The Expanded Risk Score in Rheumatoid Arthritis: performance of a disease-specific calculator in comparison with the traditional prediction scores in the assessment of the 10-year risk of cardiovascular disease in patients with rheumatoid arthritis

The increased risk of cardiovascular disease has emerged as a major issue in patients with rheumatoid arthritis – it has been estimated that the cardiovascular disease burden in rheumatoid arthritis is comparable to that of diabetes mellitus

Binding properties of different categories of IDO1 inhibitors: a microscale thermophoresis study

Aim: Inhibition of IDO1 is a strategy pursued in the immune-oncology pipeline for the development of novel anticancer therapies. At odds with an ever-increasing number of inhibitors being disclosed in the literature and patent applications, only very few compounds have hitherto advanced in clinical settings. Materials & methods: We have used MicroScale Thermophoresis analysis and docking calculations to assess on a quantitative basis the binding properties of distinct categories of inhibitors to IDO1. Results: Results shed further light on hidden molecular aspects governing the recognition by the enzyme of compounds with different mechanism of inhibition. Conclusion: Results pinpoint specific binding features of distinct inhibitors to IDO1 that offer clues for the design of next-generation inhibitors of the enzyme

Toll-like receptor-4 is involved in hepatic fibrogenesis in the course of non-alcoholic fatty liver disease

Toll-like receptor-4 (TLR4) is actively involved in liver in the response to injury from a variety of etiologies. Recently TLR4 expression by hepatic progenitor cells (HPC) and biliary epithelial cells has been associated to the progression of liver damage in chronic HCV-related hepatitis (1). HPC compartment activation in ductular reaction (DR) is a feature of progressive disease also in non-alcoholic fatty liver disease (NAFLD) (2). We aimed to investigate the association among TLR4 expression, HPC compartment activation and histopathologic features of fibrotic disease progression in NAFLD. Seventy-four patients who had undergone liver biopsy were included and immunohistochemistry for TLR4 was performed on hepatic tissue samples. CK-7 was used to evaluate HPC, bile ducts (BD)/ductules of DR and intermediate hepatocytes; α-smooth muscle actin was used to quantify the activation of hepatic stellate cells (HSC) and of portal/septal myofibroblasts (MF). HPC in BD/DR were responsible for the highest TLR4 intensity of staining. TLR4-positive HPC and BD/ DR correlated with fibrosis (p<0.01 and p<0.05), activity of MF (p<0.001 and p<0.05) and HSC (p<0.001 and p<0.001), portal and interface chronic inflammation (p<0.01 and p=0.01). The present study indicates the activation of the TLR4 expressing HPC compartment as important determinant of the progressive liver damage in NAFLD. TLR4 stimulation could represent one of the mechanisms directly linking the activation of HPC to inflammation and fibrosis in NAFLD

Treg/Tcon Immunotherapy and High Dose Marrow Irradiation Ensure Full Control of Leukemia Relapse in Haploidentical Transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) is the most powerful therapy for patients with high risk of relapse. In spite of that, no matter the donor source or conditioning regimen used, leukemia relapse is still the leading cause of HSCT failure. In HLA-haploidentical HSCT, we recently applied a clinical protocol consisting of total body irradiation (TBI)-based conditioning regimen and a peripheral blood CD34+ cell graft combined with the adoptive transfer of naturally occurring regulatory T cells (Tregs) and conventional T cells (Tcons). No post-transplant pharmacologic GvHD prophylaxis was given. Such protocol was associated with low GvHD and relapse rate (Martelli et al., Blood 2014). To further reduce leukemia relapse in Treg/Tcon-based haploidentical HSCT (Treg/Tcon haplo-HSCT) we used high dose hyper-fractionated TBI (HF-TBI) in the conditioning regimen. We also extended Treg/Tcon haplo-HSCT to patients that are unfit (because of previous comorbidities) and/or too old to withstand high intensity regimens. In these patients the extra-hematologic toxicity of irradiation was reduced with the use of targeted total marrow and lymph node irradiation (TMLI). 40 patients with high risk acute leukemia (36 AML, 4 ALL) received Treg/Tcon haplo-HSCT. All but 3 patients were transplanted in complete remission. 12 younger patients (median age: 28, range: 20-43) received HF-TBI, while 28 older or unfit patients (59, 40-70) received TMLI in the conditioning regimen. HF-TBI (14.4 Gy) was administered in 12 fractions, 3 times a day for 4 days. TMLI was administered by means of Helical Tomotherapy HI-ART (9 fractions, 2 times a day for 4.5 days). Irradiation was followed by chemotherapy with Thiotepa, Fludarabine, and Cyclophosphamide. 2 × 106/kg freshly isolated CD4+CD25+FOXP3+ Tregs were transferred 4 days before the infusion of 1 × 106/kg Tcons and a mega-dose of CD34+ hematopoietic stem cells. No post-transplant pharmacologic GvHD prophylaxis was given. 38/40 patients engrafted. 12 (31%) developed aGvHD grade ³2 (10 are alive and off-therapy). 6 (16%) died because of transplant related complications (2 because of aGvHD, 2 infections, 1 veno-occlusive disease, 1 intracranial hemorrhage). Strikingly, despite the high risk diseases, no patient relapsed after a median follow up of 13 months (range 1-36, Fig. A). Further, only 1 patient developed cGvHD. Thus, cGvHD/Leukemia-free survival was 82% (Fig. B). Treg adoptive transfer allows for the safe infusion of an otherwise lethal dose of donor alloreactive Tcons in the absence of any other form of immune suppression. Our results demonstrate that the potent graft versus leukemia effect of Treg/Tcon adoptive transfer was boosted by high dose marrow irradiation. Thus, this study proves that the right combination of haploidentical Treg/Tcon immunotherapy plus a powerful conditioning regimen can fully eradicate leukemia

Portal and interface chronic inflammation are associated with the progenitor cell compartment activation during NAFLD

Background and aim: During nonalcoholic fatty liver disease (NAFLD), portal and interface chronic inflammation (PCI and ICI) are strongly associated with fibrosis by activation of hepatic stellate cell (HSC)s (Brunt et al., 2009; Vespasiani-Gentilucci et al., 2014). However, the determinants of PCI and ICI observed in NAFLD remain to be elucidated. Since portal and periportal ductular reaction is related to disease progression, we aimed to investigate if PCI and ICI are associated with hepatic progenitor cell (HPC) compartment activation. Methods: Fifty-two NAFLD patients were studied. NAFLD activity score, fibrosis, PCI and ICI were histologically evaluated. HPCs, intermediate hepatobiliary cells and bile ductules/interlobular bile ducts were evaluated by immunohistochemistry for CK-7, CK-19 and EpCAM. HSC and myofibroblast (MF) activity were determined by immunohistochemistry for α-SMA. Results: PCI and ICI strongly correlated with HPC compartment activation and with the activity of MFs (p≤0.001). Lobular inflammation, ballooning and HPC compartment activation were all associated with both PCI (p<0.01) and ICI (p<0.05) by univariate analysis. In the multivariate models, HPC compartment activation was independently associated with PCI and ICI (OR 4.4, 1.7-11.5; OR 3.4, 1.5-7.9, respectively). Conclusions: During NAFLD, PCI and ICI are strongly associated with HPC compartment activation and this association is likely one determinant subtending the strong association between PCI/ICI and fibrosis

Liver vitamin D receptor, CYP2R1 and CYP27A1 expression related to progression of metabolic and viral chronic liver damage

Background and aim: Low serum 25(OH)vitamin D3 levels were associated with the presence and prognosis of liver diseases [1]. The biological effects of 1,25(OH)2 vitamin D3 are mediated by the vitamin D receptor (VDR) and VDR has been widely detected in liver, but its expression in the course of liver disease has never been investigated [2]. We aimed to evaluate the hepatic expression of VDR and vitamin D 25-hydroxylases in patients with chronic hepatitis C (CHC) or non-alcoholic steatohepatitis (NASH) and its relationship with liver histology and serum 25(OH) vitamin D3 levels. Methods: Patients affected by CHC or NASH who had undergone liver biopsy and subjects without liver disease were included. Expression of VDR, CYP2R1 and CYP27A1 was evaluated by immunohistochemistry. Results: In CHC subjects, fibrosis stage was associated with low hepatic CYP27A1 expression, whereas in patients with VDR-negative inflammatory cells and low VDR expression on hepatocytes, the portal inflammation was significantly higher (p<0.009 and p<0.03). In NASH patients, VDR expression on cholangiocytes was inversely correlated with steatosis severity (p<0.02), lobular inflammation (p<0.01) and NAS score (p<0.03). Conclusions: The liver of patients with viral and metabolic chronic liver disease expresses VDR in a manner inversely proportional to the severity of histological lesions and a role of the vitamin D/VDR system in the progression of chronic liver damage is suggested

The hepatic expression of GH/IGF1 axis components is impaired with fibrosis progression in patients with HCV-related chronic hepatitis

Background and aim: Resistance to the action of growth hormone (GH), characterized by low serum levels of insulin-like growth factor-1 (IGF1) in the face of high concentrations of GH, frequently complicates cirrhosis (Assy et al., 2008). Physiologically, the activation of GH receptor (GHR) determines phosphorylation of signal transducer and activator of transcription (STAT)-5 and the consequent induction of IGF-1 expression. The suppressor of cytokine signalling (SOCS)-3 negatively regulates this intracellular cascade. Since, to date, the hepatic expression of the GH/IGF1 axis components has been studied mainly in animal models (Blaas L et al., 2010), we aimed to evaluate their expression in the liver of patients with HCV-related chronic hepatitis. Methods: Fifty HCV patients were studied and liver samples were histologically re-evaluated for grading and staging. The expression of GH/IGF1 axis components was assessed by immunohistochemistry. Results: At the hepatocyte level, IGF-1 and phospho-STAT5 showed a negative correlation with fibrosis stage, while SOCS3 a positive one (p<0,05 for all). Furthermore, the hepatocyte expression of IGF1 was negatively correlated with its expression by hepatic stellate cells (p<0,05). Conclusions: IGF1 expression by hepatocytes was reduced with fibrosis progression, probably due to the impairment of GHR intracellular cascade. The inverse correlation between IGF1 expressed by hepatocytes and hepatic stellate cells suggests specific roles for IGF-I produced by different hepatic cells

Reelin expression by hepatic stellate cells and ductular reaction in HCV related liver fibrosis

Reelin is a secreted extracellular glycoprotein that is thought to guide migrat- ing neurons during brain development and maturation cooperating with Disabled-1 (Dab1), an adaptor protein obligate effector of reelin signalling pathway (1). Reelin is also expressed in human liver by hepatic stellate cell(HSC)s that following liver injury become activated, migrating and fibrogenic cells (2). The cross-talk between HSCs and other cells such as those of ductular reaction (DR) is believed to rule liver fibrogenesis leading to cirrhosis (3). In order to better understand the role of ree- lin in human liver tissue with ongoing fibrosis, we aim to analyse the hepatic reelin expression and its relationship with the main histological determinants of the dis- ease activity and severity. Eighty-one liver biopsies of patients with chronic hepatitis C were studied. The expression of Reelin, Dab1, and HSC markers was investigated by immunohistochemistry and immunofluorescence. The Knodell histology activity index and DR score were evaluated. Activated HSC were frequently reelin positive and a statistical correlation was found between the number of reelin positive HSCs and Knodell’s stage (r= 0,3; p<0,05). Dab1 was expressed by cells of DR and the number of reelin positive HSCs correlated with DR score in mild/moderate fibrosis (r=0,4; p<0,05). Since reelin expression by HSCs correlates with increasing fibrosis and DR, whose cells in turn express Dab1, it might act as mediator in DR activation by HSCs. Further studies are needed to test reelin as useful biomarker for liver fibrosis assess- ment

Toll-like receptor-4 expression by hepatic progenitor cells and biliary epithelial cells in HCV-related chronic liver disease

Background. Toll-like receptor-4 (TLR4) is a transmembrane pattern recognition receptor that plays a key role in innate immunity by triggering inflammatory responses to Gram negative bacteria lipopolysaccharide (LPS) (1). Since liver is the main clearance organ for LPS, which is excreted in large amounts in the bile (2), it is not surprising that TLR4 has been involved in the pathogenesis of most liver diseases (3). Numerous evidences suggest a role for TLR4 in the pathogenesis of chronic hepatitis C virus (HCV) infection (4) and hepatic fibrosis (5), but the localization and level of TLR4 expression in the liver of patients with hepatitis C have never been investigated. Aim and methods. We aimed to evaluate, by means of immunohistochemistry (IHC) and real-time polymerase chain reaction (rt-PCR), hepatic TLR4 expression in patients with chronic HCV infection. Sixty-one patients with chronic HCV infection, and 12 controls free of liver disease, were included in the study. Each case was analyzed by IHC for TLR4, α–smooth muscle actin (αSMA) and cytokeratin-7 (CK-7), and a subgroup of patients and all controls by rt-PCR for TLR4. A score of activation of portal/septal myofibroblasts and lobular hepatic stellate cells (HSCs) was evaluated by IHC for α-SMA, whereas IHC for CK-7 was analysed in order to count hepatic progenitor cells (HPCs), interlobular bile ducts and intermediate hepatocytes. Results. The parenchimal elements responsible for the highest TLR4 level of expression were HPCs and biliary epithelial cells (BECs) of interlobular bile ducts in the infected group. Double-labeling experiments with anti-TLR4, anti-CK7 and anti-CD133 confirmed this finding. TLR4-positive HPCs and interlobular bile ducts were significantly correlated with the stage of liver disease (p<0.001), the grade of inflammation (p<0.001), and with the activity of portal/septal myofibroblasts (p<0.001). Rt-PCR study confirmed an increased TLR4 expression in the 26 patients analyzed with respect to controls (p<0.001). TLR4 expression positively correlated with fibrosis (p<0.05) and inflammation (p<0.05). Conclusions. The expression of TLR4 in HPCs and BECs in HCV-related liver damage significantly correlates with inflammation, activation of portal/septal myofibroblasts and fibrosis. 1) Beutler. Nature 2004;430:257-63; 2) Van Bossuyt et al. J Hepatol 1988;7:325-37; 3) Seki et al. Hepatology 2008;48:322-35; 4) Machida et al. J Virol 2006;80:866-74. 5) Seki et al. Nat Med 2007;13:1324-32

Molecular and histological traits of reduced lysosomal acid lipase activity in the fatty liver

Recent studies demonstrated reduced blood lysosomal acid lipase (LAL) activity in patients with nonalcoholic fatty liver disease (NAFLD). We aimed to verify hepatic LAL protein content and activity in in vitro and in vivo models of fat overload and in NAFLD patients. LAL protein content and activity were firstly evaluated in Huh7 cells exposed to high-glucose/high-lipid (HGHL) medium and in the liver of C57BL/6 mice fed with high-fat diet (HFD) for 4 and 8 months. LAL protein was also evaluated by immunohistochemistry in liver biopsies from 87 NAFLD patients and 10 controls, and correlated with hepatic histology. Huh7 cells treated with HGHL medium showed a significant reduction of LAL activity, which was consistent with reduced LAL protein levels by western blotting using an antibody towards the N-term of the enzyme. Conversely, antibodies towards the C-term of the enzyme evidenced LAL accumulation, suggesting a post-translational modification that masks the LAL N-term epitope and affects enzymatic activity. Indeed, we found a high rate of ubiquitination and extra-lysosomal localization of LAL protein in cells treated with HGHL medium. Consistent with these findings, inhibition of proteasome triggered dysfunctional LAL accumulation and affected LAL activity. Accumulation of ubiquitinated/dysfunctional LAL was also found in the liver of HFD fed mice. In NAFLD patients, hepatic levels of non-ubiquitinated/functional LAL were lower than in controls and inversely correlated with disease activity and some of the hallmarks of reduced LAL. Fat overload leads to LAL ubiquitination and impairs its function, possibly reducing hepatic fat disposal and promoting NAFLD activity