Mechanisms of HCV pathogenesis: role of viral core+1 protein

Hepatitis C virus (HCV) was discovered in 1989 as the major causative agent of non-A, non-B hepatitis. It is an enveloped, single stranded, positive sense RNA virus and belongs to the family of Flaviviridae. Studies around the globe have identified seven major HCV genotypes and more than 100 different subtypes. It is estimated that more than 170 million people are infected worldwide, that covers about 3% of the world’s population. This virus establishes chronic infection in most acutely infected individuals, frequently leading to liver cirrhosis and hepatocellular carcinoma. The genome of HCV is 10-kb long encodes a single polyprotein that is proteolyticaly cleaved by cellular and viral proteases producing three structural (core, E1, and E2) and at least six non-structural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. In addition, several studies from independent laboratories have established the existence of another viral protein by definition that is expressed during the natural course of HCV infection. Originally this protein was found to be expressed from an alternate open reading frame (ORF) overlapping the core encoding region in the +1 frame of genotype 1a (designated as AFRP, F, or core+1/F protein). Work from Boulant et al showed that the core+1 protein was found to be expressed also in genotype 1b. In addition, a shorter form of the core+1 protein has been observed derived from internal translation initiation codons at positions 85/8. A smaller core+1/S protein and in the absence of these codons, the protein is still produced by an internal translation initiation site at codon 26. Another scientific group described another internal translation initiation codon at codon 26, though the resulting core+1 protein cannot be observed in the absence of codons 85/87. To a greater extent the true nature of the core+1 viral protein has not yet been determined, although it is believed that some of the functions attributed to the core protein may actually be due to the core+1 protein or to the combined effects of both proteins. In the present study we investigated the effect of the novel HCV core+1 protein on the transcriptional and translational regulation of several hepatic genes, such as the α-1 antitrypsin. We provide strong evidence that instead of having a modulatory effect on the α1-antitrypsin from core protein, being a transcription regulator, we observed such an effect from the novel core+1 protein. This effect is mediated through C/EBP, /a major transcription factor for liver. Furthermore we tried to generate transgenic mouse lines that will express independently HCV core and core+1 proteins, in order to observe any regulatory differences in the mouse hepatic cells.Ο ιός της ηπατίτιδας Γ αποτελεί ένα από τα συχνότερα αίτια χρόνιας ηπατικής νόσου παγκοσμίως. Στην Ελλάδα ο επιπολασμός της χρόνιας λοίμωξης από τον ιό υπολογίζεται σε 1,5-2%. Αν και ελαττώθηκε η λοίμωξη μετά τα τέλη της δεκαετίας του 1980, παλαιότερα μη διαγνωσμένα περιστατικά αναμένεται να αυξήσουν σημαντικά τον αριθμό ασθενών με χρόνια λοίμωξη την επόμενη δεκαετία. Ο ιός της ηπατίτιδας Γ μεταδίδεται κυρίως παρεντερικά και η χρήση ενδοφλέβιων ναρκωτικών αποτελεί σήμερα το συχνότερο τρόπο διασποράς του. Η οξεία ιϊκή λοίμωξη είναι κυρίως ασυμπτωματική και μεταπίπτει σε χρόνια σε 60-80% των ασθενών. Άνω του 90% των ασθενών με χρόνια λοίμωξη αναπτύσσουν χρόνια ηπατίτιδα και ίνωση του ήπατος και σε ποσοστά 20-30% εμφανίζουν αυξημένο κίνδυνο κίρρωσης του ήπατος, ηπατική ανεπάρκεια, ή και ηπατοκυτταρικό καρκίνο εντός 20ετίας. Αντι-ιϊκή θεραπεία πεγκυλιωμένης ιντερφερόνης-άλφα και ριμπαβιρίνης βελτιώνει αποτελεσματικά τις ηπατικές βλάβες και αναστέλλει την εξέλιξη της χρόνιας ηπατίτιδας Γ σε κίρρωση και ηπατοκυτταρικό καρκίνο. Το γονιδίωμα του ιού της ηπατίτιδας Γ φέρει ένα κανονικό ανοιχτό και ένα εναλλακτικό +1 πλαίσιο ανάγνωσης, που κωδικοποιούν πολυπρωτεΐνη και πρωτεΐνη F ή ARFP ή core+1, αντίστοιχα. Το εναλλακτικό αυτό πλαίσιο ανάγνωσης +1 στερείται κωδικού έναρξης, υποδηλώνοντας ότι στην έκφρασή του εμπλέκονται ασυνήθιστοι μηχανισμοί μετάφρασης και συμμετέχει με αμινοξικά κατάλοιπα στην δημιουργία χιμαιρικών πρωτεϊνών με τμήματα καψιδιακής (core) πρωτεΐνης. Απόδειξη μετάφρασης της πρωτεΐνης core+1 σε φυσική μόλυνση με τον ιό έδωσε η ανίχνευση ειδικών αντισωμάτων έναντι επιτόπων core+1 σε ορρούς ασθενών με οξεία ή χρόνια λοίμωξη. Επίσης, το 2008 οι Vassilaki et al. έδειξαν ότι τα εναρκτήρια κωδικόνια 85 και 87 είναι συντηρημένα στους γονότυπους 1α και 1β του ιού με αποτέλεσμα η πρωτεΐνη core+1/S να είναι η επικρατέστερη core+1 μορφή. Στη παρούσα μελέτη διερευνήθηκαν μηχανισμοί παθογένειας του ιού της ηπατίτιδας Γ και ιδιαίτερα ο ρόλος των ιϊκών πρωτεινών core και core+1 στη ρύθμιση της μεταγραφικής ενεργότητας συγκεκριμένων υποκινητών ηπατικών γονιδίων, που αλληλεπιδρώντας με μεταγραφικούς παράγοντες του ξενιστή μπορεί να οδηγήσουν σε ηπατική βλάβη και καρκίνο. Επιπλέον, ένας άλλος στόχος της παρούσας μελέτης ήταν η δημιουργία διαγονιδιακών ποντικών που εκφράζουν ξεχωριστά τις πρωτεΐνες core και core+1 στο ήπαρ, ώστε να συγκριθεί η ογκογόνος δράση και η ρυθμιστική λειτουργία τους στα ηπατικά κύτταρα των πειραματοζώων in vivo

Asymptomatic hyperuricemia and chronic kidney disease: Narrative review of a treatment controversial

Today there is plausible evidence both on experimental and epidemiological basis, that hyperuricemia represents a risk factor for the development and progression of chronic kidney disease (CKD). Nevertheless, the role of serum uric acid lowering treatment in CKD is still a matter of serious controversy. Review of randomised controlled trials, suggests that there may be an improvement of renal function with allopurinol treatment in CKD stage 3–5. However, these studies have included a relatively limited number of participants and provide insufficient information on adverse events and on the incidence of the end stage renal disease. Therefore, before adequately powered randomised, placebo-controlled trials are completed we cannot recommend treating asymptomatic hyperuricemia in patients with CKD

Crystalline silica activates the T-cell and the B-cell antigen receptor complexes and induces T-cell and B-cell proliferation

Silicosis is an occupational fibrotic lung disease, which is associated with an increased incidence of autoimmune diseases. The effect of crystalline silica on the immune system is thought to be mediated by the antigen presenting cells. However, the direct effect of silica on T-cells and B-cells has not been evaluated adequately. For this purpose, CD4(+)T-cells and B-cells from 10 healthy individuals were isolated and cultured with or without Min-U-Sil 5. Cell proliferation was assessed with BrdU assay. In cell proliferation experiments, tacrolimus, an inhibitor of the signal transduction derived from the activation of the T-cell or the B-cell antigen receptor (BCR) complex, was also used. The levels of phosphorylated zeta and phosphorylated Igα, indicative of the T-cell and BCR complex activation respectively, and of the transcription factor c-Myc, required for cell proliferation, were assessed by Western blotting. Crystalline silica triggered CD4(+)T-cell and B-cell proliferation, while tacrolimus significantly decreased the silica-induced proliferation in both cell types. Crystalline silica enhanced the level of phosphorylated zeta and phosphorylated Igα in CD4(+)T-cells and B-cells, respectively. In both cell types, treatment with silica increased c-Myc expression. Thus, crystalline silica may induce T-cell and B-cell proliferation by activating T-cell and BCR complexes. It is likely that the direct activation of CD4(+)T-cells and B-cells by silica crystals detected in this study circumvents many self-tolerance check-points and offers a mechanistic explanation for the crystalline silica-induced autoimmune diseases

Cell Death Patterns Due to Warm Ischemia or Reperfusion in Renal Tubular Epithelial Cells Originating from Human, Mouse, or the Native Hibernator Hamster

Ischemia⁻reperfusion injury contributes to the pathogenesis of many diseases, with acute kidney injury included. Hibernating mammals survive prolonged bouts of deep torpor with a dramatic drop in blood pressure, heart, and breathing rates, interspersed with short periods of arousal and, consequently, ischemia⁻reperfusion injury. Clarifying the differences under warm anoxia or reoxygenation between human cells and cells from a native hibernator may reveal interventions for rendering human cells resistant to ischemia⁻reperfusion injury. Human and hamster renal proximal tubular epithelial cells (RPTECs) were cultured under warm anoxia or reoxygenation. Mouse RPTECs were used as a phylogenetic control for hamster cells. Cell death was assessed by both cell imaging and lactate dehydrogenase (LDH) release assay, apoptosis by cleaved caspase-3, autophagy by microtubule-associated protein 1-light chain 3 B II (LC3B-II) to LC3B-I ratio, necroptosis by phosphorylated mixed-lineage kinase domain-like pseudokinase, reactive oxygen species (ROS) fluorometrically, and lipid peroxidation, the end-point of ferroptosis, by malondialdehyde. Human cells died after short periods of warm anoxia or reoxygenation, whereas hamster cells were extremely resistant. In human cells, apoptosis contributed to cell death under both anoxia and reoxygenation. Although under reoxygenation, ROS increased in both human and hamster RPTECs, lipid peroxidation-induced cell death was detected only in human cells. Autophagy was observed only in human cells under both conditions. Necroptosis was not detected in any of the evaluated cells. Clarifying the ways that are responsible for hamster RPTECs escaping from apoptosis and lipid peroxidation-induced cell death may reveal interventions for preventing ischemia⁻reperfusion-induced acute kidney injury in humans

The effect of anti‑HLA class I antibodies on the immunological properties of human glomerular endothelial cells and their modification by mTOR inhibition or GCN2 kinase activation

In antibody‑mediated rejection (ABMR), the graft endothelium is at the forefront of the kidney transplant against the assault from the recipient's humoral immune system, and is a target of the latter. The present study investigated the effect of antibodies against human leukocyte antigen (HLA) class I (anti‑HLAI) on the immunological properties of human glomerular endothelial cells. Additionally, the effect of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) inhibitor (everolimus), or the general control nonderepressible 2 kinase (GCN2K) activator (halofuginone) on anti‑HLAI antibody‑mediated alterations was assessed. Cell integrity was examined, an lactate dehydrogenase (LDH) release assay was performed and cleaved caspase‑3 levels were determined. Furthermore, cell proliferation was analyzed by performing a bromodeoxyuridine assay and the cellular proteins involved in signal transduction or immune effector mechanisms were assessed via western blotting. IL‑8, monocyte chemoattractive protein‑1 (MCP‑1), von Willebrand factor (vWF) and transforming growth factor‑beta 1 (TGF‑β1) were assayed via ELISA. The results revealed that anti‑HLAI triggered integrin signaling, activated mTOR and GCN2K, preserved cell integrity and promoted cell proliferation. Additionally, by increasing intercellular adhesion molecule 1 (ICAM‑1), HLA‑DR, IL‑8 and MCP‑1 levels, anti‑HLAI enhanced the ability of immune cells to interact with endothelial cells thus facilitating graft rejection. Contrarily, by upregulating CD46 and CD59, anti‑HLAI rendered the endothelium less vulnerable to complement‑mediated injury. Finally, by enhancing vWF and TGF‑β1, anti‑HLAI may render the endothelium prothrombotic and facilitate fibrosis and graft failure, respectively. According to our results, mTORC1 inhibition and GCN2K activation may prove useful pharmaceutical targets, as they prevent cell proliferation and downregulate ICAM‑1, IL‑8, MCP‑1 and TGF‑β1. mTORC1 inhibition also decreases vWF

A role for human renal tubular epithelial cells in direct allo-recognition by CD4+ T-cells and the effect of ischemia-reperfusion

Direct allorecognition is the earliest and most potent immune response against a kidney allograft. Currently, it is thought that passenger donor professional antigen-presenting cells (APCs) are responsible. Further, many studies support that graft ischemia-reperfusion injury increases the probability of acute rejection. We evaluated the possible role of primary human proximal renal tubular epithelial cells (RPTECs) in direct allorecognition by CD4+ T-cells and the effect of anoxia-reoxygenation. In cell culture, we detected that RPTECs express all the required molecules for CD4+ T-cell activation (HLA-DR, CD80, and ICAM-1). Anoxia-reoxygenation decreased HLA-DR and CD80 but increased ICAM-1. Following this, RPTECs were co-cultured with alloreactive CD4+ T-cells. In T-cells, zeta chain phosphorylation and c-Myc increased, indicating activation of T-cell receptor and co-stimulation signal transduction pathways, respectively. T-cell proliferation assessed with bromodeoxyuridine assay and with the marker Ki-67 increased. Previous culture of RPTECs under anoxia raised all the above parameters in T-cells. FOXP3 remained unaffected in all cases, signifying that proliferating T-cells were not differentiated towards a regulatory phenotype. Our results support that direct allorecognition may be mediated by RPTECs even in the absence of donor-derived professional APCs. Also, ischemia-reperfusion injury of the graft may enhance the above capacity of RPTECs, increasing the possibility of acute rejection

Serpin Family E Member 1 Tag Single-Nucleotide Polymorphisms in Patients with Diabetic Nephropathy: An Association Study and Meta-Analysis Using a Genetic Model-Free Approach

Background: Many lines of evidence highlight the genetic contribution on the development of diabetic nephropathy (DN). One of the studied genes is SERPINE1 whose the role in the risk of developing DN remains questionable. In order to elucidate the contribution of SERPINE1 in DN progression in the context of type 2 diabetes mellitus (T2DM), we conducted an association study and meta-analysis of SERPINE1 genetic variants. Materials and Methods: A total of 190 patients with DN, 150 T2DM (type 2 diabetes mellitus) patients without DN and 238 healthy controls were recruited. We selected five tag single-nucleotide polymorphisms (SNPs) from the HapMap. The generalized odds ratio (ORG) was calculated to estimate the risk on DN development. Subgroup analyses based on ethnicity and type of diabetes were also performed. Results: Both the present association study regarding SERPINE1 SNPs (rs2227667, rs2070682, rs1050813, rs2227690, rs2227692) did not found any significant association between SERPINE1 variants and DN and the meta-analysis of variant 4G>5G (rs1799889) did not also reveal a significant association between 4G>5G variant and DN in main and subgroup analyses. Discussion: In conclusion, the present association study and meta-analysis provides strong evidence that SERPINE1 genetic variant 4G>5G is not implicated in the risk or development of DN in Caucasians. Further studies in other populations remain to further investigate the role of this variant in the course of DN