Combination of "low-dose" ribavirin and interferon alfa-2a therapy followed by interferon alfa-2a monotherapy in chronic HCV-infected nonresponders and relapsers after interferon alfa-2a monotherapy

AIM To report on the efficacy, safety and tolerability of interferon alfa-2a combined with a "low dose" of ribavirin for relapsers and non responders to alpha interferon monotherapy.METHODS Thirty-four chronic hepatitis C virus-infected non-responders to interferon alfa2a monotherapy (a course of at least 3 months treatment) and 13 relapsers to interferon alfa 2a monotherapy (a dose of 3 to 6 million units three times per week for at least 20 weeks but not more than 18 months) were treated with the same dose of interferon alfa-2a used before (3 to 6 million units three times per week) and ribavirin (10 mg/ kg daily) for 6 months. In complete responders, interferon alfa-2a was administered for further 6 months at the same dose used before as monotherapy.RESULTS Seven (20.6%) of 34 non-responders stopped the combined therapy due to adverse events, including two patients with histological and clinical Child A cirrhosis. In 17/27 (63%)non-responders, the combined therapy was stopped after three months because of non-response. Ten of the 27 non-responders completed the 1;2-month treatment course. At a mean follow up of 28 months (16- 37 months)after the treatment, 4/10 (15%) previous non-responders still remained complete responders,All 13 previous relapsers completed the 12-month treatment course. At a mean follow up of 22months (9 - 36 months) after treatment, 6/13(46%) the previous relapsers were stillsustained complete responders.CONCLUSION Our treatment schedule of the combined therapy for 6 months of interferon alfa2a with a low dose of ribavirin (10 mg/kg/day)followed by 6 months of interferon alfa-2amonotherapy is able to induce a sustainedcomplete response rate in 15% of non-responders and 46% of relapsers with chronic hepatitis C virus-related liver diseases comparable to those obtained with the standarddoses of ribavirin 1000 - 1200 mg/day.Randomized prospective controlled trials using lower total amounts of ribavirin in combination with interferon should be performed.

Diet high in fructose leads to an overexpression of lipocalin-2 in rat fatty liver

AIM:To explore lipocalin-2(LCN-2)expression and its possible role and mechanism(s)of production in rat models of diet-inducible fatty liver.METHODS:Fatty liver was triggered in male SpragueDawley rats fed either with liquid Lieber-DeCarli(LDC)or LDC+70%cal fructose(L-HFr)diet for 4 or 8 wk.Chow-nourished animals served as controls.Hepatic expression of LCN-2 and other metabolic and inflammatory mediators was assessed by quantitative reverse transcription polymerase chain reaction and Western blotting.Serum LCN-2,fasting leptin,and lipid profile were evaluated via Enzyme-Linked Immunosorbent Assay,Radioimmunoassay,and colorimetric assays,respectively.The localization of LCN-2 in the liver was detected by using immunofluorescence staining.Furthermore,HE stain was used to evaluate hepatic fatdegeneration and inflammation.RESULTS:Both LDC-fed and L-HFr-fed rat histologically featured fatty liver.In the liver,mRNA transcriptions of Mcp-1,a2-m,Il-8 and Glut5 were increased in the L-HFr group at both time points(P<0.001),while the transcription of Tlr4,Inos,and Tnf-a was significantly up-regulated at week 4.Interestingly,hepatic Lcn-2 expression was 90-fold at week 4 and 507-fold at week 8 higher in L-HFr-subjected rats vs control(P<0.001).In contrast to HDL-cholesterol,systemic levels of LCN-2,fasting leptin and triglycerides were elevated in the L-HFr regimen(P<0.001).Moreover,protein expression of hepatic LCN-2,CD14,phosphoMAPK,caspase-9,cytochrome c and 4-hydroxynonenal was increased in the L-HFr group.Conversely,the hepatic expression of PGC-1a(a mitochondrial-biogenic protein)was reduced in the L-HFr category at week 8.The localization of LCN-2 in the liver was predominantly restricted to MPO+granulocytes.CONCLUSION:Fructose diet up-regulates hepatic LCN-2 expression,which correlates with the increased indicators of oxidative stress and mitochondrial dysfunction.The LCN-2 may be involved in liver protection.

Initial steroid-free immunosuppression after liver transplantation in recipients with hepatitis c virus related cirrhosis

AIM: Steroids can increase hepatitis C virus (HCV) replication. After liver transplantation (LTx), steroids are commonly used for immunosuppression and acute rejection is usually treated by high steroid dosages. Steroids can worsen the outcome of recurrent HCV infection. Therefore,we evaluated the outcome of HCV infected liver recipients receiving initial steroid-free immunosuppression.METHODS: Thirty patients undergoing LTx received initial steroid-free immunosuppression. Indication for LTx included 7 patients with HCV related drrhosis. Initial immunosuppression consisted of tacrolimus 2x0.05 mg/kg.d po and mycophenolate mofetil (MMF) 2x15 mg/kg.d po. The tacrolimus dosage wasa djusted to trough levels in the target range of 10-15 μg/Ldudng the first 3 mo and 5-10 μg/L thereafter. Manifestations of acute rejection were verified histologically.RESULTS: Patient and graft survival of 30 patients receiving initial steroid-free immunosuppression was 86% and 83% at 1 and 2 years. Acute rejection occurred in 8/30 patients,including 1 HCV infected recipient. All HCV-infected patients had HCV genotype II (lb). HCV seropositivity occurred within the first 4 mo after LTx. The virus load was not remarkably increased during the first year after LTx. Histologically, grafts had no severe recurrent hepatitis.CONCLUSION: From our experience, initial steroid-free immunosuppression does not increase the risk of acute rejection in HCV infected liver recipients. Furthermore, none of the HCV infected patients developed serious chronic liver diseases. It suggests that it may be beneficial to avoid steroids in this particular group of patients after LTx.

Differential gene expression of chemokines in KRAS and BRAF mutated colorectal cell lines:Role of cytokines

AIM:To study KRAS/BRAF mutations in colorectalcancer(CRC)that influences the efficacy of treatment.To develop strategies for overcoming combination of treatment.METHODS:Five colonic cell-lines were investigated:DLD-1 with KRAS(G13D)mutation,HT 29 and Colo205 with BRAF(V600E)mutation as well as the wild type(Wt)cell-lines Caco2 and Colo-320.DLD-1(KRAS),HT-29(BRAF)and Caco2(Wt)cell lines were treated with cytokines(TNFα50 ng,IL-1β1 ng and IFNγ50ng)and harvested at different time points(1-24 h).KRAS inhibition was performed by the siRNA-approach.Two colorectal cancer cells DLD-1 and Caco2 were used for KRAS inhibition.About 70%confluency were confirmed before transfection with small interferring RNA(siRNA)oligonucleotides.All the synthetic siRNA sequences were designed in our laboratory.Total RNA and protein was isolated from the cells for RT-PCR and Western blotting.Densitometry of the Western blotting was analyzed with the Image J software(NIH).Results are shown as mean±SD.RESULTS:RT-PCR analysis in non-stimulated cells showed a low basal expression of TNFαand IL-1βin the DLD-1 KRAS-mutated cell-line,compared to Caco2wild type.No detection was found for IL-6 and IFNγin any of the studied cell lines.In contrast,pro-angiogenic chemokines(CXCL1,CXCL8)showed a high constitutive expression in the mutated cell-lines DLD-1(KRAS),HT-29 and Colo205(BRAF),compared to wild type(Caco2).The anti-angiogenic chemokine(CXCL10)showed a high basal expression in wild-type,compared to mutated cell-lines.KRAS down-regulation by siRNA showed a significant decrease in CXCL1 and CXCL10gene expression in the DLD-1(KRAS)cell-line in comparison to wild type(Caco2)at 72 h after KRAS silencing.In contrast,the specific KRAS inhibition resulted in an up-regulation of CXCL1 and CXCL10.The results of our study show a higher expression of pro-angiogenic chemokines at basal level in mutated cell-lines,which was further increased by cytokine treatment.CONCLUSION:To summarize,basal chemokine gene expression for pro-angiogenic chemokines was high in mutated as compared to wild type cell-lines.This reflects the likely existence of a different microenvironment in tumours consistent of wild type or mutated cells.This may help to rationalize the choice of molecular targets for suitable therapeutic investigation in clinical studies.

Adult-to-adult right lobe living donor liver transplantation:Comparison of endoscopic retrograde cholangiography with standard T2-weighted magnetic resonance cholangiography for evaluation of donor biliary anatomy

AIM: To compare the value of endoscopic retrograde cholangiography (ERC) and standard T2-weighted magnetic resonance cholangiography (MRC) in the evaluation process as adult-to-adult right lobe living donor liver transplantation (LDLTx) demands a successful outcome, and exact knowledge of the biliary tree is implicated to avoid biliary complications, postoperatively.METHODS: After starting the LDLTx program, 18 liver transplant candidates were selected for LDLTx by a stepwise evaluation process. ERC and standard T2-weighted MRC were performed to evaluate the biliary system of the donor liver. The anatomical findings of ERC and MRC mapping were compared using the Ohkubo classification. RESULTS: ERC allowed mapping of the whole biliary system in 15/15 (100%) cases, including 14/15 (93.3%) with biliary variants while routine MRC was only accurate in 2/13 (15.4%) cases. MRC was limited in depicting the biliary system proximal of the hepatic bifurcation. Postoperative biliary complications occurred in 2 donors and 8 recipients. Biliary complications were associated with Ohkubo type C, E or G in 6/8 recipients, and 2/3 recipients with biliary leak received a graft with multiple (≥2) bile ducts. CONCLUSION: Pretransplant ERC is safe and superior over standard MRC for detection of biliary variations that occur with a high frequency. However, precise knowledge of biliary variants did not reduce the incidence of postoperative biliary complications.

Endotoxin receptor CD14 gene variants and histological features in chronic HCV infection

AIM:To analyze the correlation between CD14 rs2569190/C-159T single nucleotide polymorphism (SNP) and disease progression in chronic hepatitis C.METHODS: Liver biopsy specimens from a total of 137 and 349 patients with chronic hepatitis C were separately evaluated with respect to necroinflammatory activity (grading) and architectural changes (staging). In one group, further histological lesions characteristic for hepatitis C, hepatitis C virus subtypes, and biochemical parameters of liver disease were also investigated. Samples of genomic DNA were genotyped for the respective SNP by 5'-nuclease assays using fluorescent dye-labeled allele-specif ic probes.RESULTS: Genotype distribution did not deviate from the Hardy-Weinberg equilibrium. In the first group, patients homozygous for the variant allele T were found to be younger than C allele carriers (39.6±12.5 vs 45.7±11.5, P=0.008). Among the histological lesions studied, portal lymphoid aggregates were more frequently observed among TT homozygotes than among C carriers (21/37 vs 32/100, P=0.008). The presence of portal lymphoid aggregates was closely correlated with hepatic inflammation (P=0.003) and with bile duct damage (P<0.001). The degree of fibrosis, in contrast, was not found to be related to the CD14 gene C-159T polymorphism.CONCLUSION: The data suggest a possible relationship between CD14 C-159T polymorphism and the formation of portal lymphoid aggregates, but not liver fibrosis progression in chronic hepatitis C.

Spontaneous elimination of hepatitis C virus infection: A retrospective study on demographic, clinical, and serological correlates

AIM: To fi nd correlates to spontaneous clearance of hepatitis C virus (HCV) infection, this study compared individuals with self-limited and chronic infection with regard to clinical, demographic, and serological pa-rameters. METHODS: Sixty-seven anti-HCV positive and repeatedly HCV RNA negative individuals were considered to have resolved HCV infection spontaneously. To determine the viral genotype these patients had been infected with HCV serotyping was performed. For comparison reasons, 62 consecutive patients with chronic hepatitis C were enrolled. Cases and controls were compared stratifi ed for age and sex. RESULTS: Retrospective analysis showed (1) a lower humoral reactivity to HCV in patients with self-limited compared to chronic HCV-infection and (2) that younger age, history of iv drug use, and acute/post-acute hepatitis A or B co-infections, but not viral genotypes, are independent correlates for spontaneous HCV clearance. CONCLUSION: The stronger humoral reactivity to HCV in patients with persistent infections and in those with a history of iv drug use is supposed to be due to continuous or repeated contact(s) to the antigen. Metachronous hepatitis A or hepatitis B infections might favor HCV clearance.

Reabsorption of iron into acutely damaged rat liver:A role for ferritins

AIM To studied iron metabolism in liver, spleen, and serum after acute liver-damage, in relation to surrogate markers for liver-damage and repair.METHODS Rats received intraperitoneal injection of the hepatotoxin thioacetamide(TAA), and were sacrificed regularly between 1 and 96 h thereafter. Serum levels of transaminases and iron were measured using conventional laboratory assays. Liver tissue was used for conventional histology, immunohistology, and iron staining. The expression of acute-phase cytokines, ferritin light chain(FTL), and ferritin heavy chain(FTH)was investigated in the liver by q RT-PCR. Western blotting was used to investigate FTL and FTH in liver tissue and serum. Liver and spleen tissue was also used to determine iron concentrations.RESULTS After a short initial decrease, iron serum concentrations increased in parallel with serum transaminase(aspartate aminotransferase and alanine aminotransferase) levels, which reached a maximum at 48 h, and decreased thereafter. Similarly, after 48 h a significant increase in FTL, and after 72 h in FTH was detected in serum. While earliest morphological signs of inflammation in liver were visible after 6 h, increased expression of the two acute-phase cytokines IFN-γ(1 h) and IL-1β(3 h) was detectable earlier, with maximum values after 12-24 h. Iron concentrations in liver tissue increased steadily between 1 h and 48 h, and remained high at 96 h. In contrast, spleen iron concentrations remained unchanged until 48 h, and increased mildly thereafter(96 h). Although tissue iron staining was negative, hepatic FTL and FTH protein levels were strongly elevated. Our results reveal effects on hepatic iron concentrations after direct liver injury by TAA. The increase of liver iron concentrations may be due to the uptake of a significant proportion of the metal by healthy hepatocytes, and only to a minor extent by macrophages, as spleen iron concentrations do not increase in parallel. The temporary increase of iron, FTH and transaminases in serum is obviously due to their release by damaged hepatocytes.CONCLUSION Increased liver iron levels may be the consequence of hepatocyte damage. Iron released into serum by damaged hepatocytes is obviously transported back and stored via ferritins.

Treatment of genotype 2 and 3 chronic hepatitis C virus-infected patients

AIM: Before pegylated interferon alpha (IFN) was introduced for the therapy of chronic hepatitis C virus(HCV)-induced hepatitis, conventional thrice weekly IFN therapy was supplemented by ribavirin. Also, at that time,higher and more frequent doses of IFN were expected to be more effective than the standard regimen of 3 MU thrice weekly. As ribavirin significantly increases side effects and negatively influences the quality of life particularly in young patients, we started a prospective non-randomized study with a daily IFN-2a monotherapy as an initial treatment for chronic hepatitis C.METHODS: Forty-six consecutive chronic HCV-infected patients received 3 MU IFN-2a per day as an initial treatment. Patients with genotype 2 or 3 (n = 12) were treated for 24 wk, and patients with genotypes other than 2 or 3 (n = 34) for 48 wk. Treatment outcome was followed up for 48 wk after the end of treatment (EOT).Virological response was defined as the absence of detectable serum HCV-RNA. Patients without virological response at 12 wk after the start of treatment received RESULTS: During treatment, three genotype 3 patients were excluded from the study due to incompliance. The remaining patients (n = 9) infected with genotype 2 or 3showed an initial virological response rate of 100%. Six patients (66.7%) were still found to be virus-free at the end of follow-up period. In these patients, initial virological response was evident already after 2 wk of treatment. In contrast, initial virological response occurred first after 4 wk of treatment in the three patients who relapsed(33.3%). In comparison, patients infected with genotypes other than 2 or 3 (n = 34) showed an initial virological response rate of only 23.5% (n = 8), and even in combination with ribavirin a sustained virological response(SVR) rate of only 11.8% (n = 4) could be achieved.CONCLUSION: In chronic HCV-infected patients with genotype 2 or 3, a SVR can be expected after 24 wk of daily dose IFN-2a treatment without ribavirin, if initial virological response develops early. This finding is worth to be confirmed in a prospective randomized study with pegylated IFN.

Is laparoscopy an advantage in the diagnosis of cirrhosis in chronic hepatitis C virus infection?

AIM: To evaluate the potential of laparoscopy in the diagnosis of cirrhosis and outcome of interferon treatment in HCV-infected patients.METHODS: In this retrospective study, diagnostic laparoscopy with laparoscopic liver biopsy was performed in 72 consecutive patients with chronic HCV infection. The presence or absence of cirrhosis was analyzed macroscopically by laparoscopy and microscopically by liver biopsy specimens. Clinical and laboratory data and outcome of interferon-alfa treatment were compared between cirrhotic and noncirrhotic patients.RESULTS: Laparoscopically, cirrhosis was seen in 29.2%(21/72) and non-cirrhosis in 70.8% (51/72) of patients.Cirrhotic patients were significantly older with a significant longer duration of HCV infection than noncirrhotic patients.Laboratory parameters (AST, y-GT, y-globulin fraction) were measured significantly higher as well as significantly lower (prothrombin index, platelet count) in cirrhotic patients than in non-cirrhotic patients. Histologically, cirrhosis was confirmed in 11.1% (8/72) and non cirrhosis in 88.9% (64/72). Patients with macroscopically confirmed cirrhosis (n=21) showed histologically cirrhosis in 38.1% (8/21) and histologically noncirrhosis in 61.9% (13/21). In contrast, patients with macroscopically non-cirrhosis (n=51) showed histologically non cirrhosis in all cases (51/51). Thirty-nine of 72 patients were treated with interferon-alfa, resulting in 35.9% (14/39)patients with sustained response and 64.1% (25/39) with non response. Non-responders showed significantly more macroscopically cirrhosis than sustained responders. In contrast, there were no significant histological differences between non-responders and sustained responders.CONCLUSION: Diagnostic laparoscopy is more accurate than liver biopsy in recognizing cirrhosis in patients with chronic HCV infection. Liver biopsy is the best way to assess inflammatory grade and fibrotic stage. The invasive marker for staging, prognosis and management, and treatrnent outcome of chronic HCV-infected patients need further research and clinical trials. Laparoscopy should be performed for recognition of cirrhosis if this parameter is found to be of prognostic and therapeutic relevance in patients with chronic HCV infection.

Measurements of 5-FU Plasma Concentrations in Patients with Gastrointestinal Cancer: 5-FU Levels Reflect the 5-FU Dose Applied

Introduction: 5-Fluorouracil (5-FU) is the basis of most combination chemotherapies for gastrointestinal tumors. It is generally well tolerated, but side-effects might require dose-adjustment. As adverse events are not specific to the 5-FU component of the chemotherapy-combination, i.e. neutropenia, diarrhea or cardiotoxicity, the knowledge of 5-FU serum levels might help to attribute these side effects to the 5-FU compound. The optimal concentration-range (AUC, area under the curve) has been described to be within 20-25 mgh/l. The aim of this study was to analyse the intra- and interindividual variability of 5-FU AUC-levels in patients with 5-FU infusion therapy. Methods: 230 blood samples were obtained from 31 different gastrointestinal cancer patients (esophagus (8), stomach (10), ileum (1), colorectum (12)) treated with 5-FU-infusional regimes, based on a 24- or 48-hour AIO treatment-schedule. 5-FU plasma concentrations were measured using an immunolinked Elisa assay (Saladax 5-FU PCMTM). Intra- and interindividual differences were analysed before (0 h;n = 115), at 2 - 3 hours after the start of infusional 5-FU treatment (n = 19) (early sampling) and towards the end of the infusion (n = 96) (late sampling). Results: Early blood sampling resulted in low 5-FU plasma concentrations (541 ± 127 g/ml) due to saline prefilling (2 - 3 ml) of the Baxter pump. Blood sampling at the later time-point resulted in reproducible values (971 ± 81 ng/ml). 5-FU concentrations were dose-dependent with low intra- and interindividual variability. However, care has to be taken, as the results can be influenced by inaccurate blood sampling: too early or late sampling (when the folfusor-pump is empty), delayed centrifugation of the tube or hemolysis. Conclusions: With critical analysis of the measurements and correct performance of blood sampling, the measurement of 5-FU plasma concentrations with the immunoassay may in the future allow to optimize 5-FU dosing and to identify the cause of toxicity. Changes of 5-FU clearance in long-term therapy still have to be studied.

Hypoalbuminemia: an underestimated, vital characteristic of hospitalized COVID-19 positive patients?

The COVID-19 pandemic has led to the greatest worldwide health crisis in decades.The number of infected patients with severe SARS-CoV-2(COVID-19)disease has overwhelmed the capacity of almost all health care systems around world.Hypoalbuminemia has now been reported in patients with severe disease seeking help in the emergency room because of COVID-19 infection.In the past,hypoalbuminemia was considered to be a negative prognostic marker,not only in patients with chronic liver disease,but also in patients with SARS and MERS infections.Albumin is the major serum protein synthesized by the liver.A low serum albumin level is an ominous clinical sign.Introduction of amino acids to a patient's diet is of fundamental importance to hepatic albumin synthesis in different clinical situations.This highlights the importance of nutritional support during the early phases of COVID-19-infection.Furthermore,albumin synthesis in the hepatocyte is downregulated at a pretranslational level by the direct interaction of the major acute-phase cytokines which are released into the circulation during the cytokine"storm"induced by the viral effects on the lungs.Both mechanisms contribute to severe hypoalbuminemia which,combined with massive fluid losses due to the fever,is responsible for severe hypovolemia and shock commonly observed in patients with COVID-19 in critical care settings.