Predisposing factors for advanced liver fibrosis in patients with sickle cell disease.

Sickle cell disease (SCD) is one of the most common monogenic disorders worldwide and liver complications are common in this group of patients. Our study aims to highlight the prevalence of chronic liver complications and the main predisposing factors for advanced liver fibrosis in SCD patients. For this purpose, 219 patients from eight Thalassemia and Sickle Cell Units across Greece enrolled in our study and history of liver related disease complications was recorded, as well as a full laboratory and imaging analysis concerning their liver function. 13.6% of the patients had advanced liver fibrosis. The presence of liver fibrosis was significantly correlated with advanced age, male gender, cholelithiasis and higher LDH, γ-GT, INR, direct and indirect bilirubin levels. These patients had exhibited significantly more episodes of liver crises and acute intrahepatic cholestasis. No correlation was observed with right heart failure or previous viral hepatitis. Patients with advanced liver fibrosis were receiving a more intensive transfusion therapy for a longer period of time and had higher Liver Iron Concentration levels. Our study shows that liver complications and cirrhosis is a significant cause of morbidity in patients with SCD and it is primarily associated with intravascular hemolysis and vaso-occlusive phenomena and secondarily with iron overload.

Bone disease in patients with cirrhosis of different etiology and severity; are Klotho protein and osteoprotegerin potential biomarkers?

BACKGROUND AND AIMS: Cirrhosis is associated with increased risk for osteoporosis and osteopenia. This study aims to further investigate this relationship by examining if etiology and severity of cirrhosis are independent predictors of bone mineral density (BMD) loss. Furthermore we examined the serum levels of osteoprotegerin (OPG) and Klotho proteins that have been involved in bone metabolism. METHODS: Seventy-four patients with cirrhosis of different etiology and 25 matched healthy controls were included in this study. Bone mineral densitometry at both lumbar spine and femoral neck was measured. Serum total OPG, Klotho protein and vitamin D levels were also determined. Comparisons were performed according to etiology and severity of cirrhosis. RESULTS: Decreased bone density was observed in cirrhotic patients compared to healthy controls with T = -1.46 and T = -1.37 in lumbar spine and femoral bone respectively compared to T = -0.396 and T = -0.672 in the control group. In the cirrhotic group, osteopenia was observed in 46% in lumbar spine and 51% in femoral bone whereas osteoporosis was observed in 20% in lumbar spine and 9% in femoral bone. Decreased bone density was confirmed, regardless of cirrhosis etiology or stage of liver function. Patients were found to have higher levels of OPG than the control group (136 pg/ml vs. 67 pg/ml, p < 0.001), but lower levels of Klotho protein (1051 pg/ml vs. 1842 pg/ml, p < 0.001) regardless etiology and severity of cirrhosis. High OPG levels were found to be associated with low femoral bone density. CONCLUSIONS: BMD is lower in cirrhotic patients regardless etiology and severity of liver disease with osteopenia and osteoporosis be present in 50% and 20%, respectively. Higher levels of OPG and lower levels of Klotho protein were observed in cirrhotic patients regardless etiology and severity in comparison to matched healthy group.

Immune System and Hepatocellular Carcinoma (HCC): New Insights into HCC Progression.

According to the WHO's recently released worldwide cancer data for 2020, liver cancer ranks sixth in morbidity and third in mortality among all malignancies. Hepatocellular carcinoma (HCC), the most common kind of liver cancer, accounts approximately for 80% of all primary liver malignancies and is one of the leading causes of death globally. The intractable tumor microenvironment plays an important role in the development and progression of HCC and is one of three major unresolved issues in clinical practice (cancer recurrence, fatal metastasis, and the refractory tumor microenvironment). Despite significant advances, improved molecular and cellular characterization of the tumor microenvironment is still required since it plays an important role in the genesis and progression of HCC. The purpose of this review is to present an overview of the HCC immune microenvironment, distinct cellular constituents, current therapies, and potential immunotherapy methods.

Clinical, histological and molecular profiling of different stages of alcohol-related liver disease.

OBJECTIVE: Alcohol-related liver disease (ALD) ranges from never-decompensated ALD (ndALD) to the life-threatening decompensated phenotype, known as alcohol-related hepatitis (AH). A multidimensional study of the clinical, histological and molecular features of these subtypes is lacking. DESIGN: Two large cohorts of patients were recruited in an international, observational multicentre study: a retrospective cohort of patients with ndALD (n=110) and a prospective cohort of patients with AH (n=225). Clinical, analytical, immunohistochemistry and hepatic RNA microarray analysis of both disease phenotypes were performed. RESULTS: Age and mean alcohol intake were similar in both groups. AH patients had greater aspartate amino transferase/alanine amino transferase ratio and lower gamma-glutamyl transferase levels than in ndALD patients. Patients with AH demonstrated profound liver failure and increased mortality. One-year mortality was 10% in ndALD and 50% in AH. Histologically, steatosis grade, ballooning and pericellular fibrosis were similar in both groups, while advanced fibrosis, Mallory-Denk bodies, bilirubinostasis, severe neutrophil infiltration and ductular reaction were more frequent among AH patients. Transcriptome analysis revealed a profound gene dysregulation within both phenotypes when compare to controls. While ndALD was characterised by deregulated expression of genes involved in matrisome and immune response, the development of AH resulted in a marked deregulation of genes involved in hepatocyte reprogramming and bile acid metabolism. CONCLUSIONS: Despite comparable alcohol intake, AH patients presented with worse liver function compared with ndALD patients. Bilirubinostasis, severe fibrosis and ductular reaction were prominent features of AH. AH patients exhibited a more profound deregulation of gene expression compared with ndALD patients.

Cellular, Molecular and Proteomic Characteristics of Early Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers. Early detection/diagnosis is vital for the prognosis of HCC, whereas diagnosis at late stages is associated with very low survival rate. Early diagnosis is based on 6-month surveillance of the patient and the use of at least two imaging modalities. The aim of this study was to investigate diagnostic markers for the detection of early HCC based on proteome analysis, microRNAs (miRNAs) and circulating tumor cells (CTCs) in the blood of patients with cirrhosis or early or advanced HCC. We studied 89 patients with HCC, of whom 33 had early HCC and 28 were cirrhotic. CTCs were detected by real-time quantitative reverse transcription PCR and immunofluorescence using the markers epithelial cell adhesion molecule (EPCAM), vimentin, alpha fetoprotein (aFP) and surface major vault protein (sMVP). Expression of the five most common HCC-involved miRNAs (miR-122, miR-200a, miR-200b, miR-221, miR-222) was examined in serum using quantitative real time PCR (qRT-PCR). Finally, patient serum was analyzed via whole proteome analysis (LC/MS). Of 53 patients with advanced HCC, 27 (51%) had detectable CTCs. Among these, 10/27 (37%) presented evidence of mesenchymal or intermediate stage cells (vimentin and/or sMVP positive). Moreover, 5/17 (29%) patients with early HCC and 2/28 (7%) cirrhotic patients had detectable CTCs. Patients with early or advanced HCC exhibited a significant increase in miR-200b when compared to cirrhotic patients. Our proteome analysis indicated that early HCC patients present a significant upregulation of APOA2, APOC3 proteins when compared to cirrhotic patients. When taken in combination, this covers the 100% of the patients with early HCC. miR-200b, APOA2 and APOC3 proteins are sensitive markers and can be potentially useful in combination for the early diagnosis of HCC.

Iron Chelators, Such as Deferasirox, When Combined With Hydroxyurea, Provide an Additional Benefit of Iron Chelation in Patients Receiving Chronic Transfusion Therapy.

Red blood cell (RBC) transfusions have been established as one of the primary therapies in treating sickle cell anemia. However, they are not free of side effects, with overloading the body with iron being one of the most important. Iron chelation therapy greatly reduces the iron load of the body. In addition, hydroxyurea (HU), an oral chemotherapeutic drug also has a significant role in the treatment of the disease with beneficial effects on many of the clinical problems that arise, mainly in reducing painful crises. The aim of this study was to investigate the effect of synergistic transfusion therapy and HU on the response to deferasirox (DFX) chelation therapy. Eighteen patients with sickle cell disease were divided into two groups based on their treatment, either with simple transfusions and DFX or with a combination of transfusion therapy, DFX and HU, and were evaluated with magnetic resonance imaging (MRI) for liver iron concentration (LIC) and biochemistry. All patients completed the study. The results of the study showed improvement in serum ferritin (FER) levels and LIC after 12 months of therapy in both groups, especially in the group receiving the combination therapy with HU. In addition, there was a noteworthy improvement in serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and lactate dehydrogenase (LDH) levels with serum creatinine (Cr) levels remaining stable during the study in both groups. Hydroxyurea, when combined with iron chelators such as DFX, provides an additional benefit of iron chelation in patients receiving chronic transfusion therapy.

Acute Chest Syndrome in Sickle Cell Disease: Clinical Presentation and Outcomes. The Experience of a Single Thalassemia and Sickle Cell Unit in a University Hospital.

Acute chest syndrome (ACS) is a common cause of death for sickle cell disease patients. This syndrome is defined as: respiratory symptoms, new X-ray findings developed and/or fever; ACS requires prompt treatment to avoid clinical deterioration and death in adults with sickle cell disease. Sixteen episodes of acute chest syndrome were studied in 16 adults with sickle cell disease. The clinical and radiological findings, treatment, response and outcome of the episode were evaluated respectively. The patient's past history and comorbidities were taken into account in the outcome and days of hospitalization. Fourteen patients recovered with no sequelae; one patient who required mechanical ventilation also recovered; one patient died due to pulmonary emboli. The mean hospitalization days were 7.43.

Differential Expression of the Host Lipid Regulators ANGPTL-3 and ANGPTL-4 in HCV Infection and Treatment.

Host lipid metabolism reprogramming is essential for hepatitis C virus (HCV) infection and progression to severe liver disease. Direct-acting antivirals (DAAs) achieve a sustained virological response (SVR) in most patients, but virus eradication does not always protect against hepatocellular carcinoma (HCC). Angiopoietin-like protein-3 (ANGPTL-3) and angiopoietin-like protein-4 (ANGPTL-4) regulate the clearance of plasma lipids by inhibiting cellular lipase activity and possess emerging roles in tumourigenesis. We used ELISA and RT-qPCR to investigate ANGPTL-3 and ANGPTL-4 expression in HCV patients with characterised fibrosis throughout the natural history of hepatitis C and in long-term HCV infection in vitro, before and after DAA treatment. ANGPTL-3 was decreased in patients with advanced fibrosis compared to other disease stages, while ANGPTL-4 was progressively increased from acute infection to cirrhosis and HCC, peaking at the advanced fibrosis stage. Only ANGPTL-3 mRNA was down-regulated during early infection in vitro, although both ANGPTLs were increased later. DAA treatment did not alter ANGPTL-3 levels in advanced fibrosis/cirrhosis and in HCV infection in vitro, in contrast to ANGPTL-4. The association between ANGPTLs and fibrosis in HCV infection was underlined by an inverse correlation between the levels of ANGPTLs and serum transforming growth factor- ß (TGF-ß). Collectively, we demonstrate the pivotal role of advanced fibrosis in defining the expression fate of ANGPTLs in HCV infection and after treatment and propose a role for ANGPTL-3 as a contributor to post-treatment deregulation of lipid metabolism that could predispose certain individuals to HCC development.

High prevalence of antibodies to core+1/ARF protein in HCV-infected patients with advanced cirrhosis.

Hepatitis C virus (HCV) possesses a second open reading frame (ORF) within the core gene encoding an additional protein, known as the alternative reading frame protein (ARFP), F or core+1. The biological significance of the core+1/ARF protein remains elusive. However, several independent studies have shown the presence of core+1/ARFP antibodies in chronically HCV-infected patients. Furthermore, a higher prevalence of core+1/ARFP antibodies was detected in patients with HCV-associated hepatocellular carcinoma (HCC). Here, we investigated the incidence of core+1/ARFPantibodies in chronically HCV-infected patients at different stages of cirrhosis in comparison to chronically HCV-infected patients at earlier stages of disease. Using ELISA, we assessed the prevalence of anti-core+1 antibodies in 30 patients with advanced cirrhosis [model for end-stage liver disease (MELD) ≥15] in comparison with 50 patients with mild cirrhosis (MELD <15) and 164 chronic HCV patients without cirrhosis. 28.7 % of HCV patients with cirrhosis were positive for anti-core+1 antibodies, in contrast with 16.5 % of non-cirrhotic HCV patients. Moreover, there was significantly higher positivity for anti-core+1 antibodies in HCV patients with advanced cirrhosis (36.7 %) compared to those with early cirrhosis (24 %) (P<0.05). These findings, together with the high prevalence of anti-core+1 antibodies in HCV patients with HCC, suggest that core+1 protein may have a role in virus-associated pathogenesis, and provide evidence to suggest that the levels of anti-core+1 antibodies may serve as a marker for disease progression.

Treatment of chronic hepatitis C with direct-acting antivirals in patients with ß-thalassaemia major and advanced liver disease.

Interferon-based regimens for chronic hepatitis C (CHC) were often deferred in patients with ß-thalasaemia major (ß-TM) due to poor efficacy and tolerance. Current guidelines recommend direct-acting antivirals (DAAs) for these patients. The aim of this study was to assess the safety and efficacy of DAAs in patients with ß-TM and advanced liver disease due to CHC. Patients were recruited from eight liver units in Greece. The stage of liver disease was assessed using transient elastography and/or liver histology. Five regimens were used: sofosbuvir (SOF) + ribavirin (RBV); SOF + simeprevir ± RBV; SOF + daclatasvir ± RBV; ledipasvir/SOF ± RBV and ombitasvir/paritaprevir-ritonavir + dasabuvir ± RBV. Sixty-one patients (median age 43 years) were included. The majority of patients was previously treated for hepatitis C (75%) and had cirrhosis (79%). Viral genotype distribution was: G1a: n = 10 (16%); G1b: n = 22 (36%); G2: n = 2 (3%); G3: n = 14 (23%); G4: n = 13 (22%). The predominant chelation therapy was a combination of deferoxamine and deferiprone (35%). Overall sustained virological response rates were 90%. All treatment regimens were well tolerated and no major adverse events or drug-drug interactions were observed. Approximately half of the patients who received RBV (7/16, 44%) had increased needs for blood transfusion. Treatment of CHC with DAAs in patients with ß-TM and advanced liver disease was highly effective and safe.

Liver innervation and hepatic function: new insights.

The hepatic nervous system has a well-known impact on the regulation of liver function and organism homeostasis. The aim of this review is to summarize the new available data regarding the role of hepatic nerves. In the last decade, studies have shown that hepatic nerves exert subtle but significant modifications on the regulation of glucose and lipid metabolism, food intake, and liver regeneration. They also play a role in liver disease pathogenesis, and hepatic denervation has beneficial results to liver graft ischemia-reperfusion injury. Available data are still limited, and further research toward neural pathways involving the liver that can modify response to disease is required.

Hepatitis C virus modulates lipid regulatory factor Angiopoietin-like 3 gene expression by repressing HNF-1α activity.

BACKGROUND & AIMS: HCV relies on host lipid metabolism to complete its life cycle and HCV core is crucial to this interaction. Liver secreted ANGPTL-3 is an LXR- and HNF-1α-regulated protein, which plays a key role in lipid metabolism by increasing plasma lipids via inhibition of lipase enzymes. Here we aimed to investigate the modulation of ANGPTL-3 by HCV core and identify the molecular mechanisms involved. METHODS: qRT-PCR and ELISA were used to assess ANGPTL-3 mRNA and protein levels in HCV patients, the JFH-1 infectious system and liver cell lines. Transfections, chromatin immunoprecipitation and immunofluorescence delineated parts of the molecular mechanisms implicated in the core-mediated regulation of ANGPTL-3 gene expression. RESULTS: ANGPTL-3 gene expression was decreased in HCV-infected patients and the JFH-1 infectious system. mRNA and promoter activity levels were down-regulated by core. The response was lost when an HNF-1α element in ANGPTL-3 promoter was mutated, while loss of HNF-1α DNA binding to this site was recorded in the presence of HCV core. HNF-1α mRNA and protein levels were not altered by core. However, trafficking between nucleus and cytoplasm was observed and then blocked by an inhibitor of the HNF-1α-specific kinase Mirk/Dyrk1B. Transactivation of LXR/RXR signalling could not restore core-mediated down-regulation of ANGPTL-3 promoter activity. CONCLUSIONS: ANGPTL-3 is negatively regulated by HCV in vivo and in vitro. HCV core represses ANGPTL-3 expression through loss of HNF-1α binding activity and blockage of LXR/RXR transactivation. The putative ensuing increase in serum lipid clearance and uptake by the liver may sustain HCV virus replication and persistence.

Liver regeneration: immunohistochemical study of intrinsic hepatic innervation after partial hepatectomy in rats.

BACKGROUND: We examined the intrinsic hepatic innervation after partial hepatectomy (PH) in rats and the presence and pattern of neural sprouting in regenerating liver. METHODS: Male Wistar rats (age 9-13 weeks-w, weight 204-356 g), were submitted to two-thirds PH. Rats were sacrificed at postoperative days (d) 1, 3, 5, 7, at 2 and 4 w, and at 3 and 6 months (m) (6-7 animals/group, control group n = 4). Immunohistochemistry for the pan-neural marker protein gene product 9.5 (PGP9.5) and growth-associated protein 43 (GAP-43), a marker of regenerating nerve axons, was performed on tissue sections from the R1 lobe of the regenerating liver. Portal tracts (PTs) with immunoreactive fibers were counted in each section and computer-assisted morphometric analysis (Image Pro Plus) was used to measure nerve fiber density (number of immuno-positive nerve fibers/mm2 (40x)). RESULTS: Immunoreactivity for PGP9.5 was positive in all groups. The number of PGP9.5 (+) nerve fibers decreased from 0.32 +/- 0.12 (control group) to 0.18 +/- 0.09 (1d post-PH group), and gradually increased reaching pre-PH levels at 6 m (0.3 +/- 0.01). In contrast, immunoreactivity for GAP-43 was observed at 5d post-PH, and GAP-43 (+) PTs percentage increased thereafter with a peak at 3 m post-PH. GAP-43 (+) nerve fiber density increased gradually from 5d (0.05 +/- 0.06) with a peak at 3 m post-PH (0.21 +/- 0.027). At 6 m post-PH, immunoreactivity for GAP-43 was not detectable. CONCLUSIONS: Following PH in rats: 1) nerve fiber density in portal tracts decreases temporarily, and 2) neural sprouting in the regenerating liver lobes starts at 5d, reaches peak levels at 3 m and disappears at 6 m post-PH, indicating that the increase in hepatic mass after PH provides an adequate stimulus for the sprouting process.

Phenotypic and functional alterations of primary human PBMCs induced by HCV non-enveloped capsid-like particles uptake.

Hepatitis C virus non-enveloped particles circulate in the serum of HCV-infected patients and are believed to be involved in viral persistence. It was previously demonstrated that recombinant HCVne particles can efficiently enter T cells. In this study we investigated the effect of this entry on the phenotype and function of PBMCs, focused on the CD4+ and CD8+ T-cells. We have generated recombinant HCVne in the absence of other viral proteins. PBMCs from healthy donors were sampled after incubation either with HCVne or the control at different time points. Levels of expression of CD107a, CD25, CTLA-4, and T regulatory cells were estimated and cytokine expression and secretion were also monitored. Peripheral T cells expressed elevated CD127. The intracellular expression of the inhibitory marker CTLA-4 (CD152) increased significantly on peripheral T cells at late hours post-treatment, compared to the respective non-treated group. Despite the fact that there was an initial immune response due to HCVne uptake, T cells were driven to a partial exhausted phenotype. A significant induction of CD4+CD25+(hi)CD127-regulatory T cells at late hours was observed. Consistently, Foxp3+CD4+ T cells were also increased. In parallel, a significant transcriptional activation and increased secretion of IL-2, IL-10, and IFN-γ, was recorded. Moreover, mRNA transcription of TGF-ß was considerably elevated. HCVne particles have the potential to shape the immune response by modifying specific phenotypic and functional markers mainly on CD4+ T cells and driving them to partial exhaustion as well as to Treg expansion.

Addition of Dulaglutide or Empagliflozin to Standard-of-Care Treatment: Effect on Liver Steatosis in Patients With Type 2 Diabetes Mellitus.

Background Patients with liver steatosis and diabetes mellitus can benefit from medications like glucagon-like peptide 1 receptor agonists or sodium-glucose co-transporter 2 inhibitors, as far as both hyperglycemia and fatty liver are concerned. Studies comparing members of both these families have not yet been published. We aimed to compare the effects of Empagliflozin and Dulaglutide, focusing primarily on liver steatosis. Methodology This prospective, observational, controlled study enrolled 78 patients from two centers in Athens, Greece. Adults with type 2 diabetes mellitus (DM2) and nonalcoholic fatty liver disease were assigned to one of three groups and received either Empagliflozin or Dulaglutide or any other medical treatment deemed appropriate by their physician. The primary endpoint was the reduction in liver fat fraction, assessed using magnetic resonance imaging-proton density fat fraction. Additionally, we evaluated the proportion of patients achieving a relative reduction above 30% of their initial liver fat concentration. Results The Empagliflozin group exhibited a reduction in liver fat fraction. Furthermore, the percentage of patients with a relative reduction of liver steatosis, >30%, was significantly larger in this group, compared to the Dulaglutide and Control groups. Significant body weight reduction was observed in all three groups, but no improvement in fibrosis assessing scores was noted. Conclusions Empagliflozin is effective in improving liver steatosis, while Dulaglutide does not exhibit a similar effect. Larger studies, comparing these or related agents, are necessary, to further assess benefits in patients with DM2 and nonalcoholic fatty liver.

Liver disease in adult transfusion-dependent beta-thalassaemic patients: investigating the role of iron overload and chronic HCV infection.

BACKGROUND: Iron overload and hepatitis-C virus (HCV) infection, have been implicated in the evolution of liver disease, in patients with transfusion-dependent beta-thalassaemia major (BTM). However, the impact of these factors in late stages of liver disease in adults with BTM, has not been extensively studied. AIMS: To investigate serum indices of iron overload, HCV infection and liver disease, in a cohort of 211 adult Greek patients with BTM, in relation with the findings from liver biopsies. METHODS: In this cross-sectional study, 211 patients with BTM were enrolled and studied, in relation with HCV infection, ferritin, transaminases, chelation treatment and antiviral treatment. Based on 109 patients biopsied, we correlated liver fibrosis, haemosiderosis and inflammation, with serum indices and HCV status RESULTS: Among all patients, 74.4% were anti-HCV positive (HCV+). Ferritin was positively correlated with transaminases and negatively correlated with age, while it was not significantly different among HCV+ and HCV- patients. Among the HCV+ patients, 55.4% reported antiviral treatment, while genotype 1 predominated. In a subfraction of 109 patients, in which liver biopsy was performed, 89% were HCV+ and 11% HCV-. Fibrosis was significantly correlated with age (P = 0.046), AST (P = 0.004), ALT (P = 0.044) and inflammation (P < 0.001). Advanced fibrosis was present with even minimal haemosiderosis, independently of ferritin values or HCV history. CONCLUSIONS: These data suggest that in the late stages of liver disease in BTM patients, iron overload may be the critical determinant, since fibrosis is related to the minimal haemosiderosis, independently of HCV history.

Co-culture of primary human tumor hepatocytes from patients with hepatocellular carcinoma with autologous peripheral blood mononuclear cells: study of their in vitro immunological interactions.

BACKGROUND: Many studies have suggested that the immune response may play a crucial role in the progression of hepatocellular carcinoma (HCC). Therefore, our aim was to establish a (i) functional culture of primary human tumor hepatocytes and non-tumor from patients with hepatocellular carcinoma (HCC) and (ii) a co-culture system of HCC and non-HCC hepatocytes with autologous peripheral blood mononuclear cells (PBMCs) in order to study in vitro cell-to-cell interactions. METHODS: Tumor (HCC) and non-tumor (non-HCC) hepatocytes were isolated from the liver resection specimens of 11 patients operated for HCC, while PBMCs were retrieved immediately prior to surgery. Four biopsies were obtained from patients with no liver disease who had surgery for non malignant tumor (normal hepatocytes). Hepatocytes were either cultured alone (monoculture) or co-cultured with PBMCs. Flow cytometry measurements for MHC class II expression, apoptosis, necrosis and viability (7AAD) were performed 24 h, 48 h and 72 h in co-culture and monocultures. RESULTS: HCC and non-HCC hepatocytes exhibited increased MHC-II expression at 48h and 72h in co-culture with PBMCs as compared to monoculture, with MHC II-expressing HCC hepatocytes showing increased viability at 72 h. PBMCs showed increased MHC-II expression (activation) in co-culture with HCC as compared to non-HCC hepatocytes at all time points. Moreover, CD8+ T cells had significantly increased apoptosis and necrosis at 48h in co-culture with HCC hepatocytes as compared to monocultures. Interestingly, MHC-II expression on both HCC and non-HCC hepatocytes in co-culture was positively correlated with the respective activated CD8+ T cells. CONCLUSIONS: We have established an in vitro co-culture model to study interactions between autologous PBMCs and primary HCC and non-HCC hepatocytes. This direct interaction leads to increased antigen presenting ability of HCC hepatocytes, activation of PBMCs with a concomitant apoptosis of activated CD8+ T cells. Although, a partially effective immune response against HCC exists, still tumor hepatocytes manage to escape.

Liquid Biopsies, Novel Approaches and Future Directions.

Cancer is among the leading causes of death worldwide. Early diagnosis and prognosis are vital to improve patients' outcomes. The gold standard of tumor characterization leading to tumor diagnosis and prognosis is tissue biopsy. Amongst the constraints of tissue biopsy collection is the sampling frequency and the incomplete representation of the entire tumor bulk. Liquid biopsy approaches, including the analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating miRNAs, and tumor-derived extracellular vesicles (EVs), as well as certain protein signatures that are released in the circulation from primary tumors and their metastatic sites, present a promising and more potent candidate for patient diagnosis and follow up monitoring. The minimally invasive nature of liquid biopsies, allowing frequent collection, can be used in the monitoring of therapy response in real time, allowing the development of novel approaches in the therapeutic management of cancer patients. In this review we will describe recent advances in the field of liquid biopsy markers focusing on their advantages and disadvantages.

The Epigenetic Controller Lysine-Specific Demethylase 1 (LSD1) Regulates the Outcome of Hepatitis C Viral Infection.

Hepatitis C virus (HCV) alters gene expression epigenetically to rearrange the cellular microenvironment in a beneficial way for its life cycle. The host epigenetic changes induced by HCV lead to metabolic dysfunction and malignant transformation. Lysine-specific demethylase 1 (LSD1) is an epigenetic controller of critical cellular functions that are essential for HCV propagation. We investigated the putative role of LSD1 in the establishment of HCV infection using genetic engineering and pharmacological inhibition to alter endogenous LSD1 levels. We demonstrated for the first time that HCV replication was inhibited in LSD1-overexpressing cells, while specific HCV proteins differentially fine-tuned endogenous LSD1 expression levels. Electroporation of the full-length HCV genome and subgenomic replicons in LSD1 overexpression enhanced translation and partially restored HCV replication, suggesting that HCV might be inhibited by LSD1 during the early steps of infection. Conversely, the inhibition of LSD1, followed by HCV infection in vitro, increased viral replication. LSD1 was shown to participate in an intriguing antiviral mechanism, where it activates endolysosomal interferon-induced transmembrane protein 3 (IFITM3) via demethylation, leading endocytosed HCV virions to degradation. Our study proposes that HCV-mediated LSD1 oscillations over countless viral life cycles throughout chronic HCV infection may promote epigenetic changes related to HCV-induced hepatocarcinogenesis.

Clinical significance of mutational variants in beta and alpha genes in patients with hemoglobinopathies from two large Greek centers: a complex interplay between genotype and phenotype.

Hemoglobinopathies affect patients in the wider Mediterranean area consisting of 4 distinct subgroups: beta thalassemia major (TM), beta thalassemia intermedia (TI), sickle cell disease (SCD) and hemoglobin H disease (alpha thalassemia). The clinical spectrum varies from mild to severe. Complex interactions between genes and environmental factors form the clinical manifestations. There is an unmet need to clarify these multifactorial mechanisms. This is the first Greek study describing mutational alleles (HBB and HBA1/HBA2 gene variants) in 217 patients with hemoglobinopathies of two large centers in Greece (Larissa and Athens) and associating particular genotypes or gene variants with clinical manifestations (transfusion frequency, complications). Thus, the complex interplay between corresponding genotypes and phenotypes was investigated. Our results are in accordance with previous national studies with limited variations, due to regional prevalence of specific gene variants, as expected. It is also a description of the prevalence of hemoglobinopathies in the Greek population. The type and prevalence of beta and alpha globin gene variants differ significantly among countries. We also confirm the well-known observation of many studies that in our beta thalassemic or SCD patients, co-inheritance of variants in the alpha globin genes, leading to absence or reduction of alpha globin synthesis were associated with milder clinical course, whereas the inheritance of additional alpha genes (triplication) led to a more severe clinical phenotype. In cases in whom the genotype and phenotype did not correlate, factors like the function or modification of possible regulatory genes or additional nutritional-environmental effects should be investigated. KEY MESSAGES: • This is the first Greek study, fully molecularly defining the beta and alpha mutational alleles in 217 patients with hemoglobinopathies of two large centers in Greece and correlating particular genotypes or gene variants with clinical manifestations (transfusion frequency, complications). • In the beta thalassemic or SCD patients of our cohort, co-inheritance of variants in the alpha globin genes, leading to absence or reduction of alpha globin synthesis were associated with milder clinical course (confirmation of a well-known previous observation). • The inheritance of additional alpha genes (triplication) led to a more severe clinical phenotype (confirmation of a well known previous observation). • The function or modification of possible regulatory genes should be investigated in cases in whom the genotype and phenotype did not correlate.