Relationship between ischemia/reperfusion injury and the stimulus of fibrogenesis in an experimental model: comparison among different preservation solutions

Background and Aims

Orthotopic liver transplantation (OLT) has been the standard treatment for end-stage acute and chronic liver disease. Ischemia-reperfusion (I/R) injury is one of the major causes of poor graft function early after OLT, and adversely influencing graft and patient survivals. It is unknown whether I/R injury influences liver fibrogenesis.

Materials and Methods

Livers from 25 adult male Wistar rats were randomly assigned into 5 experimental groups according to the preservation solution: saline solution (SS); University of Wisconsin (UW) solution; Fructose 1, 6-biphosphate (FBP); S-Nitroso-N-Acetylcysteine (SNAC): or UW + SNAC (SNAC+UW). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) were determined in preservation solution samples at 2, 4, and 6 hours. After 6 hours of cold ischemia, ex situ reperfusion was applied to the liver for 15 minutes. Serum AST, ALT, LDH, and renin levels were determined. Fresh liver slices were processed for histological studies, determination of thiobarbituric acid reactive substances, catalase, and glutathione, and expression of TGF-β1 and angiotensin II AT1 receptor.

Results

AST was significantly lower during cold storage with UW than with the older media (P = .001); ALT was lower in the FBP group (P = .023) and LDH was lower in the FBP and SNAC groups (P = .007). After reperfusion, serum AST, ALT, LDH, and TBARS showed no significant differences among the groups. Catalase was significantly lower in the SS and FBP groups (P = .008 and P = .006, respectively). Compared with UW, glutathione concentrations were significantly higher in SS, FBP, and SNAC 200 (P = .004). Renin levels were significantly lower in the FBP group (P = .022). No histological signs of preservation injury were observed in the hepatic sample. No expressions were detected of TGF-β1 or AT1 receptor.

Conclusion

In this experimental model of early reperfusion injury, preservation changes related to higher levels of renin, which suggest its role in fibrogenesis. FBP was associated with lower renin levels than other solutions including UW.     

How are HCV-infected patients being identified in Brazil: a multicenter study

Background

Hepatitis C is an important health problem. In Brazil, 1–2 million people are infected. Despite this expressive number, and the availability of very successful treatment, many patients remained undiagnosed mainly because of the asymptomatic nature of the infection.

Association of interferon lambda-4 rs12979860 polymorphism with hepatocellular carcinoma in patients with chronic hepatitis C infection

BACKGROUND Hepatitis C virus(HCV) infection is a public health concern worldwide.Several factors,including genetic polymorphisms,may be evolved in the progression of HCV infection to liver diseases.Interferon lambdas(IFNLs) modulate the immune response during viral infections.IFNLs induce antiviral activity,interfering in the viral replication by promoting the expression of several genes that regulate immunological functions.The interferon lambda-4(IFNL4) rs12979860 polymorphism,which is characterized by a C to T transition in intron 1,is associated with spontaneous and treatment-induced clearance of HCV infection and may play a role in the development of HCV-associated liver diseases,including hepatocellular carcinoma(HCC).AIM To investigate the association of IFNL4 rs12979860 polymorphism with fibrosis,cirrhosis,and HCC in patients with chronic HCV infection.METHODS This study was comprised of 305 chronic HCV-infected patients(53 fibrosis,154 cirrhosis,and 98 HCC cases).The control group was comprised of 260 HCVnegative healthy individuals.The IFNL4 rs12979860 polymorphism was genotyped using the TaqMan assay.Fibrosis was diagnosed based on liver biopsy findings,while cirrhosis was diagnosed through clinical,laboratory,anatomopathological,and/or imaging data.HCC was diagnosed through imaging tests,tumor,and/or anatomopathological markers.RESULTS The T allele was observed in the three groups of patients(fibrosis,cirrhosis,and HCC) at a significantly higher frequency when compared with the control group(P=0.047,P 0.001,and P=0.01,respectively).Also,genotype frequencies presented significant differences between the control group and cirrhosis patients(P 0.001) as well as HCC patients(P=0.002).The risk analysis was performed using the codominant and dominant T allele models.In the codominant model,it was observed that the CT genotype showed an increased risk of developing cirrhosis in comparison with the CC genotype [odds ratio(OR)=2.53;95%confidence interval(CI):1.55-4.15;P 0.001] as well as with HCC(OR=2.54;95%CI:1.44-4.56;P=0.001).A similar result was observed in the comparison of the TT vs CC genotype between the control group and cirrhosis group(OR=2.88;95 % CI:1.44-5.77;P=0.001) but not for HCC patients.In the dominant T allele model,the CT+TT genotypes were associated with an increased risk for progression to cirrhosis(OR=2.60;95%CI:1.63-4.19;P 0.001) and HCC(OR=2.45;95%CI:1.42-4.31;P=0.001).CONCLUSION These findings suggest that the T allele of IFNL4 rs12979860 polymorphism is associated with the development of cirrhosis and HCC in chronic HCV-infected patients.     

Low levels of Lysosomal Acid Lipase (LAL) activity increases necroinflammation in adult patients with biopsy-proven metabolic associated fatty liver disease

Introduction and objectiveMetabolic associated fatty liver disease (MAFLD), characterized by intra-hepatic fat accumulation, will soon be the leading cause of end-stage liver disease. Lysosomal Acid Lipase (LAL) is a key enzyme in lipid metabolism. We investigated its activity in patients with biopsy-proven MAFLD.MethodsProspective cross-sectional study in patients with biopsy-proven MAFLD. Blood LAL-activity (pmol/punch/h) was measured with dried blood spot extracts using Lalistat 2. Demographic, clinical, and laboratory data were collected.Results101 adult patients were recruited. Among them, 11.9% had a diagnosis of MAFLD without steatohepatitis and 88.1% had MAFLD with steatohepatitis. The median of LAL-activity in patients with MAFLD was 76.8 pmol/punch/h. MAFLD patients with steatohepatitis showed an increase in gamma-glutamyl transferase (p = 0.042), insulin (p = 0.001), homeostatic model assessment for insulin resistance (HOMA-IR, p = 0.001) and advanced liver fibrosis (p < 0.001), compared to cases of MAFLD without steatohepatitis. There was no statistical difference in LAL-activity between the cases (p = 0.296). When considering LAL-activity above and below 77 pmol/punch/h as a cut-off value, patients with reduced LAL-activity had a significant increase in necroinflammatory activity according to the METAVIR score (p = 0.040), and NAFLD activity score (NAS, p = 0.031) compared to cases with higher LAL-activity.ConclusionOur findings suggest that reduced LAL-activity is associated with increased necroinflammatory activity and severity of the NAS. A better knowledge of the role of LAL may provide new insights into the pathogenesis and progression of MAFLD.     

Chromosomal instability and cytotoxicity induced by ribavirin: comparative analysis in cell lines with different drug-metabolizing profiles

Ribavirin is an important component of the treatment for hepatitis C virus (HCV) infection and, in combination with the new direct-acting antiviral (DAA) agents, comprises the major current therapeutic regimens. This study evaluated the cytotoxicity and chromosomal instability induced by ribavirin using the in vitro cytokinesis-block micronucleus cytome (CBMN-Cyt) assay in two cell lines with different expression levels of drug-metabolizing enzymes: human hepatocellular carcinoma cells (HepG2) and Chinese hamster ovary (CHO-K1) cells. HepG2 cells were treated with nine concentrations (from 15.3?μg/ml to 3.9?mg/ml) and CHO-K1 cells were exposed to eight concentrations (from 15.3?μg/ml to 1.9?mg/ml) of ribavirin for 24?h. Ribavirin inhibited cell proliferation in both cell lines, but at different concentrations: 3.9?mg/ml in HepG2 and 244.2?μg/ml in CHO-K1 cells. No significant differences were observed regarding aspects of cell death in HepG2 and CHO-K1 cells, reflecting the absence of cytotoxic effects associated to ribavirin. Ribavirin did not increase the frequency of nucleoplasmic bridges (NPBs) and nuclear bud (NBUD). However, when compared to the negative control, a significant increase in micronuclei (MNi) frequency was observed in both cell lines. However, chromosomal instability was induced by higher concentrations of ribavirin in HepG2 cells (from 61.1 to 976.8?μg/ml), compared with CHO-K1 cells (15.3 and 30.5?μg/ml). These results demonstrate the potential of ribavirin to promote chromosomal instability, and suggest that cells with different expressions of drug-metabolizing enzymes show different susceptibility to ribavirin effects.     

Comparison Between Handgrip Strength, Subjective Global Assessment, Anthropometry, and Biochemical Markers in Assessing Nutritional Status of Patients with Crohn’s Disease in Clinical Remission

Introduction Crohn’s disease (CD) may lead to protein and calorie malnutrition (PCM) secondary to impaired digestive and absorptive functions of the small intestine and sometimes to the influence of diet taboos. The earlier the PCM is diagnosed, the higher are the odds of reversal. The objective of this study was to compare different methods of nutritional assessment in patients with CD and correlate them with the disease characteristics. Sample The sample comprised 75 patients with CD from the Gastroenterology Service at the Hospital de Clínicas de Porto Alegre; 37 were male, with a mean age of 38.2 years old (SD = 13.3). All patients had been in clinical remission (CDAI <150) for over 3 months. They were not receiving enteral or parenteral nutrition. The nutritional assessment was considered: body mass index (BMI), triceps skin fold (TSF), arm circumference (MAC), arm muscle circumference (MAMC), subjective global assessment (SGA), non-dominating handgrip strength (HGS) and food intake inquiries. Results When comparing the different nutritional assessment methods, 26.7% of the patients were malnourished according to the MAC, 29.3% according to the MAMC, 18.7% according to the SGA, 6.7% according to the BMI, 37.3% according to the TSF and 73.3% according to the HGS. No statistically significant associations were found for disease location, its behavior, drugs utilized, ESR, CRP, age of patients and disease time with the nutritional state verified by HGS, TSF, MAMC and SGA. Conclusion The prevalence of malnutrition is significant in patients with CD, even in clinical remission. The BMI should not be used as reference in this population. The HGS detected a high prevalence of nutritional risk in patients with CD in remission. Studies are required that correlate it with more sensitive methods, for the patients’ effective nutritional state assessment.     

Hepatocellular carcinoma staging systems: Hong Kong liver cancer vs Barcelona clinic liver cancer in a Western population

BACKGROUND Despite being the world's most widely used system for staging and therapeutic guidance in hepatocellular carcinoma(HCC) treatment, the Barcelona clinic liver cancer(BCLC) system has limitations, especially regarding intermediate-grade(BCLC-B) tumors. The recently proposed Hong Kong liver cancer(HKLC) staging system appears useful but requires validation in Western populations.AIM To evaluate the agreement between BCLC and HKLC staging on the management of HCC in a Western population, estimating the overall patient survival.METHODS This was a retrospective study of HCC patients treated at a university hospital in southern Brazil between 2011 and 2016. Demographic, clinical, and laboratory data were collected. HCC staging was carried out according to the HKLC and BCLC systems to assess treatment agreement. Overall survival was estimated based on the treatment proposed in each system.RESULTS A total of 519 HCC patients were assessed. Of these, 178(34.3%) were HKLC-I; 95(18.3%) HKLC-IIA; 47(9.1%) HKLC-IIB; 29(5.6%) HKLC-IIIA; 30(5.8%) HKLCIIIB; 75(14.4%) HKLC-IV; and 65(12.5%) HKLC-V. According to the BCLC, 25(4.9%) were BCLC-0; 246(47.4%) BCLC-A; 107(20.6%) BCLC-B; 76(14.6%) BCLCC; and 65(12.5%) BCLC-D. The general agreement between the two systems was80.0%-BCLC-0 and HKLC-I(100%); BCLC-A and HKLC-I/HKLC-II(96.7%);BCLC-B and HKLC-III(46.7%); BCLC-C and HKLC-IV(98.7%); BCLC-D and HKLC-V(41.5%). When sub-classifying BCLC-A, HKLC-IIB, HKLC-IIIA and HKLC-IIIB stages according to the up-to-7 in/out criterion, 13.4, 66.0, 100 and36.7%, respectively, of the cases were classified as up-to-7 out.CONCLUSION In a Western population, the general agreement between the two systems was80.0%, although in BCLC-B cases the agreement was low, suggesting that some individuals could be candidates for the curative treatment recommended by the HKLC. The authors suggest that the BCLC system should be routinely employed,although for BCLC-B cases it should be associated with the HKLC system.     

HLA-G 3′UTR haplotype analyses in HCV infection and HCV-derived cirrhosis,hepatocellular carcinoma and fibrosis

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. Chronic HCV infection is also an important cause of hepatic fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV has the capacity to evade immune surveillance by altering the host immune response. Moreover, variations in immune-related genes can lead to differential susceptibility to HCV infection as well as interfere on the susceptibility to the development of hepatic fibrosis, cirrhosis and HCC. The human leucocyte antigen G (HLA-G) gene codes for an immunomodulatory protein known to be expressed in the maternal–foetal interface and in immune-privileged tissues. The HLA-G 3′ untranslated region (3′UTR) is important for mRNA stability, and variants in this region are known to impact gene expression. Studies, mainly focusing in a 14 bp insertion/deletion polymorphism, have correlated HLA-G 3′UTR with susceptibility to viral infections, but other polymorphic variants in the HLA-G 3′UTR might also affect HCV infection as they are inherited as haplotypes. The present study evaluated HLA-G 3′UTR polymorphisms and performed linkage disequilibrium test and haplotype assembly in 286 HCV infected patients who have developed fibrosis, cirrhosis or HCC, as well as in 129 healthy control subjects. Haplotypes UTR-1, UTR-2 and UTR-3 were the most observed in HCV+ patients, in the frequencies of 0.276, 0.255 and 0.121, respectively. No statistically significant difference was observed between HCV+ and control subjects, even when patients were grouped according to outcome (HCC, cirrhosis or fibrosis). Despite that, some trends in the results were observed, and therefore, we cannot rule out the possibility that variants associated to high HLA-G expression can be involved in HCV infection susceptibility.