cryoablation was superior in achieving local tumor control for tumors >2 cm. While additional large trials are needed, cryoablation may become a first-line ablative therapy for medium-sized tumors.Emerging ablation methods.Irreversible electroporation (IRE) is emerging as an excellent approach for HCC tumors near vital structures. A percutaneous approach is used to position electrodes around the tumor which give multiple, millisecond high voltage (>500 V/cm) electrical pulses. This results in the irreversible breakdown of cell membranes and hemorrhagic necrosis [62]. Since IRE is not temperature-dependent, it can be used on tumors adjacent to blood vessels. Importantly, IRE has been shown to effectively ablate tumors within the liver hilum while preserving the structure and functionality of the hepatic artery, portal vein, and bile duct [62,143,144]. There are no clinical trials yet comparing IRE to other ablation techniques; however, prospective studies suggest outcomes are similar [149]. For patients with tumors <3 cm in locations that would be too risky to treat with other ablative techniques, IRE seems to be a promising approach.Another percutaneous approach is laser ablation (LA). LA uses thin fibers (~300 μm) to deliver near-infrared light to tumor tissue. Upon absorption, the light is converted to heat, resulting in hyperthermia-induced cell death [63]. Retrospective studies have shown LA is safe and, more recently, a RCT showed LA was equivalent to RFA in achieving complete ablation of tumors <4 cm [145,150]. One significant advantage of LA is its low cost (70% less expensive compared RFA) [145].Unlike other modalities, high-intensity focused ultrasound (HIFU) is completely extracorporeal and lacks the risks of bleeding and tumor seeding with the direct puncture of tumors. Under MRI guidance, ultrasound beams are focused to the depth of the tumor and the absorption of acoustic energy generates heat, which results in tumor coagulation necrosis [64]. A group at the University of Hong Kong has published multiple small prospective studies suggesting that, in addition to being non-invasive, HIFU is safe for patients even with advanced liver disease (Child-Pugh class C) and may be considered as a bridging treatment to transplantation [146,148] (Table 3).Comparison of ablation methods.Abbreviations: +, recommended; −, not recommended/no evidence supporting use; ±, may be considered; PEI, percutaneous ethanol injection; RFA, radiofrequency ablation; MWA, microwave ablation; TACE, transarterial chemoembolization; TARE, transarterial radioembolization; CRYO, cryoablation; IRE, irreversible electroporation; Laser, laser ablation; HIFU, high intensity focused ultrasound.More than 70% of patients with HCC present with advanced disease and are poor candidates for anatomical targeting (resection, transplantation, or locoregional therapies) [33]. Historically targeting HCC tumor biology with chemotherapy has been ineffective. HCC is inherently chemoresistant and the altered drug metabolism of cirrhotic livers makes many chemotherapies highly toxic [78]. However, in 2007, sorafenib, a multi-kinase inhibitor targeting VEGFR, PDGFR-B, c-kit, FLT3, and cRAF, became the first systemic therapy approved for advanced HCC (Child-Pugh A/B, unresectable, metastatic or with vascular invasion) [79]. In the SHARP study, a phase III, randomized, double-blind, placebo-controlled trial, patients treated with sorafenib vs. placebo had a significantly improved OS (10.7 vs. 7.9 months p < 0.001) [77]. The side effect profile of sorafenib was tolerable and included hand-foot skin reactions (8%), diarrhea (8%), and fatigue (3%) [77]. Due to the impressive response of HCC to sorafenib, multiple studies are investigating the efficacy of sorafenib in other stages of disease. In particular, trials are under way using sorafenib following liver transplantation, resection, TACE, and TARE [76,78,151].The FDA-approval of sorafenib represents a great leap for the treatment of advanced HCC; yet therapeutics are needed for patients with intolerance or acquired resistance to sorafenib. Over the past five years, phase III RCTs have compared the efficacy of multiple other kinase inhibitors. In the first-line setting, anti-angiogenic agents (sunitinib, brivanib, and linifanib) targeting VEGFR and PDGFR or combinations of sorafenib with anti-proliferative agents (erlotinib) targeting EGFR failed to show superiority to single-agent sorafenib with respect to overall survival [152,153,154,155]. Likewise, in the second-line setting, the VEGFR/FGFR inhibitor brivanib and the mTOR inhibitor everolimus did not meet primary endpoints of demonstrating superiority in overall survival when compared to placebo [156,157].The disappointing results of numerous phase III RCTs using kinase inhibitors could be due to wrong molecular targets, high toxicities in patients with underlying liver cirrhosis, or poor patient selection [80]. Based on trials using brivanib and erlotinib, one might assume that inhibiting FGFR or EGFR pathways would have low antitumoral potency in HCC. On the contrary, FGFR and EGFR might still be effective targets, but only in selected patients whose tumors exhibit activation of such pathways. A recent phase II trial using tivantinib, a c-MET tyrosine kinase inhibitor, in the second-line setting provides rationale for treatment stratification based on tumor biomarkers [158]. Initial analyses showed the time to progression was similar between the tivantinib vs. placebo-treated group (1.6 vs. 1.4 months). However, when the tivantinib-treated group was stratified based on immunohistochemical detection of c-MET expression, patients with high c-MET-expressing tumors had a significantly longer time to progression (2.7 months). To enable better patient selection, predictive biomarkers are being identified for other therapies. Examples include high AFP levels as an indicator of response to the VEGFR2 inhibitor, ramucirumab, and genomic amplifications of VEGFA or FGF3/4 as markers of sensitivity to sorafenib [81,159,160].Despite numerous treatment options, HCC is still one of the most lethal cancers worldwide. Anatomical targeting of HCC is the most effective treatment option; however, less than 30% of patients are eligible due to advanced tumor stage at diagnosis. Yet, even if eligible for surgical and/or locoregional therapies, the high recurrence rate of HCC impedes long-term survival. Although sorafenib was recently approved for advanced HCC, the median survival of patients is only one year. Hence, there is a critical need to: (1) improve methods for early detection so that more patients are eligible for curative therapies; (2) identify prognostic markers to improve patient selection and surveillance postsurgical or locoregional therapies; and (3) investigate the molecular mechanisms driving HCC progression to identify new therapeutic targets (Figure 1).Diagnosis of HCC without pathologic confirmation is currently based on serum AFP and imaging (ultrasound, MRI, CT). AFP levels are associated with tumor size, and only about two-thirds of HCC patients with tumors <3 cm will have elevated AFP levels [161]. Moreover, the specificity of AFP for HCC is low since elevated AFP is also detected in the serum of patients with cirrhosis and hepatitis [10]. MRI and CT are expensive, and ultrasound, while specific, is highly operator-dependent and has poor sensitivity in detecting HCC in patients with underlying cirrhosis [162]. Due to risks associated with biopsies in patients with cirrhosis and the need for cost-effective tests, research has been focused on identifying proteins, nucleic acids, and metabolites that could enable HCC diagnosis through serological testing.Proposed integration of biomarkers into HCC treatment. (Left) Few patients present with HCC tumors amenable to curative therapies, so emphasis has been placed on biomarkers to detect early-stage HCCs. Proteins, nucleic acids, and metabolites released by the tumor into circulation can provide a non-invasive method of early detection. (Middle, Right) Most treatment decisions for HCC patients are currently based on tumor size and liver function. However, these parameters cannot accurately predict optimal therapies for all patients and, in particular, those with metastatic disease. Tumor gene expression signatures can characterize tumor biology and aid in predicting prognosis and treatments.The search for serum protein markers for early detection of HCC has identified numerous promising candidates including: des-gamma-carboxy-prothrombin (PIVKA-II), glypican-3, the ratio of glycosylated AFP (L3 fraction) to total AFP, alpha fucosidase, Dickkopf-1 (DKK1), and osteopontin [163,164,165,166,167,168]. The majority of these candidates have yet to show superiority to AFP. However, a recent retrospective trial comparing serum DKK1 to AFP found DKK1 could enhance the diagnostic accuracy of HCC [167]. Serum DKK1 was not only able to distinguish HCC from chronic liver disease, but could also detect HCC in early-stage patients who had normal AFP levels [167]. While the data for DKK1 is intriguing, additional studies are needed before it can be accepted as a valid marker for HCC screening. Rather than reliance on just one serum marker, a combinations of markers (i.e., AFP, osteopontin, and DKK1) may provide superior sensitivity and specificity in detecting HCC [169].The detection of circulating nucleic acids, particularly microRNAs (miRNAs), has the potential to be used in HCC diagnosis. miRNAs are small, non-coding RNAs which negatively regulate gene expression and have important roles in hepatocarcinogenesis [170]. In addition to their presence in HCC tumor tissue, some miRNAs can be found in systemic circulation, providing rationale to investigate their use in HCC diagnosis. In a large cohort study of 934 patients (healthy, chronic HBV, cirrhosis, and HBV-related HCC), a panel of seven miRNAs (miR-122, miR-192, miR-21, miR-223, miR26a, miR-27a, and miR-801) had a sensitivity of 83% and specificity of 94% in diagnosing HCC [171].Nomograms combining clinical, radiology, laboratory, and pathology results can also predict HCC recurrence after resection or transplant. These predictions can guide adjuvant therapy and tumor surveillance.In comparison to hepatocytes in cirrhotic or normal livers, metabolic profiling of HCC cells has identified alterations in pathways associated with phospholipid, fatty acid, and bile acid metabolism [172,173]. These differences have instigated the search for cancer-associated metabolites in body fluids for predicting/detecting HCC development. Using a mass spectrometry-based approach, Wang and colleagues profiled the serum metabolites in patients with HCC, liver cirrhosis, and normal livers [172]. The metabolite canavaninosuccinate was significantly increased in the serum of patients with HCC, but decreased in patients with cirrhosis. Furthermore, canavaninosuccinate was able to predict HCC with a sensitivity of 80% and specificity of 100%, which is much higher than AFP. All patients in this study had HBV-associated HCC, and it will be important to investigate if the same metabolites are increased in HCC associated with other etiologies.HCC tumor recurrence occurs in approximately 70% of patients treated with resection or ablative therapies [34]. This major clinical dilemma underscores the need for predictive markers to aid in patient selection and in guiding surveillance/adjuvant treatment (Figure 1). Pathological characteristics such as vascular invasion and multifocality are independent predictors of recurrence, but are difficult to evaluate preoperatively [35]. Serum biomarkers such as AFP, circulating DNA, and miRNAs are also being investigated for their predictive and prognostic potential [174,175]. Perhaps the most promising biomarkers for predicting recurrence are molecular signatures based on HCC tumors or the tumor microenvironment.Gene expression profiling of resected HCC tumors has identified molecular signatures with prognostic potential. Over 20 gene signatures associated with HCC have been reported, including genes associated with survival [176], metastasis [36], and early recurrence [177]. The clinical utility of these signatures is controversial as their prognostic power has yet to be validated in large studies [35]. Nault et al., published a five-gene score based on the expression of HN1, RAN, RAMP3, KRT19, and TAF9 that could predict prognosis (overall survival, early tumor recurrence, and risk of death after recurrence) in patients after resection more accurately than previously reported molecular signatures [44]. The authors also developed a “clinico-molecular” nomogram combining the five-gene score, Barcelona clinic liver cancer (BCLC) classification, and microvascular invasion to stratify patients based on low, medium, and high rates of recurrence. While still needing validation in prospective studies, the five-gene score and nomogram might be applicable in selecting candidates for liver transplantation. For instance, if a patient is outside of Milan criteria but the tumor has a low risk five-gene score, consideration of a liver transplant may still be warranted and vice versa.One-third of HCC recurrences will occur more than two years after surgical resection [178]. Such late recurrences are considered to be de novo tumors rather than metastasis from the primary tumor [179]. Molecular signatures derived from the surrounding non-tumor cirrhotic tissue instead of the primary tumor can predict late recurrence. Budhu et al. used non-cancerous hepatic tissue from patients with venous metastasis to identify a 17-gene signature enriched in immune and inflammatory response genes [180]. This signature could predict metastasis, overall survival, and tumor recurrence more accurately than clinical parameters such as microvascular invasion and the Child-Pugh score. However, the 17-gene signature was derived from patients only with HBV-associated HCC tissue. Using tissues from patients with different HCC-associated etiologies (HBV, HCV, and EtOH), Hoshida et al. reported a 186-gene poor-survival signature enriched in adjacent tumor tissue which can predict overall survival and recurrence [179]. The question then arises as to which tissue should be subjected to molecular profiling to predict recurrence. Perhaps a combination of both: profiling the tumor might be best for predicting early recurrence, while profiling the adjacent non-tumor tissue could predict late recurrence [35].Since HCC occurs in the background of diverse etiologies, a thorough understanding of the precise molecular factors driving the disease has been difficult. In a European cohort of 24 HCC tumors, genomic sequencing identified five to 121 mutations per tumor [6]. In addition to the heterogeneity in the number of mutations, there was no common mutation found in the majority of tumors. Identification of a common druggable molecular target, similar to BRAFV600E in melanoma or BCR-ABL in chronic myelogenous leukemia, is unlikely in HCC. Nevertheless, subtypes of HCC are becoming apparent through large-scale genomic and transcriptomic sequencing. Targeting-altered cellular pathways in these subtypes may yield novel therapeutic strategies.Whole genome and exome sequencing of ~400 tumors identified TERT, TP53, B-catenin, and ARID1A as the most frequently mutated genes in HCC [6,181,182,183,184]. The prevalence of some of these mutations is associated with etiology. TP53 mutations occur in >50% of HBV-related HCC, while B-catenin mutations are more frequent in the background of EtOH-associated HCC [6,181,182]. Common chromosomal gains are less prevalent but result in amplifications of cyclin D1 (11q13), FGF19 (11q13), VEGFA2 (6p21), Myc (8q), and Met (7q31) genes [185,186,187]. Chromosomal losses of CDKN2A (9p) and IGF2R (6p) have also been reported [188,189]. Functional classification of these mutated/amplified genes has found key pathways altered in HCC. Pathways include: Wnt/B-catenin, PI3K-AKT-mTOR, MAPK, telomere maintenance, cell cycle regulation, chromatin remodeling/epigenetic regulation, IGF signaling, and Il-6/JAK-STAT [174,181]. Evidence from mouse models of HCC have validated the functional importance of the Wnt/B-catenin pathway, and Myc, Met, and cyclin D1 genes [190,191,192,193]. Which of these pathways are oncogenic “drivers” and should be therapeutic targets for HCC remains unknown.Sequencing has identified genomic alterations in HCC, but the question still remains as to how these molecular analyses can translate into effective therapeutic strategies (Table 4). One approach is to target mutationally activated pathways with specific pathway inhibitors. Since RAS mutations drive activation of the RAF-MAPK-MEK cascade, a trial is ongoing targeting RAS-mutated HCC with the MEK inhibitor, refametinib (NTC01915589) [80]. Many of the pathways activated in HCC such as the Wnt/B-catenin pathway or TP53 alterations are not currently considered druggable targets. An alternative approach is to use the pathway activation or molecular subgroup information as a biomarker to predict response. For instance, Finn et al. used the signatures published by Lee et al. to classify human HCC cell lines into subgroups, having either a hepatoblast (HB) or hepatocyte (HC) signature [176,194,195]. Interestingly, the HB cell lines were sensitive to the SRC/ABL tyrosine kinase inhibitor, dasatinib, while HC cell lines were not. Hence, one could hypothesize that patients with tumors having the HB signature would respond to dasatinib. Since specific genetic alterations are associated with different environmental exposures (EtOH, HBV), it will also be useful to determine if HCC cells respond to targeted therapeutics in an etiology-specific manner.Emerging therapeutic targeting approaches for HCC.Abbreviations: CT, clinical trials; HDAC, histone deacetylase inhibitor; ab, antibody.Immunotherapy is emerging as a treatment approach for HCC (Table 4). Since the liver does not metabolize most immunotherapeutic drugs, they are appealing for patients with cirrhosis [212]. Promising immunotherapeutics for HCC include oncolytic viruses, CTLA-4 blockade, and tumor-antigen specific antibodies. The oncolytic virus JX-594 specifically infects and lyses tumor cells and expresses GM-CSF to stimulate an anti-tumor T cell response [212]. In a phase II trial of 30 patients with advanced HCC, an intra-tumoral injection of high dose JX-594 was associated with a longer median survival compared to low-dose JX-594 (14.1 vs. 6.7 months) [213]. CTLA-4 blockade can increase tumor-specific T cell activity by preventing T cell exhaustion. In patients with advanced HCV-associated HCC, the CTLA-4 antibody tremelimumab had a tolerable toxicity profile and was associated with a 76% disease control rate [210]. The surface glycoprotein, glypican-3 (GPC3) is overexpressed in more than 70% of HCC tumors and functions as a regulator of both Yap and Wnt signaling pathways [207,208,209]. Blocking GPC3 with an antibody was shown to be safe in patients with advanced HCC. In addition, patients with high-expressing GPC3 tumors vs. low-expressing GPC3 tumors had a prolonged time to progression (26 vs. 7.1 weeks) [211].Selecting the optimal approach to target HCC is challenging. Reasons include aggressive tumor biology, presentation in the context of severe liver disease, and the lack of universally accepted treatment guidelines. As we have reviewed, the majority of clinical decisions are currently based on tumor size, number of lesions, and liver function. For patients with small tumors and preserved liver function, treatments of choice are either resection or PEI/RFA. In decompensated cirrhotics having tumors within the Milan criteria, liver transplantation is the best treatment modality. Large or multifocal tumors are usually targeted first with TACE and poor responders or those with severe liver impairment are offered sorafenib. While this clinical approach has proven effective in some cases—in particular using the Milan criteria for selecting transplant candidates—the dismal prognosis for most patients with HCC highlights the need for new therapeutics and better patient selection for established treatment options. Fulfilling this need is the emergence of nomograms, and protein, nucleic acid, and metabolic biomarkers, which can accurately predict prognosis in order to guide treatment choices, post-treatment surveillance, and adjuvant therapies. Furthermore, genomic analyses have stratified HCC tumors into subtypes, and determining if these subtypes respond uniquely to therapy will be a tremendous advance to guiding treatment decisions. Combination approaches (surgery and targeted therapy) or immunotherapy hold promise to reduce rates of recurrence or act as bridges to resection or transplantation. Overall, it is clear that treatment strategies for HCC are evolving, and we are optimistic HCC will eventually change from being a death sentence to a manageable disease.C.F., R.W.B. and A.Z. wrote the manuscript. All authors discussed, edited and approved the final version.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Vitamin D has several extra calcemic effects. Vitamin D deficiency is highly prevalent in chronic obstructive pulmonary disease (COPD) patients but little is known about it’s association with lung function. Objective: To investigate whether supplementation with vitamin D could improve pulmonary function in COPD patients. Design: Before and after, double center, clinical trial. Setting: Hazrat Rasoul University Hospital, Tehran, and Imam Khomaini University Hospital, Ahvaz, Iran. Participants: 24 patients with mild to very severe COPD. Intervention: Loading dose of 300,000–600,000 International Units (IU) of vitamin D, then 50000 IU weekly for 12 weeks. Measurements: The outcomes included forced expiratory volume in one second (FEV1), forced vital capacity (FVC), vital capacity (VC), forced expiratory flow between 25%–75% of forced vital capacity (FEF 25%–75%), exercise capacity according to the six minute walk test(6MWT) and the saturation of oxygen during exercise. Results: The mean FEV1 (p-value = 0.866), FVC (p-value = 0.475) and VC (p-value = 0.425) were not significantly different before and after intervention. FEF 25%–75% did not improve with this intervention (p-value = 0.555). The vitamin D supplementation did not have any significant effect on the exercise capacity (p-value=0.175) or the saturation of oxygen (p-value = 0.635). Conclusion: Pulmonary function and exercise capacity did not improve with vitamin D supplementation in COPD patients.Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation due to inhalation of noxious particles or gases such as cigarette smoke. Airway inflammation is prominent in COPD [1]. With disease progression, FEV1 declines [2]. No treatment has been proven to inhibit COPD progression except smoking cessation [3].Vitamin D and parathyroid hormone are the regulators of calcium and phosphate but low concentration of vitamin D is associated with many diseases other than osteoporosis, including hypertension, ischemic heart diseases, type I diabetes, cancer, autoimmune diseases and infections. Also, there are significant associations between vitamin D status and death caused by diseases of the respiratory system, the digestive system, and endocrine, nutritional and metabolic diseases [4,5,6,7,8,9,10,11,12,13].There are some associations between vitamin D deficiency and COPD. Skaaby et al. reported an inverse association between vitamin D deficiency and COPD and vitamin D deficiency is present in 60% to 75% of patients with severe COPD [14,15].There is a strong relationship between serum concentration of 25-hydroxyvitamin D and FEV1 and FVC [16], but to the best of our knowledge there are few studies that have evaluated the effect of vitamin D supplementation on the improvement of lung function in COPD patients.The aim of this study is to investigate the role of vitamin D supplementation in improving lung function, exercise capacity and the saturation of oxygen during exercise in COPD patients.Our study was a before and after clinical trial. Eligible patients were current or ex-smokers or were exposed to noxious inhalents, and had a diagnosis of COPD according to the Global Initiative for chronic obstructive Lung Disease (GOLD) definition. They had no history of renal stone or renal failure or other diseases that interact with vitamin D metabolism and did not take vitamin D or calcium supplements. They had no recent history of exacerbation before inclusion.Baseline characteristics included serum levels of 25-hydroxy vitamin D, severity of airflow obstruction, six-minute walk test, and O2 saturation. We excluded patients who needed continued oxygen therapy during exercise, because we did not have suitable portable oxygen for continues oxygen therapy during 6MWT.The spirometery was performed before and after intervention with a standard spirometer (Ganshorn Company, Niederlauer, Germany).If the patients had a low level of serum 25-hydroxyvitamin D, they took a loading dose of 300,000 of vitamin D for insufficient (<30 ng/mL) and 600,000 IU for deficient (<20 ng/mL) levels intramuscularly and all patients took 50,000 IU weekly for 12 weeks per oral [17,18]. The serum level of vitamin D was checked afterwards and all of the variables were tested again. We evaluated hyper calcuria only in toxic levels of vitamin D (more than 80 ng/mL) [19]. The patients were excluded from the study if they did not want to continue the treatment or were noncompliant.We calculated the sample size with consideration of α = 0.05 and β = 0.8. A total of 18 patients were needed for rich statistically significant results. The included patients were 24. One patient died before termination of trial because of severe multi lobar pneumonia. Twenty-three patients continued the study. Paired sample t-test was used to analyze the results.The patients provided written, informed consent. The study was approved by the local ethics committee of the Iran University of Medical Sciences and registered with Iranian Clinical Trails Center (NCT 2015012720828N1).A total of 59 participants aged 35 to 78 years participated in this study. Forty-two patients had insufficient or deficient levels of 25-hydroxyvitamin D. We were able to follow 23 of them to initiate supplementation. The mean age of patients was 62 (SD = 12). Three patients were not cigarette smokers: one was a female water pipe smoker and the two others were exposed to noxious inhalants.One of the patients was in mild stage, eight in moderate stage,13 in severe and one in very severe stage of COPD. The mean serum level of vitamin D was 14 ng/mL (SD = 5) (Table 1).Baseline characteristics.Paired sample t-test analysis showed no significant differences before and after supplementation between FEV1 (p-value = 0.866), FVC (p-value=0.475), VC (p-value = 0.425) and FEF 25%–75% (p-value = 0.555; Table 2) (Figure 1).The mean distance that participants walked during 6MWT was 350 meters before and 360 meters after vitamin D supplementation, which was not significantly different (p-value = 0.175). The saturation of oxygen during 6MWT did not improve with supplementation.End points.Means of FEV1 (A), FVC (B), VC (C) and FEF 25%–75% (D) before and after supplementation.The purpose of this study was to evaluate the role of correction of serum vitamin D level on the pulmonary function of COPD patients. We could not show any effect of this intervention.A cross-sectional study by Peter N. Black and Robert Scragg reported a relationship between serum 25-hydroxyvitamin D and FEV1 and FVC but there was no difference in the FEV1/FVC ratio between the highest and lowest quintiles of serum level of vitamin D. In spite of a greater difference in FEV1 between the highest and lowest quintiles of vitamin D in those with diagnosis of chronic bronchitis or emphysema than for other participants, the interaction between the diagnosis and the serum vitamin D was not significant [16].This study had comparable results with a randomized clinical trial by A. Lehouck et al. [20]. The main goal of their study was the reduction of COPD exacerbation with a high dose of vitamin D. The annual rate of exacerbation in their study was 2.8. Frequent exacerbations may be associated with a more rapid decline of FEV1 [21] and may interfere with the likely effect of vitamin D supplementation on pulmonary function. Also, most of our participants were screened during outpatient visits and did not have exacerbation long before their inclusion and most of them had no history of hospitalization for COPD exacerbation before and during the study. Only one of our patients had exacerbation at the initiation of the study. However, we used a higher dose of vitamin D supplementation. In fact, there are some suggestions that higher doses of vitamin D are required to increase serum25-hydroxyvitamin D levels, which may be needed for extra calcemic effects [22,23].The result of a cohort study by K.M. Kunisaki et al. supports our study [24].Their cases and controls were continued smokers with rapid and slow lung function decline respectively. The baseline serum 25-hydroxy vitamin D levels were not predictive of subsequent lung function decline in a six-year follow up. Moreover, the results of some trials of vitamin D supplementation on its effect in other chronic diseases have been disappointing [25].There are some hypotheses for these results. There are several mechanisms of action from vitamin D in COPD: (1) calcemic effects; (2) lung tissue remodeling effects; (3) antimicrobial effects; (4) immune modulation effects; and (5) peripheral muscle function effects.The calcemic effect of vitamin D on the pulmonary function is related to its kyphosis effect, and its reversal needs a long time period in order to allow for lung tissue remodeling. Therefore, the short term follow up period of our study could not evaluate the vitamin D effects on these mechanisms.The absence of the immune modulatory effect of vitamin D may be due to the obstruction of small and larger airways in COPD, which is associated with the remodeling process, and seems to be irreversible. The presence of airway obstruction in the absence of accumulation of some type of inflammatory cells in stage 3 and 4 of COPD despite a cessation of smoking nine years earlier supports this idea [1].There were no significant differences in saturation of oxygen during exercise before and after vitamin D supplementation. This may be confounded by the exclusion of hypoxemic patients for the reason we described. Also, the mean distances that the patients could walk after the intervention were not longer, which supports the results of previous similar studies in heart failure patients [21,23].Our study had limitations. One was small sample size. In addition, this study was a before and after study. In this type of studies, the intervention is confounded by the Hawthrone effect. This effect can lead to an overestimation of the effectiveness of an intervention. However, we could not show any effect of our intervention and it seems that our results were not confounded by this effect.The technique of doing 6MWT did not fulfill the ATS guidelines. We did not use the Borg scale of dyspnea and the only end point in this test was the distance that the patient walked without severe dyspnea in his or her sense. Only one of our patients could not continue the test and stopped because of dyspnea.The other limitation was the short follow up period. Some of our patients were from other cities, or were from villages and we had difficulties with maintaining the study follow up. We believe the likely mechanisms of effect of vitamin D on the outcomes which we described would probably not have prevented us from arriving at our end points in the short follow up period.In conclusion, a high dose of vitamin D supplementation had no effects on the improvement of pulmonary function and the exercise capacity of COPD patients.We would like to thank our colleagues Amir Jamshidnezhad and Mohammadreza Akhond for their assistance in this article.Seied Ali Javad Moosavi and Maryam Haddadzadeh Shoushtari, performed the experimental work; Maryam Haddadzadeh Shoushtari wrote the manuscript. All authors discussed, edited and approved the final version.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Hepatocellular carcinoma (HCC) is highly resistant to currently available chemotherapeutic agents. The clinical outcome of HCC treatment remains unsatisfactory. Therefore, new effective and well-tolerated therapy strategies are needed. Natural products are excellent sources for the development of new medications for disease treatment. Recently, we and other researchers have suggested that the combined effect of natural products may improve the effect of chemotherapy treatments against the proliferation of cancer cells. In addition, many combination treatments with natural products augmented intracellular reactive oxygen species (ROS). In this review we will demonstrate the synergistic anticancer effects of a combination of natural products with chemotherapeutic agents or natural products against human HCC and provide new insight into the development of novel combination therapies against HCC.Hepatocellular carcinoma (HCC) represents the fifth most common cancer and third most common cause of cancer death [1]. Unfortunately, HCC tumors are highly resistant to currently available chemotherapeutic agents. The clinical outcome of HCC treatment remains unsatisfactory. Therefore, new effective and well-tolerated therapy strategies are urgently needed.The mechanisms underlying the pathogenesis and development of HCC are complex and heterogeneous. They involve multiple cellular signaling pathways, including the Wnt/β-catenin signaling pathway [2], p53 expression [3], Ras/mitogen-activated protein kinase (MAPK) pathway [4], activation of the retinoblastoma protein (pRb) [5], and signal transducer and activator of transcription 3 (STAT3) [6]. These molecular pathways modulate the expression of key genes that are involved in the regulation of cell proliferation, apoptosis, and angiogenesis and that are participants in the processes of induction, progression, and metastasis of hepatic cancer. Thus, each serves as a potential target for novel therapeutics.Natural products are excellent sources for developing new medications for treating diseases. Recently, the minimal side effects of natural products in anticancer treatments have been recognized. A significant number of drugs that are used to treat cancer are of natural origin [7,8]. The number of natural compounds with anticancer activities that have been discovered and tested in vitro and in vivo is increasing exponentially. However, many of these natural compounds have failed to gain favor as single-agent anticancer drugs because of a lack of potency. Conventional chemotherapy plays an important role in the treatment of cancers, but clinical limitations exist because of dose-limiting side effects and drug resistance. Therefore, combination treatment of chemotherapeutic agents and natural compounds are considered to be a promising therapeutic strategy with a higher clinical efficacy. Recently, more natural compounds, including genistein, curcumin [9], (−)-epigallocatechin-3-gallate, and resveratrol [10,11], have been recognized as cancer chemopreventive agents because of their anti-carcinogenic activity. They exert their anticancer effects by modulating different cell signaling pathways. Furthermore, we [12,13,14] and other researchers [15,16] have suggested that the combined effect of natural products may improve the effect of treatments against the proliferation of cancer cells. In this review we introduce a combination of natural products with chemotherapeutic agents or natural products and show synergistic anticancer effects against human HCC.Gambogic acid (GA) is a natural product that has been isolated from the gamboge of the Garcinia hanburyi tree. Gamboge is a dry resin and has been used as a coloring agent as well as in traditional Chinese medicine for the treatment of human diseases, including indigestion, inflammation, and ulcers [17]. Recent studies have reported that GA has anticancer effects via a variety of cellular signaling effects on different cancer cells, including HCC, gastric and lung carcinoma, breast cancer, and glioma cells [18,19,20,21,22,23,24,25,26]. Zhao et al. [27] examined the pharmacological toxicity of GA on the dog cardiovascular and respiratory system and mouse nervous system and showed that GA had no significant side effects on the cardiovascular, respiratory, or central nervous system at higher doses (4 mg/kg/day) than the recommended human doses (25 mg/60 kg/day). These results provided support for clinical applications of this promising natural anticancer agent.The ubiquitin-proteasome system is responsible for the degradation of most poly-ubiquitinated proteins, including regulatory proteins, that are involved in critical cellular processes, including cell cycle progression, cell development and differentiation, apoptosis, angiogenesis, and cell signaling pathways [28,29,30]. Therefore, targeting the ubiquitin–proteasome pathway has emerged as a rational approach to treat human cancers [31,32]. Bortezomib is the first Food and Drug Administration-approved proteasome inhibitor that has been used as a frontline therapy in refractory multiple myeloma [33].Huang et al. [34] investigated the combined effect of GA and a proteasome inhibitor in human leukemia K562 cells, mouse hepatocarcinoma H22 cells and H22 cell allografts. They reported that the combination of the natural product GA and proteasome inhibitor MG 132 or MG 262 results in a synergistic inhibitory effect on the growth of malignant cells and tumors in allograft animal models [34].Doxorubicin (DOX) is routinely used as a single drug for the treatment of patients with HCC [35]. It intercalates into DNA, stabilizes the topoisomerase II protein, and causes cell death via inhibition of topoisomerase II and generation of reactive oxygen species and free radicals by redox reactions [36]. Although doxorubicin is an effective antineoplastic agent and has cytotoxic effects, resistance limits its use in chemotherapy [35].(−)-Epigallocatechin-3-O-gallate (EGCG), one of the main polyphenols in green tea, has a variety of physiological and pharmacological effects. EGCG induces apoptosis and inhibits the proliferation of tumor cells [37,38]. EGCG has been extensively studied and has been reported to have chemopreventive effects for many different cancers such as liver, prostate, stomach, esophagus, colon, pancreas, bladder, skin, lung, and breast. EGCG also, has chemopreventive effects in carcinogenesis induced by UV light, chemical agents and genetic aberrations [39].Liang et al. [40] have shown that EGCG also serves as a promising chemosensitizing enhancer for DOX in HCC treatments. Liang et al. also showed that catechins inhibit the expression of multidrug resistance 1 (MDR1) mRNA and decrease the levels of P-glycoprotein, a membrane transporter that pumps a wide range of xenobiotics [41], in DOX-resistant HCC cells, suggesting that the administration of DOX in combination with EGCG or epicatechin gallate inhibits P-glycoprotein efflux pump activity and markedly enhances intracellular DOX accumulation [40]. Furthermore, they also found that a therapeutic regimen of EGCG co-treatment with DOX enhanced the antineoplastic efficacy mediated by suppressing autophagy [42]. On the other hand, clinical use of DOX is limited by cumulative cardiotoxicity [43,44]. Saeed et al. found that EGCG possesses cardioprotective actions against DOX-induced cardiotoxicity by suppressing oxidative stress, inflammation, and apoptotic signals as well as by activating pro-survival pathways [45]. These studies suggest that the combination of EGCG and DOX is a candidate for improving chemotherapeutic efficacy in HCC treatment.Zhao et al. examined whether the combination of quercetin, a natural flavonoid, with cisplatin, a conventional chemotherapeutic drug, would have synergistic suppressive effects on HCC cells [46].Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a polyphenolic flavonoid, is abundant in fruits and vegetables. It has been reported to have anti-oxidative, anti-inflammatory, and anticancer effects [47]. Many studies indicate that Quercetin can exert growth-suppressive effects in a variety of types of cancer cells, including esophageal [48], pancreatic [49], colon, and breast cancer cells [50]. In HCC cells, quercetin blocked cell cycle progression at the G1 phase by elevating cyclin-dependent kinase inhibitor p21 and p27 [51]. Tan et al. also showed that quercetin induces HCC cell apoptosis by downregulating surviving and bcl-2 [52]. Regarding the toxicity of quercetin, Heinz et al. [53] reported that quercetin (0.5 and 1 g/day) did not alter blood leukocytes subsets, granulocyte oxidative burst or phagocytes activity, IL-6, or TNF in healthy females.Cisplatin (cis-diamminedichloroplatinum(II)) is a commonly used anticancer drug [54]. It exerts its cytotoxic effect primarily by interacting with cellular DNA. Cisplatin binding to DNA alters the structure of the DNA and affects its ability to act as a template in transcription [55]. The effect then triggers apoptotic cell death. However, the clinical use of this drug is limited due to acquired resistance to cisplatin and severe side effects in normal tissues, such as neurotoxicity and acute nephrotoxicity [56].Thus, development of novel therapeutic strategies is urgently needed.Zhao et al. [46] revealed that the combination of quercetin and cisplatin synergistically inhibits cell growth and triggers apoptosis in HepG2 cells, which involves the alteration of many cell cycle and apoptosis regulators. They suggest that the inclusion of quercetin improves the outcomes of conventional chemotherapy in HCC. However, Li et al. [57] examined the effect of the combination of quercetin (20 mg/kg/day) and cisplatin (4 mg/kg/4 days) on a xenograft model of athymic BALB/C-nu nude mice injected with ovarian cancer cells and found that tumors treated with combination of quercetin and cisplatin were significantly heavier than those treated with cisplatin alone. Therefore, they suggested that quercetin treatment supplementation in ovarian cancer patients during chemotherapy may be antagonistic to the cytotoxic effects of chemotherapy [57].Hu et al. [58] investigated the effects of apigenin, the most common phytochemical consumed in the human diet, on enhancing the chemosensitivity of HCC cells and a HCC xenograft model in response to 5-fluorouracil (5-FU).Apigenin (4′,5,7-trihydroxyflavanone) is available in a wide variety of fruits, vegetables, and herbs [59]. It functions as an inhibitor of specific signal transduction pathways and has been shown to exhibit antitumor activities by inhibiting growth inhibition and inducing cell cycle arrest and apoptosis in many human cancer cells [59,60,61,62].5-FU, a fluorinated pyrimidine analogue of uracil, is widely used as an anticancer agent in the treatment of gastrointestinal tract, liver, brain, and ovary tumors [63]. Intracellular 5-FU is converted into several active metabolites, such as fluorouridine triphosphate, fluorodeoxyuridine monophosphate, and fluorodeoxyuridine triphosphate [64]. Fluorouridine triphosphate incorporates into RNA and causes the inhibition of pre-rRNA processing, blockage of rRNA post-transcriptional modifications and disruption of pre-mRNA splicing [65]. Fluorodeoxyuridine monophosphate binds to thymidylate synthase and inhibits the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate. Deoxyuridine monophosphate accumulation and fluorouridine triphosphate become misincorporated into DNA, resulting in DNA strand breaks and cell death [66].Hu et al. [58] have shown that sub-toxic concentrations of apigenin (4 µmol/L) significantly enhance the cytotoxicity of 5-FU (100 mg/mL) in HCC cells and that a combined treatment with apigenin (20 mg/kg, five times/week for three weeks) and 5-FU (20 mg/kg for five consecutive days) significantly inhibits the growth of HCC xenograft tumors in nude mice. They demonstrate that apigenin may potentiate the cytotoxicity of 5-FU in HCC by inhibiting reactive oxygen species, followed by a decrease in the mitochondrial membrane potential and activation of the mitochondrial pathway of apoptosis [58].Jiang et al. [67] examined whether genistein synergizes with arsenic trioxide (ATO), which is of limited therapeutic benefit for the treatment of solid tumors.ATO has been widely employed to treat acute promyelocytic leukemia [68]. The anticancer activity of ATO has also been tested in a variety of solid tumors, including HCC. However, the activity of ATO against solid tumors has not been as effective as in acute promyelocytic leukemia [69].Genistein, which exhibits multiple biochemical effects, has been shown to inhibit the growth of cancers of the breast, prostate, pancreas, and liver [70,71,72]. Furthermore, a recent study has shown that genistein induces apoptosis of HCC cells by increasing intracellular reactive oxygen species and inducing endoplasmic reticulum stress and mitochondrial injury [73]. Zeng et al. [74] studied a phase I human trial of genistein with 40 health subjects and showed that the area under the blood concentration–time curve (AUC) and the plasma concentration of the drug increased linearly when the close was increased from 50 to 100 mg; however, the increase was nonlinear when the dose was increased to 300 mg. Takimoto et al. [75] also studied a phase I pharmacokinetic and pharmacodynamics analysis of unconjugated soy isoflavones administered to individuals with cancer and showed that oral administration of soy isoflavones gives a plasma concentration of genistein that has been associated with anti-metastatic activity in vitro.Jiang et al. [67] incubated HCC cells (HepG2, Hep 3B, and SK-Hep-1) with ATO, genistein, or ATO + genistein for 72 h and determined cell viability using a Cell Counting Kit-8. Consequently, they found that ATO had little effect on the growth index, as compared with control untreated cells, whereas the combination of ATO and genistein led to a 30–40% inhibition of the growth index compared to the control and calculated values for a coefficient of drug interaction (CDI) based on the principles proposed by Chou and Talalay [76]. CDI is calculated according to the absorbance (MTT assay) of each group as follows; CDI = AB (A × B). AB is the ratio of the combination groups to the control group: A or B is the ratio of the single agent groups to the control group. A CDI value less than, equal to, or >1 indicates that the drugs are synergistic, additive, or antagonistic, respectively. A value <0.7 indicates a significantly synergistic effect [77]. The CDI values for HepG2, Hep3B, and SK-Hep-1 cells treated with genistein and ATO were 0.407, 0.543, and 0.448, respectively, indicating that the two drugs had significantly synergistic effects in inhibiting the viability of HCC cells [63]. They also showed that genistein synergized with a low dose of ATO (2.5 mg/kg) to significantly inhibit the growth of HepG2 tumors in mice. Ma et al. [78] confirmed that genistein not only potentiated the proliferation-inhibiting and apoptosis-inducing effect of ATO on human HCC cell lines (HepG2 and Hep3B) in vitro, but also dramatically augmented its suppressive effect on both tumor growth and angiogenesis in nude mice at a dose of 50 mg/kg/day.Guo et al. [79] showed that berberine sensitizes rapamycin-mediated HCC cell death.Rapamycin was initially characterized as a potent antifungal and was subsequently investigated as an immunosuppressant. It was later shown to exert powerful antiproliferative effects on a wide range of eukaryotic cells, including human tumor cells [80]. Rapamycin revealed the mammalian target of rapamycin (mTOR) signaling pathway, which is important for normal cell and cancer cell growth [81]. In cancer, mTOR is frequently hyperactivated and is a clinically validated target for drug development. A large number of preclinical and clinical studies have demonstrated that the inhibition of the mTOR signaling pathway using rapamycin or rapamycin analogs may be a useful therapeutic strategy for HCC [82,83]. Despite the use of rapamycin as a chemotherapeutic agent, the immunosuppressive effect simultaneously induced by rapamycin is problematic.Berberine, an alkaloid extracted from Cortis, has been studied for its multiple biological and pharmacological activities, including anticancer activity in a variety of human cancer cells [84]. Berberine can inhibit the growth of many cancer cell lines, such as the liver, lung, stomach, colon, skin, esophagus, brain, bones, and breast, by suppressing the growth cycle of tumor cells, inhibiting syntheses of DNA and protein and reducing the activity of topoisomerase. It is also reported that the alkaloid could promote tumor cell apoptosis by regulating apoptotic gene expression and decreasing the transmembrane potential of mitochondria [85]. Chu et al. [86] treated SiHa-bearing nude mice with placebo or berberine to verify the in vivo antitumor effects of berberine and found that berberine (20 mg/kg) feeding induced a 4.1-fold reduction in tumor weight by day 33 without any apparent signs of toxicity as proven by body-weight monitoring throughout the experiment.Guo et al. [79] hypothesized that the combination of rapamycin and berberine may increase the efficacy of chemotherapy for HCC by synergistically suppressing the mTOR signaling pathway. They found that the combined use of rapamycin and berberine had a synergistic cytotoxic effect. Berberine was observed to maintain the cytotoxic effect of rapamycin on HCC cells at a lower rapamycin concentration. In cells co-treated with berberine and rapamycin, overexpression of CD147 was found to significantly inhibit the downregulation of phosphorylated mTOR expression and decrease cell death. These findings suggest the possibility of developing a novel regimen that is capable of improving HCC therapy and minimizing the immunosuppression associated with rapamycin by decreasing its dose [79].In China, many people drink jujube tea instead of tea alone. They believe that the combination of Z. jujuba with green tea has synergistic effects that enhance immune function. However, the additive or synergistic effect of combining Z. jujuba with the extract of green tea on anticancer activity in vitro or in vivo has not been reported. Huang et al. investigated the effect of Z. jujuba extract and green tea extract on and their underlying mechanisms of action in HepG2 cells [12,13].Z. jujube, or the Chinese date, is scientifically known as Zizyphus jujuba Mill. It is also known as Hongzao or Dazao in China and Natsume in Japan. Believed to have various biological activities, it has been mentioned in the ancient famous Chinese medical book Sheng Nong Ben Cao Jing and has been traditionally used in Oriental medicines. For example, in Chinese traditional medicine, its dried fruits are prescribed as an anodyne, anti-tumor, pectoral, refrigerant, sedative, stomachic, styptic, and tonic. In Japan, the extracts of jujube are used to treat chronic hepatitis or distress and fullness in the chest and ribs [87].Japanese people and people throughout the world have consumed green tea beverages for centuries. Epidemiological studies have shown that the consumption of tea is effective in cancer prevention [88,89]. This effect has been attributed to green tea polyphenols, including flavanols, which are commonly known as catechins [90]. In addition, the ability of green tea catechins to improve metabolic abnormalities and reduce body weight has been reported by a number of basic and clinical studies [91,92]. Furthermore, the inhibition of carcinogenesis by tea has been demonstrated in many different animal models such as lung, skin, esophagus, and liver cancer [93,94,95].One of the authors of this review, Huang et al., found that green tea extract enhances the cytotoxic effect of jujube extract in HepG2 cells and causes cell growth inhibition. The involved mechanisms may be via two pathways. One pathway involves increased p53 and p21 proteins. The increased p21 binds with Cdk2 and prevents Cdk2 from binding to cyclin E, resulting in G1 phase arrest. Decreasing cyclin E levels might also lead to a direct decrease in cyclin E-cdk2 complex levels and cause G1 arrest [12]. Huang et al. also showed that the mechanism for the anticancer activity of the combination of jujube extract and green tea extract is via reduction of the expression of APRIL, a proliferation-inducing ligand, and involves upregulation of the p53 and p21 proteins in HepG2 cells [13]. These results suggest that the jujube extract and green tea extract mixture might provide a new drug design to treat hepatocellular carcinoma in the future.Resveratrol is a natural phenol that is produced by several plants and is mainly found in the skin of grapes and red wine. Resveratrol-containing plants, such as Rheum officinale Baill and Polygonum cuspdatum, have long been used in traditional Chinese medicine [96,97]. In 1997 Jang et al. reported that resveratrol use prevents skin cancer development in a mouse model [10]. Many researchers have also suggested that resveratrol administration prevents skin and colon cancer in animal models with artificially induced cancer [98]. Hebber et al. [99] treated male and female CD rats with high doses of resveratrol (0.3, 1.0, and 3.0 g/kg/day) for a period of 28 days and studied the dose response using cDNA stress assays coupled with drug-metabolizing enzyme assay. Then they found that at low doses, i.e., 0.3 and 1.0 g/kg/day, there were less significant changes over control rats and at the highest dose (3 g/kg/day) there were changes in gene expression that may be attributed to the toxicity. In addition, many clinical trials on resveratrol in the broad content of inflammation-associated disorder can be found on http://www.clinicaltrials.gov/ (Homepage of A service of the U.S. National Institutes of Health).Recently, it has been reported that resveratrol has anticancer activity against HCC [100]. The response of HCC to resveratrol includes upregulation of Sirt1 expression [101], increase of JNK and ERK1/2 MAP kinase activity [102], inhibition of VEGF expression [103], and downregulation of cyclin D1 [104]. These molecular mechanisms may underlie the resveratrol-induced apoptosis or self-protection response of the HCC cells.Curcumin is a polyphenol and major component of the spice turmeric. Turmeric is derived from the rhizome of the Indian plant Curcuma longa, which is a member of the Zingiberacae (ginger) family and used in various food preparations. Curcumin inhibits cell proliferation and induces apoptosis in numerous types of cancer cells, including prostate [105], breast [106], colon [107], and liver cancer [108]. The safety, tolerability, and nontoxicity of curcumin at a high dose (8 g/day) are well established by human-clinical trials [109,110]. However, its low bioavailability due to poor absorption and rapid metabolism has been shown to limit its therapeutic efficacy [111]. Therefore, it is necessary to work on improvement strategies.The combination of curcumin and resveratrol was found to demonstrate a synergistic anticancer effect in colon cancer [112]. In addition, Du et al. evaluated the combined effect of curcumin and resveratrol against HCC cells [15]. They demonstrated that the combination treatment of curcumin and resveratrol elicits a synergistic anticancer effect in Hepal-6 HCC cells via extrinsic and intrinsic apoptosis and is associated with reactive oxygen species (ROS) generation and downregulation of X-linked inhibitor of apoptosis protein (XIAP) and survivin, an anti-apoptosis gene. Their study suggested that a combination of curcumin and resveratrol is a promising novel anticancer treatment strategy for liver cancer.Artemisinin, a sesquiterpene lactone, is a natural product that is isolated from the plant Artemisia annual or sweet wormwood [113,114]. It contains an endoperoxide moiety that reacts with atomic iron to form cytotoxic free radicals. Artemisinin has been widely used as an antimalarial compound [115]. Artemisinin and its derivatives showed anti-proliferative effects on various tumor cell lines, including cancers of the breast, prostate, colon, and liver [116,117,118]. Artemisinin primarily induces apoptosis via activation of caspase-3 and increasing the Bax/Bcl-2 ratio and polyADP-ribose polymerase [119]. It has also been reported that artemisinin inhibits TNF-α-induced production of proinflammatory cytokines via inhibition of NF-κB and PI3 kinase/Akt signaling pathways [120].Hou et al. studied the in vivo antitumor activity of artemisinin in mouse HepG2 and Hep3B xenograft models. In their study, animals were treated with artemisinin at oral doses of 50 and 100 mg/kg/day, when mean tumor mass reached 100 ± 40 mg and artemisinin showed a dose-dependent inhibitory effect on tumor growth [121].Li et al. confirmed that combining artemisinin with resveratrol generated a synergistic effect in an in vitro model with HeLa and HepG2 cells and showed that the combination of artemisinin and resveratrol exhibited the strongest effect at the ratio of 1:2 (artemisinin to resveratrol) [16]. Furthermore, the following fluorescent microscopy measurements and cytometry demonstrated that artemisinin and resveratrol effectively inhibited the proliferation of cancer cells and enhanced migration, apoptosis, necrosis and ROS levels. These results suggest that combining artemisinin with resveratrol is a hopeful strategy for a clinical therapy for solid tumors.Matrine, an alkaloid extracted from Sophora flavescens Ait, is a natural compound of traditional Chinese medicine and exhibits many biological activities, such as anti-inflammatory [122], anti-virus [123], anti-fibrosis [124], and anti-arrhythmia effects [125], as well as immunosuppression [126]. Recently, some studies showed that matrine had potent anticancer activities by inhibiting proliferation and inducing apoptosis of gastric cancer [127], lung cancer [128], HCC [129], breast cancer [130], and melanoma [131] cells. In addition, Ma et al. [132] evaluated an in vivo antitumor efficacy of matrine in murine hepatocellular carcinoma H22 inoculated BALB/c mice and showed that seven doses of matrine at 50 mg/kg/dose inhibited 60.7% of tumor growth.Ou et al. examined the effect of the combined treatment of resveratrol and matrine on HepG2 cells and found that the combined treatment significantly enhanced the anti-proliferative effect compared with either agent alone [133]. In addition, resveratrol-induced apoptosis was significantly enhanced by matrine, which could be attributed to the activation of caspase-3 and caspase-9, downregulation of survivin, induction of ROS generation, and disruption of mitochondria membrane potential [133]. These results suggest that the combination treatment of resveratrol and matrine is a promising novel anticancer strategy for liver cancer.Li et al. have reported that two natural compounds, ginsenoside Rh2 and betulinic acid, synergistically induce apoptosis in human cervical adenocarcinoma (HeLa), human lung cancer A549, and hepatoma HepG2 cells [134].Betulinic acid, a pentacyclic triterpenoid, can be directly isolated from various plants that are widespread in the tropics. Betulinic acid was initially reported to be selectively cytotoxic to melanoma cells [135]. It was then demonstrated that betulinic acid exhibited several biological activities, including antibacterial, antimalarial, anti-HIV and anticancer effects. Betulinic acid inhibits cell proliferation and induces apoptosis in numerous types of cancer cells [136]. Fulda et al. reported that betulinic acid-induced apoptosis was not associated with the activation of ligand/receptor systems, such as CD95, and did not involve p53. Betulinic acid-induced apoptosis was mediated via direct effects on mitochondria [137] Regarding the in vivo anticancer activity of betulinic acid, Damle et al. [138] demonstrated the effect of betulinic acid on a MCF-7 human breast adenocarcinoma-induced tumor in athymic nude mice and found that betulinic acid also effectively suppressed growth of the tumors by delaying the development of MCF-7 tumors in a dose-dependent manner.Ginsenoside Rh2 is isolated from the root of Panax ginseng and has been shown to have anticancer effects [139]. It is reported that Ginsenoside Rh2 induces cell death in human hepatoma SK-HEP-1, MCF-7 human breast cancer, human leukemia THP-1, and human lung adenocarcinoma A549 cells.Ginsenoside Rh2 has been reported to induce apoptosis in a caspase 3,8-dependent manner [140] or by activating cyclin A-cdk2 with caspase 3-mediated cleavage of p21 [141]. Other studies have also demonstrated that Ginsenoside Rh2 inhibits proliferation by inducing the protein expression of p21 and reducing the protein levels of cyclin D, reducing pRB phosphorylation, and inhibiting E2F release [142] or modulating MAP kinase [143] in various cancer cells. Recently, Kim et al. have reported that the degree of Ginsenoside Rh2-induced activation of AMP-activated protein kinase correlated with differences in sensitivity to apoptosis in cancer cell lines [144]. However, it is not ideal to use ginsenoside Rh2 singly as a chemotherapeutic agent.Li et al. have shown that two natural compounds, Ginsenoside Rh2 and betulinic acid, synergistically induce apoptosis in human cervical adenocarcinoma (HeLa), human lung cancer A549, and human hepatoma HepG2 cells [134]. They found that co-treatment with Ginsenoside Rh2 and betulinic acid triggered caspase-8 processing and cleavage and sensitized tumor cells by a Bax-dependent mechanism, followed by caspase-9 and -3 processing and apoptosis. These results suggest that co-treatment with Ginsenoside Rh2 and betulinic acid could be a novel strategy to enhance the efficacy of betulinic acid-based therapy.Kato et al. have reported the synergistic effect of 1′-acetoxychavicol acetate and sodium butyrate on the death of human HCC cells [14].1′-Acetoxychavicol acetate (ACA) naturally occurs in the rhizomes and seeds of Zingiberaceae plants, such as Languas galangal and Alpinia galangal. Southeast Asia residents are traditionally exposed to ACA when using plants as a spice or medicine in everyday life. ACA exhibits chemopreventive effects on chemical-induced tumors in mouse skin and rat oral, colonic, esophageal, and pancreatic tumors [145,146]. ACA also exerts anticancer activity by inducing apoptosis in various tumor cells, such as Ehrlich ascites tumor cells [147], rat and human HCC cells, human colon cancer cells, and human myeloid leukemia cells. Furthermore, we showed that ACA induced apoptosis in Ehrlich ascites tumor cells by decreasing intracellular polyamines and inducing caspase-3 activity [147].Sodium butyrate has multiple effects on tumor cells cultured in vitro by inducing inhibition of cell proliferation and apoptosis [148,149], as well as initiating the differentiation of various carcinoma cells [150,151]. Butyrate also alters the transcription of several genes related to tumor growth and invasiveness and suppresses the growth of tumors implanted in nude mice [152]. Sodium butyrate is an inhibitor of histone deacetylase, which is a class of proteins that can inhibit malignant cell growth in vitro and in vivo, reverse oncogene-transformed cell morphology, induce apoptosis, and enhance cell differentiation.Kato et al. [14] evaluated the combination of ACA and sodium butyrate on the growth of human HCC HepG2 cells and found that treatment had a synergistic inhibitory effect. The number of HepG2 cells was synergistically decreased via apoptosis induction when the cells were treated with both ACA and sodium butyrate. Furthermore, the intracellular reactive oxygen species (ROS) levels and NADPH oxidase activities were significantly increased in the ACA- and sodium butyrate-treated cells. AMP-activated protein kinase (AMPK), a cellular energy sensor, plays an essential role in controlling processes related to tumor development. The combined treatment of ACA and sodium butyrate significantly induced AMPK phosphorylation. This induction improved when cells were pretreated with catalase [14]. These results suggest that the increase in intracellular ROS is involved in the increase of AMPK phosphorylation. These findings may provide new insight into the development of novel combination therapies against HCC.HCC is highly resistant to the currently available chemotherapeutic agents. Therefore, new effective and well-tolerated therapy strategies are needed. This literature review summarizes the current literature on combination treatments with natural products and chemotherapeutic agents or natural products for inhibiting the growth of HCC. As shown in Table 1, a growing body of combination treatments with natural products has been reported to synergistically prevent tumor growth, which has provided new insight into the development of novel combination therapies against HCC. Many combination treatments with natural products can generate intracellular ROS and then induce mitochondrial depolarization and a permeability transition. The formation of ROS could be classified into two general categories: ROS derived from mitochondrial oxygen consumption or ROS that are mitochondrial-independent. It is necessary to elucidate the exact molecular mechanisms for the formation of ROS. In addition, it is important to understand the interaction of these natural products in signaling pathways. Because quercetin has been reported to act as an antagonist to cisplatin, it is also necessary to further examine the effect of the combination treatment of tumor growth in in vivo animal models. Then we can respond to a growing demand for testing these natural products in clinical trials, which will be possible after gaining insight into their interactions with exact signaling pathways and the bioavailability as well as the cytotoxicity of these natural products. Furthermore, as with most preclinical leads, positive in vitro data does not directly correlate to positive in vivo data due to poor solubility and minimal accumulation at the target site, leading to significantly increased systemic toxicity. Regarding this problem, Ling et al. [153] have described the interesting report that nanoformulation of natural product facilitated uptake into the tumor, and specifically tumor cells, leading to a further increase in efficacy while mitigating systemic toxicity. Therefore, it is also important to discover and develop more effective therapeutic strategies against HCC.Summary of synergistic anticancer activities of natural products in HCC.Main text paragraph O’Neil.A. K.-Y., X. H. and I. M.-Y. wrote the manuscript. A. K.-Y. edited the manuscript. All authors read and approved the final manuscript.The authors declare no conflicts of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Celiac disease (CD) is a systemic immune-mediated disorder caused by the ingestion of gluten-containing grains in genetically susceptible persons. It is one of the most common lifelong disorders, affecting approximately 1% of the general population. The prevalence of CD has increased in developed countries over recent decades, pointing to the role of additional environmental triggers other than gluten. It has been hypothesized that intestinal infections, the amount and quality of gluten, the intestinal microbiota, and early nutrition are all possible triggers of the switch from tolerance to an immune response to gluten. Two recent randomized controlled trials have been performed to clarify the relationship between the age at which gluten is introduced to a child’s diet and the risk of CD, showing that timing of gluten introduction does not modify the risk of CD. Both trials also showed that breastfeeding compared with no breastfeeding or breastfeeding duration or breastfeeding during gluten introduction have no effect on the risk of CD. The two trials, although not designed to address this issue, have shown that intestinal infections seem not to influence the risk of CD. Further studies are still needed to explore the missing environmental factors of CD for future prevention.Celiac disease is a systemic immune-mediated disorder caused by the ingestion of gluten-containing grains (wheat, rye, and barley) in genetically susceptible persons. It is one of the most common lifelong disorders on a worldwide basis, affecting approximately 1% of the general population [1,2]. The frequency of celiac disease has increased in developed countries over the last decades, a finding that points out the causal role of possible environmental triggers additional to gluten [3].The development of celiac disease is determined by both environmental and genetic factors. Multiple lines of evidence favoring a genetic contribution to the pathogenesis of celiac disease have been suggested by epidemiologic data: a familial aggregation is found in 5%–15% of celiac disease patients and a striking 83%–86% concordance rate was observed among monozygotic twin pairs [4]. The genetic determinants that confer susceptibility to the disease are, however, not yet fully understood. The most important genetic factor identified is the human leukocyte antigen (HLA) locus. The HLA-DQ2 (DQA1*0501-DQB1*0201) haplotype is expressed in the majority of affected patients (90%), the DQ8 haplotype (DQA1*0301-DQB1*0302) is expressed in 5%, and 5% carry at least one of the two DQ2 alleles (usually the DQB1*0201) [5]. An increased risk of celiac disease has been observed among persons who carry two DQB1*02 alleles [6,7]. The ability of these alleles in conferring individual susceptibility to celiac disease is related to their peculiar capacity to bind negatively charged peptides such as gliadin peptides resulting from the deamidation of gluten by the antitransglutaminase. The HLA antigen results in the activation of T lymphocytes, whose secretion products play a key role in causing mucosal lesions [6]. The associations found in non-HLA genome-wide linkage and association studies are much weaker. This might be because a large number of non-HLA genes contributes to the pathogenesis of celiac disease [8]. Hence, the contribution of a single predisposing non-HLA gene might be quite modest [4].Gluten is the environmental factor required to trigger the disease, but other factors may be involved in a model of a complex multifactorial disease [7]. The total prevalence of celiac disease has, indeed, doubled in Finland during the last two decades (1.05% in 1978–1980 and 1.99% in 2000–2001) [9], and the increase cannot simply be attributed to a better rate of detection. Recently, it has been shown that celiac disease autoimmunity doubled between 1974 (one in every 501 subjects) and 1989 (one in every 219 subjects) within an American population followed since 1974. This trend apparently continued in the following years. In a different sample of the adult American population in 2001, a celiac disease prevalence of one in 105 subjects was reported. Therefore, during the last 30 years, the prevalence of celiac disease among adults in the US appeared to increase by five-fold, doubling approximately every 15 years [10]. According to the “hygiene hypothesis,” the “cleaner” environment found nowadays in developed countries led to a lower frequency of early childhood infections and differences in the spectrum of microorganisms populating the gut. These changes could modify the immune response and be responsible for a higher risk of different autoimmune disorders such as celiac disease [11]. However, the rising prevalence of adult onset of celiac disease that was observed in the US study can hardly be explained by hygienic changes occurring in childhood. The reasons for these changes are still unclear, but have to do with the environmental components of celiac disease, since genetic changes are too slow to drive these phenomena [3].It has been recently hypothesized that all the following environmental factors are possibly involved in the switches of the tolerance–immune response balance: the amount and the quality of ingested gluten, the type and duration of wheat dough fermentation, early infant feeding, the spectrum of intestinal microorganisms and how they change over time, intestinal infections, and stressors in general [7]. However, more research is needed to determine whether and how these factors can cause loss of gluten tolerance. Recent randomized control trials have been performed to clarify the role of some of these factors on the later development of celiac disease. In the present review we provide an overview of the recent research in this field.The introduction of gluten at six months of age is a long-standing practice, and although it is a rule deeply rooted in many developed countries, the optimal time of introduction of gluten in the diet of a child had never been rigorously tested. In clinical practice, many pediatricians believed that the introduction of gluten in the diet of children who have a family risk of celiac disease should be delayed to allow the maturation of the intestinal barrier and the immune response. However, investigations following the real “epidemic” of celiac disease that occurred in Sweden during the 1980s and 1990s showed that the introduction of a small amount of gluten during breastfeeding between four and six months of age reduced the risk of the disease. These data provided the basis for the hypothesis of the so-called “window of tolerance”, according to which there would be a window of time, between four and seven months of age, during which the introduction of gluten could facilitate the induction of tolerance [12,13]. The concept of the “window of tolerance” gained popularity in 2005 with the findings of a US study reporting that children at genetic risk for type 1 diabetes exposed to gluten between four and six months of age had a reduced risk of celiac disease compared to those exposed to gluten before four and after seven months of age; it is worth noting that the number of patients in this study with a diagnosis of celiac disease confirmed by intestinal biopsy was very small [14,15]. Later, a German study showed that children with a family risk of diabetes type 1 whose first dietary exposure to gluten occurred after the age of six months had an increased risk of celiac autoimmunity [16]. However, an epidemiological survey carried out in 2013 on a large Norwegian population (324 cases with celiac disease and a cohort of 81,843 healthy controls), challenged all previous observations; in particular, the results of the Norwegian study demonstrated that: (a) the introduction of gluten during breastfeeding was not protective; (b) only the delayed introduction of gluten (>6 months), but not early introduction (<4 months), was associated with an increased risk of celiac disease [17]. The main limitation of the Norwegian study was that the analysis included only children with a clinical diagnosis of celiac disease; therefore, this result could not necessarily be applied to the overall celiac population (which is at least three times larger). Another problem of this and previous case-control studies was the lack of an intervention arm.Two randomized controlled trials have been recently performed to finally clarify the relationship between the age at which gluten is introduced to a child’s diet and the risk of celiac disease [18,19]. The Risk of Celiac Disease and Age at Gluten Introduction (CELIPREV) trial is a multicenter, prospective intervention trial comparing early (six months) and delayed (12 months) introduction of gluten in infants with a familial risk of celiac disease, followed from birth until 10 years of age [18]. Infants who had a familial risk of celiac disease (i.e., infants who had at least one first-degree relative with celiac disease) were recruited in 20 centers in Italy between 2003 and 2008. Infants were randomly assigned to one of the two groups: those in group A were introduced to gluten-containing foods (pasta, semolina, and biscuits) at six months of age, and those in group B at 12 months of age. The main objective was to compare the prevalence of celiac disease according to the time of gluten introduction at five years of age. The percentage of children with celiac disease at five years of age was the same in group A and in group B (16% and 16%, p = 0.78); at 10 years there was still no significant difference between the two groups (hazard ratio at 10 years: 0.9; 95% Confidence Interval (CI): 0.6–1.4; p = 0.79). However, the average age at which celiac disease developed was 26 months in group A and 34 months in group B (p = 0.01). It is worth noting that in children with the high-risk HLA genotype (characterized by homozygosis for HLA-DQ2), the prevalence of celiac disease was higher in group A than in group B at all ages, although the difference was not significant (p = 0.51), probably due to the small size of the sample of children with this genotype. During follow-up, complications related to celiac disease (i.e., autoimmune thyroid diseases, type 1 diabetes, or both) did not develop in any child from the two groups. Therefore, the CELIPREV study has shown that postponing the introduction of gluten to 12 months of age has no effect on the risk of developing the disease in the long term: it does not reduce or increase the risk of disease. However, delaying the introduction of gluten had two potentially positive consequences: (a) to delay the development of the disease; and (b) to possibly reduce the frequency of celiac disease in the HLA-high-risk group. In fact, although this last effect did not reach statistical significance, it is worthy of further investigations. The hypothesis of the “window of tolerance” was therefore not supported, as there was no difference in the risk of celiac disease among children who were introduced to gluten at six months (during the “window” open) and those who were introduced to gluten at 12 months (when the “window” was closed).The European multicenter project Prevent Celiac Disease (PREVENT-CD) trial is a multicenter, randomized, double-blind, placebo-controlled dietary intervention study aimed at comparing the introduction of small quantities of gluten (100 mg/daily) from 16 to 24 weeks of age with a placebo, followed by standard gluten consumption after the intervention in infants with a familial risk of celiac disease, followed from birth to at least three years of age [19]. The results of this trial demonstrated that the introduction of small amounts of gluten during the window of tolerance does not reduce the risk of the disease. The cumulative incidence of celiac disease among patients three years of age was indeed similar in the gluten group and the placebo group (5.9% and 4.5%, respectively; hazard ratio at three years: 1.23; 95% CI, 0.79 to 1.91, p = 0.47).A protective role of breastfeeding against celiac disease has long been perceived, mostly based on some retrospective studies [20,21,22,23] and a systematic review of the literature [24] and a meta-analysis that summarized those results [25]. The Norwegian epidemiological investigation had instead shown that breastfeeding did not exert any protective effect against the development of celiac disease; the average duration of breastfeeding was, indeed, even longer in children with celiac disease (10.4 months) compared with controls (9.9 months), and the risk of disease was significantly higher in babies breastfed for more than 12 months [17].The study CELIPREV brought a clarification on this important aspect of child nutrition. In the cohort of infants at familial risk of celiac disease, no protective effect of breastfeeding for the development of celiac disease was observed: the average duration of breastfeeding was similar for children that developed celiac disease and for those that did not develop celiac disease (5.6 and 5.8 months, respectively); there was no significant difference in the percentage of children that developed celiac disease among children breastfed and those never breastfed (Figure 1) and among those breastfed for more or less than six months (Figure 2); finally, there was no significant difference in the percentage of children that developed celiac disease among children that were introduced gluten during breastfeeding and in those that were introduced gluten without breastfeeding (Figure 3) [18]. The same results were reported by the PREVENT-CD study [19]. Therefore, although there are many good reasons to recommend prolonged breastfeeding of newborns, prospective studies have not observed a protective effect against celiac disease.Kaplan–Meier estimates of celiac disease, according to breastfeeding (red = no breastfeeding; blue = any breastfeeding).Kaplan–Meier estimates of celiac disease, according to breastfeeding duration (red = breastfeeding < 6 months; blue = breastfeeding ≥ 6 months).Kaplan–Meier estimates of celiac disease, according to breastfeeding during gluten introduction (red = breastfeeding during gluten introduction; blue = no breastfeeding during gluten introduction).The PREVENT-CD study showed that the introduction of gluten at a dose of 200 mg/day at four to six months of age compared to the avoidance of gluten does not have an effect on the risk of celiac disease at three years of age [19]. There are no data on other amounts of gluten introduced at weaning, nor on the type of gluten introduced and later celiac disease development from the prospective interventional trial. Therefore, based on the current literature evidence, no conclusion can be made from these results.Intestinal infections (such as rotavirus among infants and Campylobacter among adults) have recently been shown to be associated with an increased risk of celiac disease [26,27]. Infections might change gut permeability, leading to the passage of immunogenic gluten peptides through the epithelial barrier. The other possibility implies that sequence similarities exist between proteins produced during rotavirus infections and proteins of gluten. Infections, especially viral infections, may also alter mucosal gene expression, specifically the expression of pattern recognition receptors, which are considered to be in a key position to determine host-environment interactions [28,29,30]. The area has not been studied much with enterocytes, but studies with other cell lines give indicative insights. Altered mucosal gene expression of tool-like receptors and their regulators has been, indeed, reported in pediatric celiac disease subjects [31].On the other hand, rates of intestinal infections have not increased markedly in recent decades, and thus are not likely to be driving the epidemic of celiac disease. In contrast, a lack of exposure to certain microbes is the hallmark of modern times [32]. The CELIPREV trial, although not designed to address this outcome, evaluated the role of all nutritional and genetic variables studied (breastfeeding, amount of gluten, age at gluten introduction, genotype, gender, celiac disease-affected first-degree relative, and intestinal infections) in influencing the risk of celiac disease by a data-mining approach with decision-tree induction. Interestingly, none of the variables studied, including intestinal infections, had a significant effect in predicting the development of celiac disease, with the exception of the HLA genotype. Indeed, the risk of celiac disease was far higher among children with high-risk HLA than among those with standard-risk HLA (26% vs. 16%, p = 0.05), confirming that predisposing HLA gene dosing is, to our knowledge, the most influential variable in increasing the risk of developing celiac disease [18]. Also, the PREVENT-CD trial evaluated the cumulative incidence of celiac disease according to the presence of gastrointestinal infection in the first 18 months of life, showing no effect on the risk of celiac disease (4.9% vs. 6.4%, p = 0.61). Moreover, the authors also found no difference in the cumulative incidence of celiac disease when comparing children that were administered a rotavirus vaccination versus those that were not administered the vaccination (2.5% vs. 6.0%, p = 0.96) [19].There is now accumulating evidence that gut microbiota plays an important role in the regulation of intestinal immune responses and in the maintenance of intestinal homeostasis. Most observational studies in children and adults have shown intestinal dysbiosis (i.e., altered gut microbiota composition or function) in celiac disease patients, untreated and treated with a gluten-free diet, compared to healthy controls [33,34,35,36,37,38,39]. Celiac disease patients with gastrointestinal symptoms are also known to have a different microbiota compared to patients with dermatitis herpetiformis and controls, suggesting that the microbiota is involved in disease manifestation [40]. Furthermore, a dysbiotic microbiota seems to be associated with persistent gastrointestinal symptoms in treated celiac disease patients, suggesting its pathogenic implication in these particular cases [41]. Nevertheless, other authors report no differences in mucosa-associated duodenal microbiome composition and diversity in celiac disease children as compared to controls [42], and there is lack of consensus and understanding of what constitutes a CD-promoting microbiota [33].Dysbiosis may be the result of both genetic and environmental factors. Specific host-genetic makeup could promote the colonization of pathobionts and reduce symbionts, thus leading to dysbiosis [33]. The genotype of infants at family risk of developing celiac disease, carrying the HLA-DQ2 haplotypes, has indeed been shown to influence the early gut microbiota composition, suggesting that a specific disease-biased host genotype may select for the first gut colonizers and could contribute to determining disease risk [43]. Recent studies also showed a significant association of celiac disease with homozigosity for a nonsense mutation in the fucosyltransferase 2 non-secretor status, which has been shown to be a major determinant for the gut microbial spectrum [44]. The non-secretor individuals were demonstrated to have an altered mucosa-associated microbiota in their intestinal tract, characterized by reduced diversity, richness, and abundance of Bifidobacterium spp., a bacterial genus that may play an important role in autoimmune disease risk [45,46]. Besides host genetics, environmental factors could also influence microbiota composition; indeed, the milk-feeding type (breast-milk vs. formula), the mode of delivery, antibiotics, and other drugs are also well-known environmental factors exerting a profound impact on the microbiota composition, potentially modifying its functional role in health and disease [33,47].Intestinal dysbiosis might contribute to the etiopathogenesis of celiac disease either by itself providing proteolytic activities that influence the generation of toxic and immunogenic peptides from gluten and by mediating host-microbe interactions, which could influence the intestinal barrier and immune function (e.g., via regulation of the cytokine network of pro-inflammatory and anti-inflammatory factors) [33,34]. Nonetheless, it still remains unclear whether the changes in the microbiota are a cause or a secondary consequence of celiac disease development. Animal models, including germ-free and gnotobiotic models, will be of critical value to study whether the composition of the gut microbiota influences the loss of tolerance to gluten in genetically susceptible hosts and the mechanisms through which microbes can influence host responses to gluten. These types of studies will be helpful in determining causality, but their direct translational value might be limited due to the associated limitations with animal models of celiac disease. Thus, clinical studies will be needed to provide translational value of basic studies [34]. Unfortunately, neither CELIPREV nor PREVENT-CD has investigated this intriguing issue in a clinical setting. Prospective studies in healthy infants at family risk of celiac disease are underway to decipher the co-evolution of the gut microbiome and the host genome in response to environmental factors and the possible causal relationships with celiac disease onset.As stated in the editorial published on this issue, “we are still in search of environmental factors that may affect the development of the disease” and “the two trials—CELIPREV and PREVENT-CD—are today a starting point rather than an end point of the research in this field” [48].A revision of the current recommendations of the European Society of Gastroenterology, Hepatology and Pediatric Nutrition (ESPGHAN) on weaning [49], which recommend the introduction of gluten from four to seven months of age, during the “window of tolerance”, and the introduction of gluten while the infant is still being breastfed in order to reduce the risk of celiac disease is strongly advised. Evidence from recent randomized controlled trials showed that neither breastfeeding nor breastfeeding during gluten introduction may reduce the risk of celiac disease, and that the age of gluten introduction into the infant's diet, whether early or late, does not influence the risk of celiac disease during childhood, and, thus, indicates that primary prevention of CD is not possible at the present timeFurther studies are needed to determine whether other environmental factors, such as the composition of the intestinal microbiota, the mode of delivery, the metabolic profile, the vaccination program, and the use of antibiotics or other drugs actually influence the balance between the immune response and tolerance to gluten. Also, the role of intrauterine and perinatal exposure is still to be explored. Moreover, the reduction of gluten antigenicity and/or the elimination of toxic peptides from gluten could be a workable strategy of primary prevention: the modifications of gluten antigenicity may be achieved in several manners, from the selection of natural cereal cultivars to the genetic modification of gluten peptide sequences or, more likely, producing genetically modified organisms, in which toxic sequences are deleted or silenced [50]. The results of all these studies could be instrumental in determining how to prevent not only the onset of celiac disease but also other autoimmune disorders.E.L. performed the review of literature and analyzed the data; E.L. and C.C. wrote the paper; E.L. and C.C. contributed to the revision of the paper.Carlo Catassi served as a consultant for Menarini diagnostics s.r.l., and for Shaer. Elena Lionetti served as a consultant for the Heinz Company.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Hepatocellular carcinoma (HCC) is a lethal cancer with limited systemic therapeutic options. Liver carcinogenesis is a complex procedure and various pathways have been found to be deregulated which are potential targets for novel treatments. Aberrant signalling through FGF19 and its receptor FGFR4 seems to be the oncogenic driver for a subset of HCCs and is associated with poor prognosis. Inhibition of the pathway in preclinical models has shown antitumour activity and has triggered further evaluation of this strategy to in vivo models. This review aims to describe the role of the FGF19/FGFR4 pathway in hepatocellular carcinoma and its role as a potential predictive biomarker for novel targeted agents against FGF19/FGFR4 signalling.In 2012 782,000 new cases of hepatocellular carcinoma (HCC) were reported worldwide from which 83% were in less developed countries and 50% just in China alone. HCC is the fifth most common cancer in men (554,000 new cases and 7.5% of all cancers) and the ninth most common cancer in women (228,000 new cases and 3.4% of all cancers). Prognosis is poor with an overall ratio of mortality to incidence of 0.95 resulting in 746,000 deaths in 2012 (9.1% of all cancer deaths) making it the second most lethal cancer after lung cancer [1].Around 80%–90% of HCC is related to chronic inflammation and cirrhosis [2]. The major risk factors for HCC are Hepatitis B virus (HBV), Hepatitis C virus (HCV), alcoholic liver disease and non-alcoholic fatty liver (NAFLD) [3]. Despite proven efficacy of HBV vaccination in preventing HCC and improvements in antiviral treatment for both HBV and HCV it is estimated that worldwide at least 53% of HCC cases are attributed to HBV and 25% to HCV [4]. Increasing incidence of obesity and diabetes in Western countries is concerning due to its strong association with NAFLD which can lead to cirrhosis and HCC, although cirrhosis may not always precede [5]. It is estimated that 30% of the US population will be affected by NAFLD during their life introducing a potentially increasing high risk population [6].Surgical resection and liver transplantation are indicated for patients with localized disease and are potentially curative treatments whilst transarterial chemoembolization (TACE) is used for patients where large and multifocal tumours who are not amenable to surgical treatments. However the majority of patients will relapse and will require systemic treatment where options are extremely limited since several chemotherapy and targeted agents have failed to show any meaningful outcomes [7,8]. Sorafenib is a multi-targeted tyrosine kinase inhibitor that interferes with tumour proliferation and angiogenesis by inhibiting the RAF kinase and the VEGFR mediated intracellular pathway. It is the only approved treatment based on the benefit seen in two phase III studies: the SHARP trial in a Western population showed a 3 month overall survival benefit versus best supportive care (10.7 vs. 7.9 months, 95% CI, 0.55–0.87, p < 0.001) [9] and a study in Asian-Pacific population showed an overall survival of 6.5 months for sorafenib versus 4.2 months for placebo (HR 0.68, 95% CI, 0.50–0.93, p = 0.014) [10].Various signaling pathways involved in proliferation, cell differentiation, angiogenesis, invasion and metastasis are deregulated in HCC and are attractive targets [11,12,13]. Multikinase inhibitors with activity against angiogenesis and FGFRs have also been tried in HCC but failed to show superiority against sorafenib. Brivanib is a multikinase inhibitor with activity against VGFR1-3 and FGFR1-2. Despite promising results in II studies [14] it failed to meet its primary endpoints in phase III studies of patients with advanced HCC compared to sorafenib in the first line [15] and placebo in the second line setting [16]. Another multikinase inhibitor that has been tested was dovitinib with activity against VEGFR1-3, FGFR1-3 and PDGFR-β and similarly failed to show superiority against sorafenib in a phase II study of patients with advanced HCC [17]. Nintedanib which has activity VEGFR1-3, FGFR1-3 and PDGFRα and PDGFRβ has been approved for the treatment of non-small cell lung cancer and idiopathic pulmonary fibrosis [18]. When compared to sorafenib in patients with advanced HCC in a phase II study no significant differences were observed between the two agents in time to progression (TTP) and overall survival (OS) [19]. Notably all of these agents were used in unselected populations without any predictive biomarkers.The need for better treatments in HCC is obvious and can only be achieved by better understanding of the molecular mechanisms that drive liver carcinogenesis. Evidence regarding the role of the members of FGF19-FGFR4 pathway in liver carcinogenesis is growing. Correlation with prognosis and in vitro experiments showing promising results with inhibition of the pathway have highlighted a subset of HCCs which are driven by FGF19/FGFR4 signaling and could potentially benefit from a selective inhibition of the pathway.The Mammalian Fibroblast Growth Factor (FGF) family consists of 22 members which are further classified to 7 subfamilies according to phylogenetic analysis. They are named FGF1-FGF23 with FGFR15 not present in humans and FGF19 not present in mice and rats. FGF15 and FGF19 are considered orthologues and are usually referred as FGF15/FGF19. Each polypeptide growth factor consists of around 150–300 amino acids with a conserved core with 30%–50% identity. There are also four FGFR genes that encode the tyrosine kinase receptors and their variants resulting in seven major FGFR proteins (1b, 1c, 2b, 2c, 3b, 3c and 4). FGF signalling proteins are present in almost all tissues and are involved in various functions like embryogenesis, tissue repair, metabolism and neural function [20].FGFs are also classified according to their cell-cell communication pattern as:
+	  
+      Intracrine: they consist of the FGF11 subfamily (FGF11, 12, 13, 14). These factors are secreted intracellularly, they do not interact with FGFRs and their main role is regulation of the electrical excitability of neurons and other excitable cells like cardiomyocytes.
+      Paracrine: they consist of the FGF1 subfamily (FGF1,2), the FGF4 subfamily (FGF4,5,6), the FGF7 subfamily (FGF3,7,10,22), the FGF9 subfamily (FGF9,17,18) and the FGF8 subfamily (FGF8,17,18). These are secreted proteins that bind to FGFRs and use heparin/heparan sulphate as a cofactor, although FGF1, 2, 3 can directly translocate to the nucleus and act as intracrine proteins. They are involved in embryogenesis and tissue repair by regulating cell proliferation, differentiation and survival.
+      Endocrine: they consist of the FGF15/19 subfamily (FGF15/19, 21, 23). Endocrine FGFs have low affinity with heparin/heparin sulphate and they bind to FGFRs using the Klotho family proteins as cofactors. They act as hormones regulating bile acid, lipid and carbohydrate homeostasis [20,21,22].Intracrine: they consist of the FGF11 subfamily (FGF11, 12, 13, 14). These factors are secreted intracellularly, they do not interact with FGFRs and their main role is regulation of the electrical excitability of neurons and other excitable cells like cardiomyocytes.Paracrine: they consist of the FGF1 subfamily (FGF1,2), the FGF4 subfamily (FGF4,5,6), the FGF7 subfamily (FGF3,7,10,22), the FGF9 subfamily (FGF9,17,18) and the FGF8 subfamily (FGF8,17,18). These are secreted proteins that bind to FGFRs and use heparin/heparan sulphate as a cofactor, although FGF1, 2, 3 can directly translocate to the nucleus and act as intracrine proteins. They are involved in embryogenesis and tissue repair by regulating cell proliferation, differentiation and survival.Endocrine: they consist of the FGF15/19 subfamily (FGF15/19, 21, 23). Endocrine FGFs have low affinity with heparin/heparin sulphate and they bind to FGFRs using the Klotho family proteins as cofactors. They act as hormones regulating bile acid, lipid and carbohydrate homeostasis [20,21,22].FGF19 specifically is involved in bile acid synthesis, gallbladder filling, glycogen synthesis, gluconeogenesis, protein synthesis and reduction of adipose tissue [23]. Following a meal bile acids are secreted from the gallbladder to the small intestine and they activate Farnesoid X Receptor (FXR) which stimulates secretion of FGF19 from the ileum [24]. FGFR4 and β-Klotho, which is the main co-factor for FGF19 activation, are both highly co-expressed in the liver [25,26]. The FGF19-mediated signaling cascade results in downregulation of bile acid synthesis through inhibition of cholesterol 7a-hydroxylase (CYP7-A1) which is the rate-limiting enzyme for bile acid synthesis, suppression of cholecystokinin which promotes gallbladder emptying [21,24], promotion of glucogen and protein synthesis and inhibition of gluconeogenesis in liver [23]. FGF19 transgenic mice have reduced adipose tissue and low levels of cholesterol and triglycerides [27] and further data suggest that FGF19 is involved in inhibition of liposynthesis and increasing fatty acid oxidation by downregulating the activity of acetyl CoA carboxylase 2 [21]. Moreover FGF19 can promote proteinosynthesis by phosphorylating several proteins which promote translation [23].FGF19 has high specificity for FGFR4 and although it can bind to FGFR4 independently, the presence of β-Klotho which is a single-pass transmembrane protein and acts as a cofactor results in a more pronounced activation of FGFR4 and its phosphorylation [28]. Activation of FGFR4 results to phosphorylation of FGF receptor substrate 2 (FRS2) and recruitment of growth factor receptor-bound protein 2 (GRB2) and eventually activation of Ras-Raf-ERK1/2 MAPK and PI3K-Akt pathways which are involved in cell proliferation and anti-apoptosis [29] (Figure 1).The FGF19/FGFR4 pathway with its main components and targets of inhibitory agents. FGF19: Fibroblast Growth Factor 19, FGFR4: Fibroblast Growth Factor Receptor 4, KLB: Klotho Beta, FRS2: Fibroblast growth factor Receptor Substrate 2, GRB2: Growth factor Receptor-Bound protein 2.Evidence supporting the role of the FGF19/FGFR4 pathway in liver carcinogenesis is accumulating. Clearly FGF19 expression is significantly higher in hepatocellular carcinoma compared to non-malignant liver [30] and has oncogenic activity. FGF19-transgenic mice which overexpress FGF19 in skeletal muscle develop multiple hepatocellular carcinomas early in their life, usually by 10–12 months, whilst other tissues are not affected [31]. Miura et al showed that addition of FGF19 recombinant protein in hepatocellular cell lines increases proliferation and invasion and inhibits apoptosis whilst decreasing FGF19 and FGFR4 expression with small interfering RNA (siRNA) had the opposite results [30].The rodent orthologue FGF15 also has mitotic and oncogenic activity. Following partial hepatectomy FGF15−/− mice have increased intrahepatic bile acid levels and high mortality. Addition of cholestyramine and adenovirally delivered FGF15 resulted in improved survival. Improved outcomes following partial hepatectomy were also seen in FGF15+/+ mice suggesting that FGF15 induces hepatocellular proliferation and is important for liver regeneration [32]. Uriarte et al used diethylnitrosamine and CCl4 which are known to induce inflammation in liver and fibrosis-mediated carcinogenesis to both FGF15−/− and FGF15+/+ mice. FGF15+/+ mice had higher tumour burden with more and bigger tumours and higher proliferation rate measured by Ki67 immunostaining supporting the oncogenic role of FGF15 [33].FGF19 also seems to have a prognostic role for FGF1. Hyeon et al reported a series of 281 patients who underwent curative resection and found that 48% of the tumours expressed FGF19 protein and this was associated with larger size of the tumour (p < 0.0001), more advanced Barcelona Clinic Liver Cancer (BCLC) stage (p = 0.001) and early recurrence (p < 0.001) [34]. An association of FGF19 m RNA expression in the tumour with disease free survival and overall survival has also been reported [30].The importance of FGFR4 in liver carcinogenesis has been showed both in vitro and in vivo. Gauglhofer et al showed that upregulation of FGFR4 in vitro promotes an aggressive phenotype of hepatocellular carcinoma by increasing invasion and tumourigenicity and downregulation of FGFR4 with si-RNA mediated knockdown or kinase-dead FGFR4 or soluble FGFR4 had the exact opposite results with decreased viability, invasion and tumour formation [35]. French et al bred FGF19 transgenic mice with FGFR4 knockout and found that only FGFR4 wild type mice could develop a hepatocellular carcinoma confirming that FGFR4 is necessary for FGF19-mediated carcinogenesis. They also used an FGFR4 neutralizing antibody which was created by immunizing FGFR4 knocked out mice with FGFR4 and showed that it had activity against hepatocellular carcinoma in mice and cell lines [36].Different groups showed that approximately one third of HCCs have high FGFR4 expression. French et al used immunohistochemistry and found moderate to increased staining for FGFR4 for 33% of HCCs and confirmed this finding in a small number of 23 samples with qualitative real-time polymerase chain reaction (q RT-PCR) where FGFR4 expression was more than 2-fold higher in 30% of the tumours [36]. Similar results were reported from Ho et al who found that FGFR4 expression was significantly higher in 31.6% of HCCs (18/57 samples) [37]. The same group also reported an interesting association between a specific single nucleotide polymorphism (SNP) of FGFR4 (G388R) and alpha fetoprotein (AFP) which is an established clinical biomarker of tumour progression; using cell lines they proved that there is a direct link between them since stimulation of FGF19 results in elevation of AFP whilst silencing of FGFR4 results in reduced AFP production [37]. Presence of G388R polymorphism has also been associated with poor prognosis for head and neck cancers [38,39] and prostate cancer [40]. Furthermore expression of FGFR4 has been associated with higher TNM stage and shorter overall survival compared to no expression [41].β- Klotho protein has been reported to be overexpressed (>2fold) in 25% of HCC samples and this finding was correlated with multifocal tumours. Unsurprisingly silencing β-Klotho resulted in decreased cell proliferation and suppression of the pathway. Interestingly, prolonged inhibition of the pathway resulted in an increased population of resistant cells with high expression of stem-cell markers like CD133 and CD44 [42].Amplification of the 11q13 amplicon is one of the most common events in cancer and has a well-recognized role in carcinogenesis of several tumours including head and neck and breast cancers [43]. The 11q13 amplicon contains Cyclin D1 which is a known oncogene but also FGF19, FGF23 and ANO1 which are tumour promoting genes. 11q13 was found amplified in 14% of 89 HCC samples and 12 HCC cell lines as reported by Sawey et al. and RNAi-mediated knockdown of Cyclin D1 or FGF19 as well as use of a neutralizing FGF19 antibody resulted in tumour growth inhibition in vitro [44]. Genomic analysis with whole exome sequencing of 231 resected HCCs (HBV related 72%) identified a number of recurrent mutations, deletions and amplifications. FGF19 amplification was present in 5% of tumours and it was significantly associated with cirrhosis (p = 0.017) [45]. Based on all these results it has been suggested that amplification of 11q13 may represent a potential biomarker to select patients who will benefit from an antiFGF19 treatments [43].The recognition of the role of FGFRs in carcinogenesis lead to discovery of several FGFR inhibitors which are under investigation for different types of cancer including breast, bladder lung and liver cancer, cholangiocarcinoma and glioblastoma [46].Pan-FGFR inhibitors are under evaluation in phase I-II trials like NVP-BGJ398 [47], and AZD4547 [48]. FINN1, FINN2 and FINN3 are covalent pan-FGFR inhibitors (FINN2 and FINN 3 are irreversible) [49]. However all of these agents have a much lower selectivity for FGFR4 compared to FGFR1-3. JNJ-42756493 is another pan-FGFR inhibitor that showed acceptable toxicity profile in a phase I study; dose limiting toxicity was hepatotoxicity with grade 3 elevation of ALT/AST and the most common side effect was hyperphosphataemia which was seen in 60% of patients [50]. LY2874455 is also a pan-FGFR inhibitor which had an acceptable toxicity profile in a phase I study with hyperphosphataemia being also common [51]. Hyperphosphataemia, nail changes and onycholysis, alopecia and hair modification, mucositis, dysgeusia and mucosal dryness, conjunctivitis, keratitis and eye dryness, asymptomatic retinal pigment epithelial detachment and osteoarticular pain, myalgias and muscle cramps seem to be FGFR-inhibition specific side effects whilst long term side effects are still unknown [46].FGFR4 selective inhibitors are also under evaluation in an effort to increase specificity and reduce side effects. Several agents are under investigation. FGF401 (Novartis) is a tyrosine kinase inhibitor with high selectivity against FGFR4 compared to FGFR1-3 and is under investigation in a phase I/II study in patients with hepatocellular carcinoma with FGFR4 and Klothoβ expression (ClinicalTrials.gov Identifier: NCT02325739). AZ709 (Astra Zeneca) is a potent and selective inhibitor of FGFR4 with antitumour activity only in cell lines that expressed high levels of FGF19 and FGFR4 (m RNA and protein) [52]. BLU9931 (Blueprint Medicines) is a potent and irreversible inhibitor of FGFR4. It is highly selective against FGFR4 because it forms a covalent bond with Cysteine 552 (Cys552) which is located near the ATP-binding site of FGFR4 and is unique for FGFR4 and not present in the other FGFRs. Similarly to previous reports BLU9931 showed activity only in HCC cell lines with a fully functional FGF19/FGFR4/Klothoβ complex. Interestingly the three most sensitive cell lines (Hep 3B, HuH7 and JHH7) had a copy-number gain of FGF19. A robust tumour inhibition was also seen in mice treated with BLU9931 with tumours that had an intact FGF19-FGFR4 pathway and treatment had an acceptable toxicity profile [53]. Blueprint Medicines has also developed another FGFR4 inhibitor called BLU554 which is currently tested on a phase I trial (Clinical Trials Gov Identifier: NCT02508467). Finally H3B6527 (H3 Biomedicine) is another highly selective FGFR4 inhibitor with potent antitumour activity in FGF19 amplified cell lines and mice. The agent was also tested in monkeys and interestingly was not associated with bile acid related side effects [54].Another approach that has been studied is that of monoclonal antibodies against FGF19 or FGFR4. Desnoyers et al produced a neutralizing anti-FGF19 monoclonal antibody which prevented development of HCC in FGF19 transgenic mice that were treated with diethylnitrosamine which can accelerate tumour progression [55]. The safety of this antibody was tested in cynomolgus monkeys which have similarities in bile acid synthesis and cholesterol metabolism with humans and treatment with anti-FGF19 antibody was associated with increased bile acid synthesis and ileal malabsorption resulting in diarrhoea [56]. LD1 is a neutralizing antibody against FGFR4 that showed activity in preclinical models and in mice but has not yet been further investigated [36] and other monoclonal antibodies against FGFR4 are currently under development. A summary of currently open trials using FGF19 and FGFR4 inhibitors in various tumours can be found in Table 1.Open trials with FGF19 and FGFR4 inhibitors in various tumours.The need for more precise and personalized treatment for HCC is still unmet. Hepatocellular carcinogenesis is complex and different oncogenic pathways are involved. The lack of predictive biomarkers may well be the reason for the failure of various agents that have been used in the past.Data for the role of FGF19/FGFR4 in liver oncogenesis is robust and oncogenomic screening has identified a subset of patients where this pathway appears to be the oncogenic driver. Several inhibitors of the pathway are under investigation and have shown promising results in the preclinical setting. Based on both in vitro and in vivo results it appears that a selective FGFR4 approach may be more promising for FGF19/FGFR4 driven HCC compared to pan-FGFR inhibitors. The optimal population for such a targeted approach needs yet to be defined. From existing data it seems that patients with tumours with detectable expression of FGF19, FGFR4 and Klothoβ would benefit from such an approach and approximately one third of all patients with HCC are expected to meet these criteria. It is critical that biomarker programmes are undertaken in parallel with clinical studies in order to identify predictive biomarkers that would enable us to select those patients that are more likely to benefit from such a therapeutic strategy.FGFR4 inhibition is not expected to have the same toxicity profile with pan-FGFR inhibitors but may be associated with an increased risk of cholestatic injury in patients who already have dysregulation of bile acid homeostasis [57]. Many of the in vivo data so far are based on mice and the differences between mice and humans need to be taken into consideration before extrapolating these data to humans. For mice peak of cholesterol and bile acid synthesis is at midnight where in humans bile acid synthesis has two peaks per day and cholesterol synthesis follows a circadian rhythm [24].Patients with advanced HCC yield a dismal prognosis and, unlike other cancer types, treatment options are extremely limited. The modest success of sorafenib has spurred on study of various targeted agents in this disease. However, all of the studies have been conducted in unselected populations without any predictive biomarkers which may be the reason for failing to show meaningful outcomes. It appears that a subset of tumours is driven by the FGF19/FGFR4 pathway and these patients could benefit from treatment with FGFR4 inhibitors. Several agents are under development and further research is eagerly awaited since it may provide a more effective treatment in one third of patients with HCC.The authors declare no conflict of interest.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death. In patients with advanced or unresectable HCC, there are few treatment options. Conventional chemotherapy has limited benefits. Sorafenib, a multi-kinase inhibitor, improves survival, but options for patients intolerant of or progressing on sorafenib are limited. There has been much interest in recent years in molecular therapeutic targets and drug development for HCC. One of the more promising molecular targets in HCC is the cellular-mesenchymal-epithelial transition (c-MET) factor receptor. Encouraging phase II data on two c-MET inhibitors, tivantinib and cabozantinib, has led to phase III trials. This review describes the c-MET/hepatocyte growth factor (HGF) signalling pathway and its relevance to HCC, and discusses the preclinical and clinical trial data for inhibitors of this pathway in HCC.Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide [1]. Treatment options in advanced HCC are limited, with conventional chemotherapy having limited efficacy. The multi-kinase inhibitor, sorafenib, was established as the standard of care in patients with advanced HCC after two randomised trials showed a significant survival benefit [2,3], but its use is generally limited to those with good hepatic reserves, and five-year survival remains dismal at less than 10%. Outside of clinical trials, there is a dearth of approved therapeutic options for patients who have disease progression on sorafenib.At least three recent phase III trials of molecularly targeted agents as second-line treatment in HCC failed to meet their primary endpoints. These trials studied brivanib (BRISK-PS) [4], everolimus (EVOLVE-1) [5], and ramucirumab (REACH) [6] compared to placebo.The tyrosine kinase receptor, cellular-mesenchymal-epithelial transition (c-MET) factor receptor, has been studied as a potential therapeutic target, and phase II data with the c-MET inhibitors, tivantinib and cabozantinib, have been encouraging, prompting ongoing phase III trials.In this review, we discuss the c-MET/HGF pathway, its relevance to HCC, and summarise the preclinical and clinical data to date regarding c-MET inhibitors in HCC.The MET proto-oncogene was first identified in an osteosarcoma cell line [7]. The gene encodes for a transmembrane receptor tyrosine kinase (RTK), also known as c-MET, for which HGF is a ligand [8].Binding of HGF to c-MET’s Sema domain leads to receptor homodimerisation, autophosphorylation of tyrosine residues in the tyrosine kinase domain, and downstream activation of the Ras/MAPK, PI3K/Akt, and Ras/Rac/Pho pathways [9]. These promote cell proliferation, survival, migration, and angiogenesis [10].Abnormal activation of HGF/c-MET signalling can occur in several ways [11,12]:(1) Overexpression of HGFElevation of HGF protein levels, both intratumoural and systemic, has been noted in many tumour types, such as lung cancer (50%), breast cancer (91%), stomach cancer (87%), colon cancer (95%), cancer of the head and neck (45%) and liver cancer (33%) [13]. Elevated plasma levels of HGF have been suggested to correlate with a poor prognosis for several forms of cancer, including HCC [14].Plasma HGF levels have been consistently shown to be higher in patients with HCC compared to controls. Biomarker analyses using samples from the SHARP trial [2] and phase II trial for tivantinib [15,16] suggest that HGF levels may have prognostic significance, with better survival in patients with lower levels, and decreasing levels suggesting disease response.(2) Overexpression of c-METOverexpression of c-MET in tumour tissue has been noted in many cancers, such as lung cancer, stomach cancer, breast cancer, kidney cancer, colon cancer, and HCC.In HCC, high c-MET expression has also been found to be a poor prognostic marker, correlating with poorly differentiated tumours and lower survival rates [15,16]. Tumour c-MET expression was also predictive of response to tivantinib.(3) MET amplificationHigh MET gene copy number can be due to general ploidy status, or true focal gene amplification. MET amplification is less common than overexpression of the protein receptor tyrosine kinase [17], but has been noted primarily in gastrointestinal cancers such as gastric cancer, oesophageal cancer [18] and colon cancer, as well as in endometrial carcinoma, medulloblastoma, non-small cell lung cancer (NSCLC) [19] and gliomas [20].(4) Activating mutationsActivating mutations in c-MET’s tyrosine kinase domain have been reported in hereditary and sporadic papillary renal cell carcinomas [21], paediatric liver cancer and squamous cell carcinoma of the head and neck. Other mutations within c-MET’s juxtamembrane region or its Sema domain, where HGF binds, have also been noted in gastric cancer, breast cancer, pleural mesothelioma, and small-cell lung cancer. In addition, c-MET also interacts with other key oncogenic signalling pathways [22].The interaction between c-MET and HER2 family members is well-documented. MET amplification has been reported to lead to EGFR tyrosine kinase inhibitor (TKI) resistance by HER3-mediated activation of PI3K/AKT signalling in NSCLC [23,24]. MET amplification has also been reported as a mechanism for resistance for colorectal cancer patients treated with anti-EGFR antibodies [25,26].c-MET/HGF signalling promotes angiogenesis through increasing vascular endothelial growth factor (VEGF)-A expression and interaction with the VEGF receptor (VEGFR) pathway [27]. It has also been shown to maintain the stem cell niche in cancer, with WNT activity in colorectal cancer stem cells described to be supported by myofibroblast-secreted HGF [28].Small molecule c-MET inhibitors can be classified as selective inhibitors, which specifically target c-MET tyrosine kinase in an ATP-competitive or non-competitive manner, or non-selective inhibitors, which target other kinases in addition to c-MET.Alternatively, blockade of the HGF/c-MET pathway can also be effected through anti-HGF neutralising antibodies, which block only HGF-dependent c-MET activation, or anti-MET antibodies (Table 1, Figure 1). Anti-HGF antibodies will not be discussed further in this paper owing to a lack of reported signal of activity in HCC.Classification of HGF/c-MET Inhibitors.Classification of HGF/c-MET inhibitors.Studies have shown the role of HGF in enhancing liver regeneration, hepatocyte survival and tissue remodelling after acute injury [29], and in suppressing hepatocyte apoptosis [28].In human HCC cell lines, c-MET positive cells were noted to have cancer stem cell-like characteristics. Treated with c-MET inhibition however, c-MET positive cells had increased apoptosis, decreased proliferation and suppressed tumour growth, while c-MET reduced cells survived the inhibition treatment. This suggests that c-MET inhibition may be an effective therapy only for selected patients with strong c-MET expression [30].c-MET pathway activation is also postulated to promote cancer metastasis by inducing epithelial-to-mesenchymal transition (EMT) [31], which causes epithelial cells to lose E-cadherin and cell-to-cell contact and acquire mesenchymal characteristics such as motility and invasion. HGF treatment has been found in murine models of HCC to induce EMT and sustain a mesenchymal phenotype [32].c-MET inhibitors are sequenced in this section by how extensively they have been investigated in HCC, in descending order. Selected active clinical trials involving these agents are summarised in Table 2.Selected Active Clinical Trials on c-MET inhibitors for HCC.All trials are for second-line therapy of advanced HCC unless otherwise indicated. † Restricted to patients with hepatic impairment; ‡ Randomised against sorafenib; § Phase II portion of study randomised against sorafenib alone.Tivantinib is an oral non-ATP-competitive selective small molecular inhibitor of c-MET. Binding of tivantinib to c-MET stabilises the receptor in its inactive conformation, hence blocking both ligand-dependent and ligand-independent receptor phosphorylation and thus reducing downstream signalling [33,34]. Tivantinib demonstrated anti-tumour activity in a wide range of tumour cell lines, as well as in xenograft models [34].Phase I studies for tivantinib, both as monotherapy [35,36,37] and as combination therapy with gemcitabine [38], erlotinib [39] and sorafenib [40], have been conducted in advanced solid tumours. The mechanism of action of tivantinib has been questioned preclinically but Yap et al. showed that c-MET decreases in tumours treated with tivantinib [36].In the phase Ib trial by Santoro et al. [41], tivantinib monotherapy was studied in previously treated HCC patients with Child-Pugh A or B liver cirrhosis. Notably, liver function did not worsen in these patients. Aside from a higher rate of neutropaenia (any grade 52%, grade 3/4 38%), tivantinib demonstrated a manageable safety profile. Out of 21 patients, none achieved any objective response, though nine achieved the best response of stable disease.The combination of tivantinib and sorafenib was examined in a phase I study of 20 HCC patients by Martell et al. [40], following the report of synergistic anti-proliferative activity with the combination in preclinical studies [42]. Of note, eight patients in the study had received sorafenib and/or sunitinib previously, and five achieved stable disease or better. The aforementioned trials suggest that tivantinib may be a promising second-line treatment for advanced HCC.Chai et al. performed a pooled analysis to summarise the outcomes of 53 patients with HCC or biliary tract cancer receiving tivantinib in phase I trials [43]. These included 23 patients receiving tivantinib monotherapy and 30 patients receiving tivantinib in combination with a second drug. The overall response rate (ORR) and disease control rate (DCR) were 6% and 62%, respectively.Based on the phase I data, tivantinib was studied as a second-line therapy for advanced HCC compared against placebo in a randomised multi-centre phase II trial in 107 patients with Child-Pugh A cirrhosis [15]. All of the patients had previously received sorafenib, except for four who had received sunitinib, and had progressed on or did not tolerate first-line treatment. Crossover to open-label tivantinib was allowed for patients on placebo at radiological progression. Of note, the initial dose of tivantinib was planned for 360 mg twice daily, but this was decreased to 240 mg twice daily after 57 patients were enrolled because of a high incidence of grade 3 and 4 neutropaenia. In the whole population, the trial met its primary end point as tivantinib improved time to tumour progression (TTP) (1.6 month vs. 1.4 month; hazard ratio, HR 0.64). Progression-free survival (PFS) and overall survival (OS) however were not statistically different.Pre-specified subgroup analysis according to c-MET expression status was performed, with MET-high defined as more than 50% of HCC cells with 2+ or stronger staining intensity on immunohistochemistry (IHC) [15]. Only the subgroup of patients with MET-high tumours showed a significant survival benefit with improvement in median TTP (2.7 months vs. 1.4 month; HR 0.43; 95% CI 0.18–0.81), median PFS (2.2 months vs. 1.4 month; HR 0.45; 95% CI 0.21–0.95) and median OS (7.2 months vs. 3.8 months; HR 0.38; 95% CI 0.18–0.81). The survival advantage for patients on the lower dose of 240 mg twice daily of tivantinib was at least equivalent to that observed among patients on the higher dose of 360 mg twice daily. Among MET-low patients, however, there was no difference in response rates or survival outcomes between tivantinib and placebo, suggesting that c-MET expression is predictive of response to tivantinib.The same authors subsequently showed that the interaction test between treatment and tumour c-MET levels in OS was positive (p = 0.0385) [16]. They also found that tumour c-MET was the only biomarker which predicted response to tivantinib.Selected phase I and II studies of tivantinib monotherapy are summarised in Table 3.Selected phase I and II clinical trials of Tivantinib monotherapy in HCC (adapted from Rimassa et al. [44]).Abbreviations: PD: progressive disease; AE: adverse events; DLT: dose-limiting toxicities; PR: partial response; SD: stable disease; TTP: time to progression; ITT: intention-to-treat; PFS: progression free survival; OS: overall survival; MTD: maximum tolerated dose; RP2D: recommended phase 2 dose; BD: twice daily.Extending from the phase II data, there are currently two phase III double-blind, randomised controlled trials that are recruiting patients with advanced HCC and high c-MET-expression to compare tivantinib as second-line treatment against placebo, namely the METIV-HCC trial in the West (ClinicalTrials.gov Identifier: NCT01755767) and the JET-HCC trial in Japan (ClinicalTrials.gov Identifier: NCT02029157).In the METIV-HCC trial, the original dose of 240 mg twice daily was reduced to 120 mg twice daily, after a higher than expected rate of neutropaenia and higher than expected drug exposure levels were observed. These were attributed to the switch in formulation from capsules used in the phase II study to tablets [45,46]. The primary endpoint of the trial is OS, with secondary endpoints defined as PFS and safety.Cabozantinib is a non-selective oral multi-kinase inhibitor targeting c-MET, VEGFR2, KIT, RET, FLT3 and TIE-2. Cabozantinib has been shown to prolong survival in a c-MET-driven transgenic mouse model of HCC and to show efficacy against human HCC xenografts grown in mice [47].A phase I dose-escalation study of cabozantinib in 85 patients with advanced solid tumours established the maximum tolerated dose (MTD) at 175 mg daily [48]. Dose-limiting toxicities (DLT) were hand-foot syndrome, mucositis, and transaminitis. The study included one patient with HCC whose disease was measurable, and in whom cabozantinib attained stable disease for at least three months.A phase II randomised discontinuation study evaluated cabozantinib in advanced solid tumours of nine different tumour types, including HCC (N = 41) [49,50]. The study design incorporated a 12-week “lead-in” treatment period with cabozantinib followed by open-label continued treatment in responders until disease progression, treatment discontinuation in patients with disease progression, and random blinded assignment between cabozantinib and placebo in those with stable disease. The most frequent grade 3 and higher adverse events associated with cabozantinib were hand-foot syndrome (15%), diarrhoea (9%), and thrombocytopaenia (9%). DCR at 12 weeks was 71% in patients with HCC [50]. Notably, 49% of these patients were sorafenib-naïve, that is, cabozantinib was the first-line therapy for these patients.Of note, this study did not evaluate for c-MET expression as a predictor of response to cabozantinib, and given the broad spectrum of targets of cabozantinib, it is unclear how much of the activity is attributable to c-MET inhibition alone. In fact, the combined inhibitory effects of c-MET and VEGF may be particularly effective, which is postulated to be due to upregulated c-MET signalling from VEGF inhibition, either from resultant hypoxia or direct interactions between VEGFR2 and MET [51].Selected phase I and II studies of cabozantinib monotherapy are summarised in Table 4.Selected phase I and II clinical trials of Cabozantinib monotherapy in HCC.Abbreviations: DLT: dose-limiting toxicities; HFS: hand foot syndrome; AE: adverse events; SD: stable disease; DCR: disease control rate; MTD: maximum tolerated dose; OD: once daily.Given the encouraging data from the phase II study, a phase III randomised double-blind study is currently recruiting to compare cabozantinib against placebo as second-line treatment for advanced HCC patients who have previously received sorafenib [52]. Enrolment started in September 2013 with a target recruitment of 760 patients. (ClinicalTrials.gov Identifier: NCT01908426). Endpoints of the study are OS (primary), PFS and ORR (secondary), with two interim analyses and a final analysis planned.Capmatinib is a highly selective c-MET inhibitor. It has demonstrated strong dose-dependent anti-tumour activity and dose-dependent reduction of phosphorylated MET (pMET) levels in c-MET-dependent murine tumour models [53].In a phase I dose-escalation study, capmatinib was tested in 33 patients with confirmed c-MET-dysregulated advanced solid tumours, with HCC representing the commonest tumour type (45%), which were refractory to current therapy or for which effective therapy was lacking [54]. Stable disease was reported in 8/33 (24%) of the entire cohort. The recommended phase II dose (RP2D) was 600 mg twice a day, with DLT of fatigue and hyperbilirubinaemia.A phase II trial is currently ongoing testing the efficacy and safety of capmatinib as first-line treatment for patients with c-MET-dysregulated advanced HCC (ClinicalTrials.gov Identifier: NCT01737827). The trial is actively recruiting patients.There was also a phase II randomised trial for capmatinib as second-line treatment for patients with advanced HCC after sorafenib, but the trial was suspended without any patient recruitment (ClinicalTrials.gov Identifier: NCT01964235).Tepotinib is a specific, reversible, ATP-competitive c-MET inhibitor.Following encouraging preclinical data in liver cancer models [55], a phase I study of tepotinib demonstrated good anti-tumour activity and tolerability in patients with advanced solid tumours [56]. DLT were asymptomatic lipase and amylase increases, nausea and vomiting, fatigue, and ALT elevation. The RP2D was 500 mg per day.There are currently two phase Ib/II trials ongoing for tepotinib. The first is a single-arm trial evaluating tepotinib as second-line treatment for MET-positive advanced HCC (ClinicalTrials.gov Identifier: NCT02115375). The other trial is a randomised open-label trial comparing tepotinib against sorafenib as upfront treatment in Asian patients with MET-positive advanced HCC (ClinicalTrials.gov Identifier: NCT01988493). MET positivity in the trial is defined as moderate or strong protein overexpression on IHC [57].Foretinib is an ATP-competitive TKI with activity against c-MET, AXL, RON, VEGFR2, TIE-2 and PDGFR. It has been shown to inhibit tumour growth and prolong mouse survival in patient-derived HCC xenograft models [58].A phase I study of foretinib showed DLT of transaminitis and elevated lipase levels, and common adverse events of hypertension, fatigue, diarrhoea and vomiting, proteinuria, and haematuria [59]. The RP2D was 250 mg given on the first five days of a 14-day cycle.Foretinib has been studied as first-line therapy in a phase I/II study in Asian patients with advanced HCC. The phase I portion of the study showed ORR of 24%, disease stabilisation rate of 79% and median time to progression of 4.2 months, with no DLT observed at 30 mg once daily [60].Golvatinib is a non-specific c-MET inhibitor, with activity also against VEGFR2, c-KIT and RON. It has been shown to promote tumour regression and prolong survival in mouse xenograft models [61].Two dose-finding phase I studies were conducted in patients with advanced solid tumours. The Japanese study found that the MTD was 200 mg twice a day [62], while the UK study determined the MTD as 400 mg once daily [63]. The DLT in both studies were similar, being derangements in liver enzymes, fatigue, and nausea and vomiting, with the former study also reporting proteinuria in 50% of the study cohort.A phase Ib/II clinical trial is currently recruiting, in which the phase II cohort will compare golvatinib plus sorafenib against sorafenib alone as first-line use in patients with advanced HCC (ClinicalTrials.gov Identifier: NCT01271504). The phase I portion of the trial suggested that the golvatinib and sorafenib combination had manageable toxicity and showed an encouraging 17% of patients with partial responses and durable stable disease in another 31% [64].Onartuzumab is a monovalent, humanised monoclonal antibody specific for an epitope in the HGF-binding domain of the c-MET receptor. It was developed to overcome the limitation of bivalent antibodies which was thought might cause receptor dimerisation [65]. Onartuzumab forms a stable bond with c-MET on the cellular surface without inducing c-MET internalisation or shedding [66].A phase I dose-escalation study of onartuzumab as a single agent and in combination with bevacizumab was carried out in patients with advanced solid malignancies. The maximum tolerated dose was not reached, while the most common drug-related adverse events included fatigue, peripheral oedema, nausea and hypoalbuminaemia [67]. A second phase I study of onartuzumab in Japanese patients with solid tumours showed no DLT when used alone or in combination with erlotinib [68]. A third phase I study of onartuzumab specific to Chinese patients with advanced or metastatic solid tumour is currently recruiting patients (ClinicalTrials.gov Identifier: NCT02031731).A phase Ib open-label study evaluating onartuzumab as a single agent and in combination with sorafenib in patients with advanced HCC has completed recruitment (ClinicalTrials.gov Identifier: NCT01897038).Emibetuzumab is a bivalent c-MET-specific monoclonal antibody that blocks HGF binding to c-MET, and neutralises and accelerates internalisation and degradation of the c-MET receptor upon binding, decreasing its level of cell surface expression [69].A phase I dose-escalation study of emibetuzumab was performed in advanced solid tumours, establishing a RP2D of 750 mg every 2 weeks [70]. No DLT were observed, with the most frequent adverse effects reported being nausea, vomiting and diarrhoea.A phase Ib/2 study examining emibetuzumab in combination with ramucirumab in advanced solid tumours including HCC is actively recruiting (ClinicalTrials.gov Identifier: NCT01602289).It is unclear whether systemic levels of HGF predict response to anti-HGF/c-MET therapies. HGF levels are also known to be elevated in many other clinical settings including infections, graft-versus-host disease, and after surgical procedures.Use of IHC for determination of c-MET protein overexpression has been extensively reviewed [71,72], but the variability among published studies in current literature suggests that standardisation of protocols is warranted [12]. Identifying MET-positive/high patients using IHC has been studied as part of various trials, such as the phase II trials of ornatuzumab or placebo with erlotinib in advanced NSCLC [73], rilotumumab or placebo combined with chemotherapy in advanced gastric or gastric oesophageal cancer [74], and tivantinib or placebo in advanced HCC [15,16]. The three trials defined different IHC cut-off criteria: at least 50% tumour cells with 2+ or 3+ staining was referred to as “MET diagnostic positive” in the ornatuzumab study and “MET-high” in the tivantinib study, whereas the rilotumumab study defined “MET-positive” as at least 25% membrane staining of tumour cells at any intensity.Determination of pMET as a biomarker has been used in vitro, but has not been validated in larger studied in histological specimens.In the onartuzumab trial [73], additional subgroup analyses were performed to determine the effect of MET copy number changes and EGFR mutational status. c-MET IHC was found to correlate with MET FISH, but benefit to c-MET-targeted therapy was seen in patients positive by IHC but negative by FISH. In the same study, pMET expression was also studied, but many cases with moderate to strong signals for c-MET by IHC were negative for pMET, suggesting that pMET is an insufficiently sensitive biomarker. In both studies, high c-MET expression was found to be prognostic, and interestingly, low c-MET expression seemed to be predictive of poorer outcome when treated with targeted therapy, emphasising the need for good assays and biomarkers to select a population suitable for targeted therapies.Another possible strategy is the assessment of MET sequence status, including MET mutations, MET amplification, and chromosome 7 polysome [75].Preclinical studies of different c-MET inhibitors revealed variable efficacy based on the specific MET mutation, e.g., PF-2341066/4217903, has greater activity against certain c-MET ATP-binding site mutations compared to c-MET kinase domain activations [76]. One such TKI, SU11274, has also showed selective inhibition for two of four identified MET mutations [77]. MET amplification (defined as MET:CEP7 ratio ≥ 2) has been found to correlate with increased clinical response of metastatic gastric cancer to foretinib in a phase II study [78]; whereas MET copy number (positives scored as ≥ four copies in ≥ 40% of cells), to correlate with increased clinical response to tivantinib with erlotinib in advanced NSCLC [79].Therapeutic options for patients with advanced HCC intolerant to or progressive on sorafenib are scant. Data from c-MET inhibitors is promising, with phase III trials in progress for tivantinib and cabozantinib.Considering that preclinical and clinical data suggest that the benefit of c-MET inhibition may be restricted to a patient subpopulation with high c-MET expression, future trials may need to be enriched by prospectively incorporating biomarker analyses to validate this hypothesis, so as to better select patients who would benefit from these therapies. Already, the METIV-HCC trial has adopted such a strategy of enrolling only patients with MET-high HCC, and its results are eagerly awaited.S.P.C. conceptualized and designed the article. J.J.X.L and J.J.C. performed research and wrote the paper. All authors read and approved the final manuscript.S.P.C.: Advisory board/received funding/consulted/speaker for Merck, Sanofi-Aventis, Bristol-Myers Squibb, Norvatis, and Bayer.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).Hepatocellular carcinoma (HCC) is the most common type of liver cancer, arising from neoplastic transformation of hepatocytes or liver precursor/stem cells. HCC is often associated with pre-existing chronic liver pathologies of different origin (mainly subsequent to HBV and HCV infections), such as fibrosis or cirrhosis. Current therapies are essentially still ineffective, due both to the tumor heterogeneity and the frequent late diagnosis, making necessary the creation of new therapeutic strategies to inhibit tumor onset and progression and improve the survival of patients. A promising strategy for treatment of HCC is the targeted molecular therapy based on the restoration of tumor suppressor proteins lost during neoplastic transformation. In particular, the delivery of master genes of epithelial/hepatocyte differentiation, able to trigger an extensive reprogramming of gene expression, could allow the induction of an efficient antitumor response through the simultaneous adjustment of multiple genetic/epigenetic alterations contributing to tumor development. Here, we report recent literature data supporting the use of members of the liver enriched transcription factor (LETF) family, in particular HNF4α, as tools for gene therapy of HCC.Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the most frequent among the primary tumors of the liver. HCCs are phenotypically and genetically heterogeneous tumors, since they often develop on the pathological background of pre-existing chronic liver diseases, including fibrosis or cirrhosis in consequence of HBV and HCV infections, alcoholic injury, or autoimmune hepatitis, that impair organ function and reduce the efficacy of common cancer therapies [1]. Moreover, most HCC patients are diagnosed at advanced stages of disease when the high tumor recurrence rate and the tendency to metastasize make current treatments ineffective and the prognosis poor [2].In recent years, intense pre-clinical and clinical research have been devoted to the development of tailored therapeutic molecules, capable of restoring the physiological cell functions lost in transformed hepatocytes, through the gene therapy of HCCs. Gene therapy is a promising approach, since it is possible to deliver vectors directly into hepatic tumors, reducing potential side effects derived from transduction in non-target cells. Molecules utilized in current protocols include genes for proteins or microRNAs (miRNAs) displaying antitumor properties (anti-proliferative, pro-apoptotic, anti-angiogenic, or immunomodulatory).Unfortunately, highly effective results have not been obtained so far, due to the low efficiency of the gene transfer [3] and to the genetic heterogeneity of HCCs [4]. For this reason, the most promising candidates would be oncosuppressor genes able to induce an efficient antitumor response without a specific correction of multiple mutations contributing to tumor development (e.g., p53) or differentiation-specific master genes, able to act as reprogramming transcriptional factors, coordinating extensive gene expression. In the context of the latter strategy, we will discuss recent progress in the knowledge of HCC biology and genetics supporting the use of Liver Enriched Transcription Factors (LETFs), and in particular of hepatocyte nuclear factor 4α (HNF4α), as promising candidates for targeted gene therapy of HCCs.In spite of the heterogeneity of HCC, tumor onset and progression have been associated with recurring cell-autonomous molecular changes [5] such as the loss of expression of differentiation genes (e.g., Hepatocyte Nuclear Factor 1 and 4, HNF1α, and HNF4α) [6,7], chromosomal instability leading either to the loss of heterozygosity in tumor suppressor genes (e.g., p. 53) [8] or to the amplification of loci for oncogenes (e.g., ERK5) [9], and aberrant activation of signaling pathways (e.g., Wnt/β-catenin pathway) [10].In addition, recent findings highlight how epigenetic alterations are commonly observed in human HCCs [11,12] and can be exploited as clinical predictors for diagnosis and prognosis [13]. These alterations include aberrant methylation of tumor suppressor genes [13], post-translational histone modifications [14,15], and altered expression profile of miRNAs [16,17].The progression of HCC toward more aggressive stages, responsible for the worst prognosis in patients, is frequently associated to the activation, in transformed hepatocytes, of a transdifferentiation process: the epithelial-to-mesenchymal transition (EMT). EMT contributes to tumor progression through the loss of epithelial/hepatocyte cell differentiation, the acquisition of motility/invasivity properties and cancer stem cell traits, the resistance to apoptosis, and metastasis (reviewed in [18]). Overexpression of EMT markers (i.e., Snail and Twist) has been reported in invasive areas of primary tumors [19] and in metastasis of aggressive hepatocarcinomas [20], and were associated with poor prognosis. Their analysis in circulating tumor cells has been recently proposed as prognostic tools for HCC patients [21]. The role of EMT master genes, in particular Snail, in tumor progression was found to be mediated by (i) the direct transcriptional repression of an extensive amount of target genes involved both in epithelial (e.g., E-cadherin) [22] and hepatic (e.g., HNF4α) [23] differentiation, (ii) the increase of mesenchymal gene expression [23], and iii) the miRNA-mediated up-regulation of stemness genes [24]. The acquisition of EMT-related stem cell characteristics has been demonstrated to positively correlate with HCC progression [25]. The presence of stemness traits in HCC tumor cells, indeed, has been associated with chemoresistance and tumor recurrence after surgery [26,27] and can contribute to the intratumoral heterogeneity of HCC tumor cells [28].An important role in HCC is also played by non-cell-autonomous cues, such as the presence of factors in the tumor niche promoting tumor growth or influencing proliferation/activation of tumor-associated fibroblasts [29,30].In particular, the role of TGFβ cytokine in the progression of HCC was largely described. In HCC patients elevated plasma levels of TGFβ have been reported, correlating with poor prognosis [31,32]. Furthermore, in late stage HCC, the TGFβ signaling pathway is constitutively activated [33] and is involved in promoting tumor invasion through stimulation of vascularization [34], acquisition of stem-like features [35], and induction of EMT [36]. The molecular signature of late stage HCC-derived cell lines, indeed, showed high levels of EMT markers (including matrix metalloproteases, vimentin and, particularly, Snail) and down-regulation of genes for liver-specific functions, indicating a reduced hepatocyte/epithelial differentiation state [37]. TGFβ signaling, moreover, can be amplified in HBV-infected HCC cells by HBx protein [38].TGFβ signaling is also closely linked to liver diseases favoring development of HCC. TGFβ can activate hepatic stellate cells (HSCs) [39] and induce an immune response, causing fibrosis and leading to HCC onset [40]. Its serum levels are also associated to virus-induced fibrosis [41,42]. Furthermore, TGFβ can induce production of CTGF (Connective Tissue Growth Factor) by cancer-associated fibroblasts promoting tumor progression [43].Recent reports highlighted a role of biophysical changes in extra-cellular matrix stiffness as microenvironmental cues influencing tumor growth and progression. Fibro-cirrhotic livers, for example, are characterized by a significant increase of ECM stiffness [44]. YAP/TAZ were recently identified as molecular relay of mechanical stimuli exerted by ECM stiffness, inhibited by the Hippo signaling pathway and involved in organ size control [45]. Dysregulation of the Hippo/YAP cascade has been recently reported for several human tumors, including HCC, and correlates with increased cell proliferation and survival, acquisition of stemness properties, and metastasis (reviewed in [46]). In particular, YAP overexpression was found in human HCC samples [47,48] and correlates with poor prognosis of HCC patients [49]. Furthermore, its inhibition in cells from advanced HCC restores hepatocyte differentiation inducing the up-regulation of master factors (i.e., HNF4α/FOXA1/FOXA3) and leading to tumor regression [50]. Interestingly, YAP protein is directly involved in switching occupancy of HNF4α on embryonic hepatoblast genes to adult hepatocyte genes [51], suggesting a direct role of YAP in influencing the function of key transcriptional factors and master genes of hepatocyte differentiation.As highlighted above, current therapies for HCC are still ineffective. Surgical liver resection efficiency is limited to small localized tumors with low risk of recurrence in non-cirrhotic patients. Conventional chemotherapy is largely unsuccessful due to tumor cell resistance and side effects of “non-selective” cytotoxic drugs. Furthermore, the immunosuppression associated to HCC (mainly subsequent to chronic HBV and HCV infections) negatively impacts on tumor recurrence. For this reason, immuno-based therapies have been proposed to ameliorate the clinical outcome of HCC patients (reviewed in [52]).Targeted approaches have also been applied, in particular for advance-stage and unresectable HCC. These treatments include oral administration of the multikinase inhibitor sorafenib, or single target agents, such as gefitinib and erlotinib, currently involved in ongoing clinical trials in the US and EU [53]. The therapy with sorafenib, in particular, showed prolonged median overall survival and delayed the median time to progression in patients with HCC, showing limited and manageable adverse effects [54]. However, chronic liver diseases that usually underlie HCC may enhance the hepatotoxicity of these agents; accordingly, the prognosis of late-stage HCC patients is still poor [53].In this context, the targeted gene therapy for the management of HCC seems to be the most promising approach. In particular, the adenoviral mediated gene therapy is well documented and included in several human clinical trials where the tolerance is high and side effects acceptable in most of the cases (reviewed in [55]). However, the efficiency of transduction and the tumor specificity still remain limiting factors for this approach. In HCC, these problems could be overcome by intratumoral administration of vectors and/or by the use of tumor-specific promoters that may restrict the delivery to hepatocytes (e.g., AFP) [56], especially improving efficacy and minimizing the toxicity of this therapeutical strategy.Among different approaches of gene therapy (restoration of oncosuppressors, delivery of suicide genes, or inhibition of oncogenes) the delivery of “differentiating” factors could achieve the best results in terms of low toxicity and maintenance of tissue homeostasis, especially compared to killing drugs or agents inducing apoptosis. The most severe consequence may be related to the damage of the stem cell compartment with the decreased number of cells (stem cells or progenitors) responsible for tissue renewal. However, in the liver, the real involvement of resident liver stem/precursor cells in hepatic regeneration after chronic injury is strongly debated since it has been recently formally proved that adult hepatocytes originate from self-duplication of other hepatocytes rather than from stem cell differentiation [57,58]. Altogether, this knowledge suggests that the ectopic expression of differentiation master genes in the liver could be tolerated and the side effects reduced.Maintenance of hepatocyte differentiation and control of liver-specific gene expression are attributed in large part to hepatocyte nuclear factors (HNFs) belonging to the LETF family, including HNF1α, HNF4α, HNF6, and FOXA2 [59]. Being reciprocal transcriptional activators, they operate cooperatively in a connected network in the liver, regulating several developmental and metabolic functions in hepatocytes [60,61].The nuclear receptor HNF4α is a key regulator of hepatocyte differentiation during embryonic development [62,63], influencing the expression of other hepatic transcription factors, and stabilizing co-regulatory networks for the maintenance of a differentiated phenotype [61]. In the adult liver, HNF4α is highly expressed in hepatocytes. HNF4α maintains hepatocyte identity both by inducing epithelial/hepatic differentiation through a direct regulation of epithelial and metabolic target genes [62,64], and by actively inhibiting mesenchymal differentiation program through a direct repression of mesenchymal and EMT master genes [65]. Accordingly, experimental HNF4α deletion in adult mouse livers has been shown to lead to dedifferentiation and proliferation of hepatocytes, hepatomegaly, and expansion of precursor cells (i.e., oval cells) [66,67].HNF4α is a strong inducer of mesenchymal-to-epithelial transition (MET). Its ectopic expression in fibroblast [62] and F9 cells [68] is sufficient to trigger epithelial gene expression and acquisition of epithelial polarity. Furthermore, HNF4α, together with FOXA1, FOXA2, or FOXA3, was found capable of inducing the direct reprogramming of mouse fibroblasts into hepatocyte-like cells [69].Importantly, in addition to the transcriptional regulation of mRNAs, HNF4α regulates the expression of miRNAs which, in turn, can act as pleiotropic elements influencing differentiation, EMT, stemness, and hepatocarcinogenesis.In particular, HNF4α (as well as other LETFs) was found to directly regulate expression of the liver-specific microRNA-122 (miR-122) [70], the most abundant miRNA in hepatocytes, and the first miRNA suggested as a tumor suppressor in the liver. Its expression, indeed, is frequently reduced in HCCs [71] and is associated with low differentiation, migration/invasivity of HCC cells [72,73], and poor prognosis in patients [72]. Mir-122 restoration in HCC cells leads to a reduction of mesenchymal markers [74], cell-cycle arrest or apoptosis [75], and sensitizes cells to antitumor agents [76,77]. Notably, miRNA-122 delivery in HCC murine models impaired tumor occurrence, growth, and progression [73,78].Recently, the transcriptional regulation of other miRNAs, i.e., members of the miR-200 family and miR-34a, by HNF4α has been described and showed to contribute to the active repression of stem cell genes [24]. Both miR-200 family members and miR-34a were suggested to function as tumor suppressors in HCCs. They appeared markedly down-regulated in HCC [79,80] and their restoration in various cancer stem cells is associated with the loss of stem cell traits, inhibition of EMT, cell differentiation, and decreased motility/invasivity [24,81,82]. However, the role of miR-34a in cancer is currently debated [83] and, in HCC, has been related to the cellular context [84].It has been recently shown that HNF4α controls the epigenetic state of differentiated hepatocytes through the miR-29-mediated DNMT3A,B down-regulation [85]. Interestingly, low levels of miR-29 and DNMT3A,B up-regulation correlate with TGFβ-induced EMT, liver fibrosis, and aggressiveness of HCC [86,87,88]. Being the epigenetic changes, including DNA methylation, sustained by the presence of high levels of DNMTs, involved in both EMT [89] and hepatocarcinogenesis [90], miR-29 could represent a good target for a therapeutic approach aimed at the epigenetic reprogramming of HCC cells.Several lines of evidence indicate HNF4α as a potential tumor suppressor of HCC. In mature hepatocytes, loss/inactivation of its function resulted in an increased risk for development of HCC. Transient inhibition of HNF4α is sufficient to initiate hepatocellular transformation in non-transformed hepatocytes and to increase invasiveness in transformed HCC cell lines through a microRNA-mediated inflammatory loop circuit [91]. This network can also contribute to the maintenance of HNF4α inactivation during hepatocellular transformation [91]. Several studies have shown a decreased expression of HNF4α in both murine models and human samples of HCC, thus indicating a critical role of this protein in the HCC onset/progression [7,92,93]. As a consequence, the restoration of HNF4α expression/function in HCCs has represented, in the last few years, an important goal for molecular approaches to HCC treatment. The whole described tumor-suppressing functions of HNF4α indicate that this protein represents a good candidate for the extensive reprogramming of tumor cells and, therefore, a promising tool for gene therapy of HCC.Several data substantiate this expectation. Forced HNF4α expression in dedifferentiated and aggressive HCC is sufficient to reduce tumor cell motility/invasivity by inducing differentiation and EMT inhibition [65,92]. Moreover, HNF4α overexpression attenuates hepatic fibrosis and, in fibrotic livers, can prevent HCC occurrence by blocking the activation of myofibroblasts [93,94]. Furthermore, overexpression of HNF4α in rodent HCC models blocks carcinogenesis and metastasis [93,95].Overall, the restoration of the HNF4α functions in invasive HCCs has been proven to be an efficient approach for the gene therapy of HCC, at least in experimental models. However, recent data have shown how microenvironment cues could reduce the efficacy of this approach. In particular, the presence of TGFβ in the tumor niche impaired HNF4α activity by inducing the displacement of the ectopic protein from its target gene promoters through the inactivation of GSK-3β activity [96]. This result suggests the need to obtain improved HNF4α proteins as tools for gene therapy, through the design of TGFβ-insensitive mutants.At the same time, the potential tumor suppressor activity of other members of the LETF family should be explored. Recently, the role in tumor suppression of HNF1α and HNF6, has been described. Similarly to HNF4α, indeed, these proteins are down-regulated in HCC and their overexpression in tumor cell lines was found to suppress EMT and invasion [92].HNF1α is a homeodomain protein that plays a critical role in hepatocyte differentiation. It contributes to the expression of products central in normal hepatic functions [97] and is required for the maintenance of the differentiated state [98]. HNF1α, moreover, together with HNF4α and HNF6, leads to the generation of functional human-induced hepatocytes (hiHeps) from fibroblasts [99] and its overexpression is necessary for the direct reprogramming of human fibroblasts to hepatocyte-like cells [100].Extensive evidence suggested that HNF1α acts as a tumor suppressor gene and that its down-regulation contributes to the development of HCC. HNF1α gene was found mutated in 84% of cases of adenomas, including familial forms [101,102], and HNF1α protein levels were found significantly reduced in moderately- and poorly-differentiated tissues from HCCs [6]. Furthermore, HNF1α knock-out mice exhibit tumor-associated characteristics, such as increased proliferation of hepatocytes, leading to a dramatic liver enlargement and liver function defects [103].Taken together, these findings suggested that restoration of HNF1α functions in HCC could restrain tumor proliferation and progression. Zeng et al. recently demonstrated that the forced re-expression of HNF1α in human hepatoma cell lines induces a re-establishment of hepatic differentiation through the significant induction of liver specific genes and the repression of cell proliferation. Most importantly, intratumoral HNF1α transduction significantly inhibits tumor growth in mice and eradicates HCC nodules after systemic delivery [104].HNF1α is not only a promising therapeutic tool for a differentiation therapy in HCC treatment but it could be also a potent anti-EMT tool, being a strong transcriptional repressor of EMT master genes as HNF4α [65]. Accordingly, suppression of HNF1α in HCC cell lines triggers expression of mesenchymal and EMT master genes, overexpression of TGFβ, and migration [105].HNF6 represents another potential molecular tool for tumor suppression in HCC. It is essential for expression of hepatic genes, also controlling the direct expression of HNF4α [106] and genes involved in glucose metabolism [107,108]. Moreover, HNF6 synergistically cooperates with HNF4α and HNF1α for the regulation of hepatocyte differentiation during development and in the adult. HNF6 is also a strong transcriptional activator of miR-122, establishing a positive feedback loop responsible for in vivo hepatocytes differentiation [109] that may contribute to prevent neoplastic transformation.HNF6, as well as other LETFs, is involved both in the maintenance of the epithelial differentiation and in the active repression of EMT program through the up-regulation of p53 tumor suppressor [110]. Furthermore, it is implicated in the inhibition of HCC progression [92].HNF6 overexpression reduced the proliferation of liver cancer cell lines [111], inhibited colony formation and cell proliferation/migration in carcinoma cells, and decreased the formation of tumors in nude mice [110]. Conversely, knockdown of HNF6 induced EMT and increased cell migration [110]. Furthermore, HNF6 has been recently shown to interfere, in vitro and in vivo, with HBV infection through the inhibition of viral gene expression and DNA replication [112].Notably, a potential inhibitory effect of HNF6 on TGFβ signaling has recently been reported. Components of TGFβ signaling pathway were activated in HNF6 knockout mice, at least in part, through the up-regulation of TGFβRII expression [113]. Interestingly, through the inhibition of TGFβ/activin signaling, HNF6 allows differentiation of precursor cells in hepatocytes [114]. These data, in light of what was previously observed for HNF4α, could indicate HNF6 as a more efficient tumor suppressor in the presence of TGFβ in the tumor microenvironment.The unsuccessful therapeutic approaches for the treatment of HCCs lead to focus the attention on molecular strategies consisting of the intra-tumoral delivery of specific proteins with tumor suppressor properties.Recent literature data discussed above demonstrates the high potential of anti-cancer therapy based on the restoration of functions of epithelial/hepatocyte differentiation master regulators belonging to the LETF family, mainly HNF4α. These proteins are able to induce cellular reprogramming, coordinating extensive gene expression either through direct transcriptional regulation or by driving epigenetic changes on regulatory regions of target genes (Figure 1). LETFs, indeed, can not only induce the terminal differentiation of tumor cells (and potentially of cancer stem cells) but they can also interfere with the EMT program responsible for tumor progression. These characteristics make LETFs promising tools for molecular therapy of HCC. The challenge is now the optimization of these tools through the creation of engineered molecules to take in account the microenvironmental cues that could influence the effectiveness of this therapeutic approach. Further studies will be necessary to achieve this result.Tumor suppressor properties of HNF4α, HNF1α, and HNF6 in HCCs. HNFs can regulate different cell functions associated with the HCC onset and progression, through the direct transcriptional activation/repression of target genes (described in the text). The reciprocal regulation among HNFs is shown.AM and MT contributed to the design, writing and editing of the review; FB and AMC contributed to the writing and editing of the review. All authors read and approved the final manuscript.The authors declare no conflict of interest.