Genome-wide DNA methylation and transcriptomic analysis of liver tissues subjected to early ischemia/reperfusion injury upon human liver transplantation.

INTRODUCTION AND OBJECTIVES: Epigenetic changes represent a mechanism connecting external stresses with long-term modifications of gene expression programs. In solid organ transplantation, ischemia-reperfusion injury (IRI) appears to induce epigenomic changes in the graft, although the currently available data are extremely limited. The present study aimed to characterize variations in DNA methylation and their effects on the transcriptome in liver transplantation from brain-dead donors. PATIENTS AND METHODS: 12 liver grafts were evaluated through serial biopsies at different timings in the procurement-transplantation process: T0 (warm procurement, in donor), T1 (bench surgery), and T2 (after reperfusion, in recipient). DNA methylation (DNAm) and transcriptome profiles of biopsies were analyzed using microarrays and RNAseq. RESULTS: Significant variations in DNAm were identified, particularly between T2 and T0. Functional enrichment of the best 1000 ranked differentially methylated promoters demonstrated that 387 hypermethylated and 613 hypomethylated promoters were involved in spliceosomal assembly and response to biotic stimuli, and inflammatory immune responses, respectively. At the transcriptome level, T2 vs. T0 showed an upregulation of 337 and downregulation of 61 genes, collectively involved in TNF-α, NFKB, and interleukin signaling. Cell enrichment analysis individuates macrophages, monocytes, and neutrophils as the most significant tissue-cell type in the response. CONCLUSIONS: In the process of liver graft procurement-transplantation, IRI induces significant epigenetic changes that primarily act on the signaling pathways of inflammatory responses dependent on TNF-α, NFKB, and interleukins. Our DNAm datasets are the early IRI methylome literature and will serve as a launch point for studying the impact of epigenetic modification in IRI.

The potential role of omentin-1 in obesity-related metabolic dysfunction-associated steatotic liver disease: evidence from translational studies.

BACKGROUND: Obesity, characterized by visceral adipose tissue (VAT) expansion, is closely associated with metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH). Recent research has highlighted the crucial role of the adipose tissue-liver axis in the development of MASLD. In this study, we investigated the potential role of omentin-1, a novel adipokine expressed by VAT, in obesity-related MASLD pathogenesis. METHODS: Through in silico analysis of differentially expressed genes in VAT from obese patients with and without MASH, we identified omentin-1 as a significant candidate. To validate our findings, we measured omentin-1 levels in VAT and plasma of lean controls and obese patients with biopsy-proven MASLD. Additionally, we assessed omentin-1 expression in the VAT of diet-induced mice MASLD model. In vitro and ex vivo studies were conducted to investigate the effects of omentin-1 on MASLD-related mechanisms, including steatosis, inflammation, endoplasmic reticulum (ER) stress, and oxidative stress. We also analyzed the impact of D-glucose and insulin on VAT omentin-1 levels ex vivo. RESULTS: Compared to the lean group, the obese groups exhibited significantly lower VAT and plasma levels of omentin-1. Interestingly, within the obese groups, omentin-1 is further decreased in MASH groups, independent of fibrosis. Likewise, VAT of mice fed with high-fat diet, showing histological signs of MASH showed decreased omentin-1 levels as compared to their control diet counterpart. In vitro experiments on fat-laden human hepatocytes revealed that omentin-1 did not affect steatosis but significantly reduced TNF-α levels, ER stress, and oxidative stress. Similar results were obtained using ex vivo VAT explants from obese patients upon omentin-1 supplementation. Furthermore, omentin-1 decreased the mRNA expression of NF-κB and mitogen-activated protein kinases (ERK and JNK). Ex vivo VAT explants showed that D-glucose and insulin significantly reduced omentin-1 mRNA expression and protein levels. CONCLUSIONS: Collectively, our findings suggest that reduced omentin-1 levels contribute to the development of MASLD. Omentin-1 supplementation likely exerts its beneficial effects through the inhibition of the NF-κB and MAPK signaling pathways, and it may additionally play a role in the regulation of glucose and insulin metabolism. Further research is warranted to explore omentin-1 as a potential therapeutic target and/or biomarker for MASLD.

Natural Compounds for Counteracting Nonalcoholic Fatty Liver Disease (NAFLD): Advantages and Limitations of the Suggested Candidates.

The booming prevalence of nonalcoholic fatty liver disease (NAFLD) in adults and children will threaten the health system in the upcoming years. The "multiple hit" hypothesis is the currently accepted explanation of the complex etiology and pathophysiology of the disease. Some of the critical pathological events associated with the development of NAFLD are insulin resistance, steatosis, oxidative stress, inflammation, and fibrosis. Hence, attenuating these events may help prevent or delay the progression of NAFLD. Despite an increasing understanding of the mechanisms involved in NAFLD, no approved standard pharmacological treatment is available. The only currently recommended alternative relies on lifestyle modifications, including diet and physical activity. However, the lack of compliance is still hampering this approach. Thus, there is an evident need to characterize new therapeutic alternatives. Studies of food bioactive compounds became an attractive approach to overcome the reticence toward lifestyle changes. The present study aimed to review some of the reported compounds with beneficial properties in NAFLD; namely, coffee (and its components), tormentic acid, verbascoside, and silymarin. We provide details about their protective effects, their mechanism of action in ameliorating the critical pathological events involved in NAFLD, and their clinical applications.

Ficolin-2 Plasma Level Assesses Liver Fibrosis in Non-Alcoholic Fatty Liver Disease.

Fibrosis is the strongest predictor for disease-specific mortality in non-alcoholic fatty liver diseases (NAFLD), but the need for liver biopsy limits its diagnosis. We assessed the performance of plasma ficolin-2 (FCN-2) as a biomarker of fibrosis identified by an in silico discovery strategy. Two hundred and thirty-five morbidly obese (MO) subjects with biopsy-proven NAFLD stratified by fibrosis stage (F0, n = 44; F1, n = 134; F2, n = 46; F3/F4, n = 11) and 40 cirrhotic patients were enrolled. The cohort was subdivided into discovery (n = 76) and validation groups (n = 159). The plasma level of FCN-2 and other candidate markers was determined. FCN-2 was inversely correlated with the stage of liver fibrosis (ρ = −0.49, p < 0.001) independently of steatosis (p = 0.90), inflammation (p = 0.57), and ballooning (p = 0.59). In the global cohort, FCN-2 level decreased significantly in a stepwise fashion from F0/F1 (median 4753 ng/mL) to F2−F3−F4 (2760 ng/mL) and in cirrhotic subjects (1418 ng/mL). The diagnostic performance of FCN-2 in detecting F ≥ 2 was higher than other indexes (APRI, FIB-4) (AUROC 0.82, 0.68, and 0.6, respectively). The accuracy improved when combined with APRI score and HDL values (FCNscore, AUROC 0.85). Overall, the FCN-2 plasma level can accurately discriminate liver fibrosis status (minimal vs. moderate/advanced) significantly improving the fibrosis diagnostic algorithms.

Modifications of IGF2 and EGFR plasma protein concentrations in NAFLD patients after bariatric surgery.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is strictly associated with the epidemic of obesity and is becoming the most prevalent liver disease worldwide. In severe obesity, bariatric surgery (BS) is the most effective treatment not only for obesity but also for the associated metabolic co-morbidities, NAFLD, among others. To date, noninvasive diagnostic/prognostic methods cannot evaluate hepatic improvements following surgery. OBJECTIVES: We aimed to measure plasma level of insulin-growth factor-2 protein (IGF2) and epithermal growth factor receptor (EGFR), and to assess their relationship with clinical and biochemical parameters during the 12 months follow-up. METHODS: Demographic, clinical-biochemical data, and plasma IGF2 and EGFR were measured in 69 patients preoperatively (T0) and 6 and 12 months (T6M and T12M, respectively) after BS. Liver biopsy was performed at T0. Relationships between IGF2, EGFR, and several biochemical parameters were performed using Pearson or Spearman correlation analysis. RESULTS: IGF2 plasma level increases during follow-up, passing from 2.5 (1.8-15.5) at baseline to 13.3 (8.6-19.1) at T12M, p < 0.001. Conversely, EGFR showed a not significant reduction. At T12M, the plasma level of both markers was comparable to those of lean subjects. The clinical-biochemical parameters (BMI, glycated hemoglobin, HOMA-IR) also return to the normal range at T12M. Correlation analysis demonstrated that IGF2 was significantly associated with total bilirubin, direct bilirubin, and albumin at T0 while with blood glucose, ALT, GGT, and AST/ALT ratio at T6M and T12M. CONCLUSIONS: IGF2 plasma levels increase after bariatric surgery, and these changes are associated with the modification of hepatic biochemical parameters, even if other clinic or metabolic improvements cannot be excluded.

Taste perception and expression in stomach of bitter taste receptor tas2r38 in obese and lean subjects.

Differences in taste perception have been related to eating behavior, nutritional status, and diseases. Recently, taste receptors have been identified in several extra-oral tissues, such as the gastrointestinal tract, where they seem to influence processes like digestion, sense of satiety as well as energy balance and intraluminal changes occurring in obesity. Our study aims to analyze differences in taste perception among 42 obese patients (OB) and 41 normal-weight subjects (LEAN). Polymorphisms in the gene codifying for the bitter taste receptor TAS2R38 and its expression on the surface of the gastric mucosa were tested and compared among OB and LEAN. Taste intensity of PROP (6-n-propylthiouracil), quinine, sucrose, citric acid and NaCl were measured on a labeled magnitude scale. DNA from peripheral whole blood was extracted and three polymorphisms in the TAS2R38 gene (rs713598, rs1726866, rs10246939) analyzed. Gastric biopsies were collected during bariatric surgery in OB and during endoscopy in LEAN. RNA was extracted and TAS2R38 gene expression assessed by RT-Real-Time qPCR. Anamnestic and anthropometric data were recorded in all participants during baseline visits. Logistic regression analysis showed that OB perceives sweet (sucrose) and bitter (PROP or 6-n-propylthiouracil) taste more intensely than LEAN (p-value = 0.02 and p-value = 0.005, respectively). While polymorphisms in TAS2R38 gene did not differ among OB and LEAN, we observed a significant increase of TAS2R38 mRNA levels in the stomach of OB compared to LEAN (p = 0.01). Our results provide new evidence of a link between obesity and altered taste perception as well as TAS2R38 expression in the stomach.

Occult hepatitis B virus infection predicts non-alcoholic steatohepatitis in severely obese individuals from Italy.

BACKGROUND & AIMS: Obesity is associated with non-alcoholic fatty liver (NAFL), which may progress towards non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). Occult hepatitis B virus infection (OBI) may contribute to hepatic damage in patients with chronic liver disease of different aetiologies (eg HCV, alcohol). However, information on the prevalence and clinical impact of OBI in obese individuals is lacking. The aims of this study were to investigate NASH prevalence and risk factors in obese people who underwent bariatric surgery. METHODS: Two-hundred and twenty-six subjects (160 females; mean age 42.9 years ±10.8 SD) without evidence of any further cause of liver disease consecutively underwent bariatric surgery in two Italian liver centers. During surgery, all patients underwent liver biopsy for histological evaluation and molecular studies. Liver DNA extracts were tested for PNPLA3, TM6SF2, MBOAT7, IRGM polymorphisms and for OBI. Univariate and multivariate analyses were used to identify predictors of NASH. RESULTS: Histology showed NASH in 115 (50.9%) and NAFL in 111 cases (49.1%). Twenty-nine/226 (12.8%) cases had OBI, 24 (82.8%) of whom had NASH and 5 (17.2%) NAFL, whereas among the 197 OBI-negative cases, 91 (46.2%) had NASH and 106 (53.8%) NAFL (P = .0002). Multivariate analysis showed that older age (P = .03, OR 1.034), alanine aminotransferase values (P = .005, OR 1.023), insulin resistance/diabetes (P = .02, OR 2.257), TM6SF2 polymorphism (P = .04, OR 3.168) and OBI (P = .004, OR 5.503) were independent predictors of NASH. CONCLUSION: NASH is highly prevalent in obese individuals undergoing bariatric surgery. OBI is one of the strongest risk factors of NASH in these patients.

Silybin Modulates Collagen Turnover in an In Vitro Model of NASH.

Silybin has been proposed as a treatment for nonalcoholic steatohepatitis (NASH). In this study, we assessed the effect of Silybin in a well-established in vitro coculture model of early-stage NASH. LX2 and Huh7 cells were exposed to free fatty acid (FFA) and Silybin as mono- or coculture (SCC). Cell viability, LX2 activation, collagen deposition, metalloproteinase 2 and 9 (MMP2-9) activity, and ROS generation were determined at 24, 96, and 144 h. Exposure to FFA induced the activation of LX2 as shown by the increase in cell viability and upregulation of collagen biosynthesis. Interestingly, while cotreatment with Silybin did not affect collagen production in LX2, a significant reduction was observed in SCC. MMP2-9 activity was reduced in FFA-treated Huh7 and SCC and cotreatment with Silybin induced a dose-dependent increase, while no effect was observed in LX2. Silybin also showed antioxidant properties by reducing the FFA-induced production of ROS in all the cell systems. Based on these data, Silybin exerts its beneficial effects by reducing LX2 proliferation and ROS generation. Moreover, MMP2-9 modulation in hepatocytes represents the driving mechanism for the net reduction of collagen in this NASH in vitro model, highlighting the importance of hepatic cells interplay in NASH development and resolution.

A simple in silico strategy identifies candidate biomarkers for the diagnosis of liver fibrosis in morbidly obese subjects.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disorder, tightly associated with obesity. The histological spectrum of the disease ranges from simple steatosis to steatohepatitis, with different stages of fibrosis, and fibrosis stage is the most significant predictor of mortality in NAFLD. Liver biopsy continues to be the gold standard for its diagnosis and reliable non-invasive diagnostic tools are unavailable. We investigated the accuracy of candidate proteins, identified by an in silico approach, as biomarkers for diagnosis of fibrosis. METHODS: Seventy-one morbidly obese (MO) subjects with biopsy-proven NAFLD were enrolled, and the cohort was subdivided according to minimal (F0/F1) or moderate (F2/F3) fibrosis. The plasmatic level of CD44 antigen (CD44), secreted protein acidic and rich in cysteine (SPARC), epidermal growth factor receptor (EGFR) and insulin-like growth factor 2 (IGF2) were determined by ELISA. Significant associations between plasmatic levels and histological fibrosis were determined by correlation analysis and the diagnostic accuracy by the area under receiver operating characteristic curves (AUROC). RESULTS: Eighty-two percentage of the subjects had F0/F1 and 18% with F2/F3 fibrosis. Plasmatic levels of IGF2, EGFR and their ratio (EGFR/IGF2) were associated with liver fibrosis, correlating inversely for IGF2 (P < .006) and directly (P < .018; P < .0001) for EGFR and EGFR/IGF2 respectively. The IGF2 marker had the best diagnostic accuracy for moderate fibrosis (AUROC 0.83), followed by EGFR/IGF2 ratio (AUROC 0.79) and EGFR (AUROC 0.71). CONCLUSIONS: Our study supports the potential utility of IGF2 and EGFR as non-invasive diagnostic biomarkers for liver fibrosis in morbidly obese subjects.

The importance of the interaction between hepatocyte and hepatic stellate cells in fibrogenesis induced by fatty accumulation.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease is characterized by an initial accumulation of triglycerides that can progress to non-alcoholic steatohepatitis, which can ultimately evolve to cirrhosis and hepatocellular carcinoma. Hepatic stellate cells play a key role in liver fibrogenesis by an increased activation and an altered profile of genes involved in the turnover of extracellular matrix components. To reproduce in-vitro the functional cell connections observed in vivo it is essential to consider cell-to-cell proximity and interaction. The aim of this study was to determine the response to free fatty acids in a simultaneous co-culture model of hepatocytes and hepatic stellate cells. METHODS: Simultaneous co-culture model and monoculture of each cell type (control) were exposed to FFA for 24 up to 144 h. Quantification of steatosis; stellate cell activation; assessment of fibrogenic response; expression and activity of metalloproteinases as well as collagen biosynthesis were evaluated. RESULTS: Free fatty acids induced comparable steatosis in simultaneous co-culture and monoculture. However, the activation of the stellate cells assessed by alpha-smooth muscle actin expression is greater when cells were in close contact. Furthermore, a time-dependent increment of tissue inhibitor metalloproteinase-2 protein was observed, which was inversely correlated with protein expression and activity of matrix-metalloproteinases, suggesting enhanced collagen biosynthesis. This behavior was absent in cell monoculture. CONCLUSIONS: These data indicate that cell-to-cell proximity between hepatocytes and stellate cells is necessary for the initiation of the fibrotic process.

Gender Differences in the Impact of a High-Fat, High-Sugar Diet in Skeletal Muscles of Young Female and Male Mice.

In the context of the increasing number of obese individuals, a major problem is represented by obesity and malnutrition in children. This condition is mainly ascribable to unbalanced diets characterized by high intakes of fat and sugar. Childhood obesity and malnutrition are not only associated with concurrent pathologies but potentially compromise adult life. Considering the strict correlation among systemic metabolism, obesity, and skeletal muscle health, we wanted to study the impact of juvenile malnutrition on the adult skeletal muscle. To this aim, 3-week-old C56BL/6 female and male mice were fed for 20 weeks on a high-fat. high-sugar diet, and their muscles were subjected to a histological evaluation. MyHCs expression, glycogen content, intramyocellular lipids, mitochondrial activity, and capillary density were analyzed on serial sections to obtain the metabolic profile. Our observations indicate that a high-fat, high-sugar diet alters the metabolic profile of skeletal muscles in a sex-dependent way and induces the increase in type II fibers, mitochondrial activity, and lipid content in males, while reducing the capillary density in females. These data highlight the sex-dependent response to nutrition, calling for the development of specific strategies and for a systematic inclusion of female subjects in basic and applied research in this field.

Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer.

Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.

Network Analysis for the Discovery of Common Oncogenic Biomarkers in Liver Cancer Experimental Models.

Hepatocellular carcinoma (HCC) is a malignancy marked by heterogeneity. This study aimed to discover target molecules for potential therapeutic efficacy that may encompass HCC heterogeneity. In silico analysis using published datasets identified 16 proto-oncogenes as potential pharmacological targets. We used an immortalized hepatocyte (IHH) and five HCC cell lines under two subtypes: S1/TGFß-Wnt-activated (HLE, HLF, and JHH6) and the S2/progenitor subtype (HepG2 and Huh7). Three treatment modalities, 5 µM 5-Azacytidine, 50 µM Sorafenib, and 20 nM PD-L1 gene silencing, were evaluated in vitro. The effect of treatments on the proto-oncogene targets was assessed by gene expression and Western blot analysis. Our results showed that 10/16 targets were upregulated in HCC cells, where cells belonging to the S2/progenitor subtype had more upregulated targets compared to the S1/TGFß-Wnt-activated subtype (81% vs. 62%, respectively). Among the targets, FGR was consistently down-regulated in the cell lines following the three different treatments. Sorafenib was effective to down-regulate targets in S2/progenitor subtype while PD-L1 silencing was able to decrease targets in all HCC subtypes, suggesting that this treatment strategy may comprise cellular heterogeneity. This study strengthens the relevance of liver cancer cellular heterogeneity in response to cancer therapies.

Proteome profiling identifies circulating biomarkers associated with hepatic steatosis in subjects with Prader-Willi syndrome.

Introduction: Prader-Willi syndrome (PWS) is a rare genetic disorder characterized by loss of expression of paternal chromosome 15q11.2-q13 genes. Individuals with PWS exhibit unique physical, endocrine, and metabolic traits associated with severe obesity. Identifying liver steatosis in PWS is challenging, despite its lower prevalence compared to non-syndromic obesity. Reliable biomarkers are crucial for the early detection and management of this condition associated with the complex metabolic profile and cardiovascular risks in PWS. Methods: Circulating proteome profiling was conducted in 29 individuals with PWS (15 with steatosis, 14 without) using the Olink Target 96 metabolism and cardiometabolic panels. Correlation analysis was performed to identify the association between protein biomarkes and clinical variables, while the gene enrichment analysis was conducted to identify pathways linked to deregulated proteins. Receiver operating characteristic (ROC) curves assessed the discriminatory power of circulating protein while a logistic regression model evaluated the potential of a combination of protein biomarkers. Results: CDH2, CTSO, QDPR, CANT1, ALDH1A1, TYMP, ADGRE, KYAT1, MCFD, SEMA3F, THOP1, TXND5, SSC4D, FBP1, and CES1 exhibited a significant differential expression in liver steatosis, with a progressive increase from grade 1 to grade 3. FBP1, CES1, and QDPR showed predominant liver expression. The logistic regression model, -34.19 + 0.85 * QDPR*QDPR + 0.75 * CANT1*TYMP - 0.46 * THOP1*ALDH1A, achieved an AUC of 0.93 (95% CI: 0.63-0.99), with a sensitivity of 93% and specificity of 80% for detecting steatosis in individuals with PWS. These biomarkers showed strong correlations among themselves and were involved in an interconnected network of 62 nodes, related to seven metabolic pathways. They were also significantly associated with cholesterol, LDL, triglycerides, transaminases, HbA1c, FLI, APRI, and HOMA, and showed a negative correlation with HDL levels. Conclusion: The biomarkers identified in this study offer the potential for improved patient stratification and personalized therapeutic protocols.

Bilirubin accumulation and Cyp mRNA expression in selected brain regions of jaundiced Gunn rat pups.

INTRODUCTION: Few data exist on regional brain bilirubin content in the neonatal period when acute bilirubin-induced neurologic damage (BIND) may occur, and no information is available on regional brain expression of cytochrome P450 monooxygenases (Cyps) that oxidize bilirubin. METHODS: Bilirubin content was analyzed by high-performance liquid chromatography and Cyp1a1, 1a2, and 2a3 mRNA expression was analyzed by quantitative PCR (qPCR) in cortex (Cx), cerebellum (Cll), superior colliculi (SC), and inferior colliculi (IC) of 17-d-old hyperbilirubinemic (jj) Gunn rat pups before and after administration of sulphadimethoxine to acutely displace bilirubin from plasma albumin. RESULTS: There was no difference in bilirubin content among brain regions in untreated rats. After intraperitoneal sulphadimethoxine, bilirubin content peaked at fourfold in Cx and SC at 1 h; but at 11- to 13-fold in Cll and IC at 24 h; returning to control levels at 72 h. The Cyp mRNA peaked at 30-70 times control at 1 h in Cx and SC, but at 3-9 times control at 24 h in Cll and IC. DISCUSSION: The close relationship in distinct brain regions between the extent of bilirubin accumulation and induction of mRNA of Cyps suggests Cyps may have a role in protecting selected brain areas from bilirubin neurotoxicity.

Kinetics and specificity of feline leukemia virus subgroup C receptor (FLVCR) export function and its dependence on hemopexin.

The feline leukemia virus subgroup C receptor (FLVCR) is a heme export protein that is required for proerythroblast survival and facilitates macrophage heme iron recycling. However, its mechanism of heme export and substrate specificity are uncharacterized. Using [(55)Fe]heme and the fluorescent heme analog zinc mesoporphyrin, we investigated whether export by FLVCR depends on the availability and avidity of extracellular heme-binding proteins. Export was 100-fold more efficient when the medium contained hemopexin (K(d) < 1 pm) compared with albumin (K(d) = 5 nm) at the same concentration and was not detectable when the medium lacked heme-binding proteins. Besides heme, FLVCR could export other cyclic planar porphyrins, such as protoporphyrin IX and coproporphyrin. However, FLVCR has a narrow substrate range because unconjugated bilirubin, the primary breakdown product of heme, was not transported. As neither protoporphyrin IX nor coproporphyrin export improved with extracellular hemopexin (versus albumin), our observations further suggest that hemopexin, an abundant protein with a serum concentration (6.7-25 mum) equivalent to that of the iron transport protein transferrin (22-31 mum), by accepting heme from FLVCR and targeting it to the liver, might regulate macrophage heme export and heme iron recycling in vivo. Final studies show that hemopexin directly interacts with FLVCR, which also helps explain why FLVCR, in contrast to some major facilitator superfamily members, does not function as a bidirectional gradient-dependent transporter. Together, these data argue that hemopexin has a role in assuring systemic iron balance during homeostasis in addition to its established role as a scavenger during internal bleeding or hemolysis.

Diagnostic management of nonalcoholic fatty liver disease: a transformational period in the development of diagnostic and predictive tools-a narrative review.

NAFLD is an emerging healthcare epidemic that is causing predictable adverse consequences for healthcare systems, societies and individuals. Whilst NAFLD is recognized as a multi-system disease with compound pathways that are both benign and pernicious in their unfolding; NASH is generally understood as a deleterious follow-on condition with path-specific tendencies that progress to cirrhosis, HCC and liver transplantation. Recent evidence is beginning to challenge this interpretation demanding more attention to the personalized nature of the disease and its pathogenesis across multiple different cohorts. This means that we need better diagnostic and prognostic tools not only to capture those 'at risk' disease phenotypes; but for better stratification and monitoring of patients according to their treatment strategies. With the advent of pipeline therapies for NASH underway, the medical profession looks to adopt more accurate non-invasive diagnostic tools that can help to delineate and eliminate NASH histology. This review looks at the search for the killer application revealing this particular moment in time as a transformational period; one that is pushing the boundaries of technology to integrate diverse panels of species through sensitive profiling and multi-omics approaches that cast wide, yet powerful diagnostic nets that have the potential to elucidate pathway specific biomarkers that are personalized and predictable.

Differentiation between stages of non-alcoholic fatty liver diseases using surface-enhanced Raman spectroscopy.

Non-alcoholic fatty liver disease (NAFLD) is a chronic disorder progressing from an initial benign accumulation of fat (NAFL) towards steatohepatitis (NASH), a degenerative form that can lead to liver cirrhosis and cancer. The development of non-invasive, rapid and accurate method to diagnose NASH is of high clinical relevance. Surface-enhanced Raman spectroscopy (SERS) of plasma was tested as a method to distinguish NAFL from NASH. SERS spectra from plasma of female patients diagnosed with NAFL (n = 32) and NASH (n = 35) were obtained in few seconds, using a portable Raman spectrometer. The sample consisted of 5 µL of biofluid deposited on paper coated with Ag nanoparticles. The spectra show consistent differences between the NAFL and NASH patients, with the uric acid/hypoxanthine band area ratio statistically different (p-value <0.001) between the two groups. The average figures of merit for a diagnostic test based on these ratios, as derived from a repeated 4-fold cross-validation of a logistic regression model, are all between 0.73 and 0.79, with an average area under the curve of 0.81. We conclude that SERS may be a reliable and rapid method to discriminate NAFLD from NASH.

Optimization of Point-Shear Wave Elastography by Skin-to-Liver Distance to Assess Liver Fibrosis in Patients Undergoing Bariatric Surgery.

BACKGROUND: Obesity is a primary limiting factor in liver stiffness measurement (LSM). The impact of obesity has always been evaluated in terms of body mass index (BMI), without studying the effects of skin-to-liver distance (SLD) on LSM. We studied the impact of SLD on LSM in a cohort of obese patients undergoing bariatric surgery and intra-operatory liver biopsy. MATERIALS AND METHODS: 299 patients underwent LSM by point-shear wave elastography (ElastPQ protocol), with two different ultrasound machines. SLD was measured as the distance between the skin and the liver capsule, perpendicular to where the region of interest (ROI) was positioned. We used the following arbitrary cut-offs: <5.7 kPa, F0-1; 5.7-7.99 kPa, F2; ≥8 kPa, F3-4. RESULTS: We developed two logistic regression models using elastography-histology agreement (EHA) as the dependent variable and SLD as the independent variable. The model based on the second machine showed strongly more performant discriminative and calibration metrics (AIC 38.5, BIC 44.2, Nagelkerke Pseudo-R2 0.894, AUROC 0.90). The SLD cut-off value of 34.5 mm allowed a correct EHA with a sensitivity of 100%, a specificity of 93%, negative predictive value of 100%, positive predictive value of 87%, an accuracy of 96%, and positive likelihood ratio of 3.56. CONCLUSION: The impact of SLD is machine-dependent and should be taken into consideration when interpreting LSM. We believe that our findings may serve as a reference point for appropriate fibrosis stratification by liver elastography in obese patients.

A transcriptome analysis identifies molecular effectors of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells.

BACKGROUND: The deposition of unconjugated bilirubin (UCB) in selected regions of the brain results in irreversible neuronal damage, or Bilirubin Encephalopathy (BE). Although UCB impairs a large number of cellular functions in other tissues, the basic mechanisms of neurotoxicity have not yet been fully clarified. While cells can accumulate UCB by passive diffusion, cell protection may involve multiple mechanisms including the extrusion of the pigment as well as pro-survival homeostatic responses that are still unknown. RESULTS: Transcriptome changes induced by UCB exposure in SH-SY5Y neuroblastoma cell line were examined by high density oligonucleotide microarrays. Two-hundred and thirty genes were induced after 24 hours. A Gene Ontology (GO) analysis showed that at least 50 genes were directly involved in the endoplasmic reticulum (ER) stress response. Validation of selected ER stress genes is shown by quantitative RT-PCR. Analysis of XBP1 splicing and DDIT3/CHOP subcellular localization is presented. CONCLUSION: These results show for the first time that UCB exposure induces ER stress response as major intracellular homeostasis in surviving neuroblastoma cells in vitro.