Hepatic IFNL4 Gene Activation in Hepatocellular Carcinoma Patients with Regard to Etiology.

Hepatocellular carcinoma (HCC) is a malignancy with a leading lethality. The etiology is quite diverse, ranging from viral infections to metabolic disorders or intoxications, and associates with specific somatic mutational patterns and specific host immunological phenotypes. Particularly, hepatitis C virus (HCV)-infected liver is featured by an activation of interferon (IFN)-stimulated genes (ISGs; IFN signature), which we suppose is driven by type III IFNL4. Taking advantage of the TCGA collection of HCC patients of various different etiologies, this study aimed at validating our previous findings on hepatic IFNL4 gene activation in HCV infection in an independent and larger cohort of patients with advanced liver disease. In a cohort of n = 377 cases, the entirety of the sequencing data was used to assess the IFNL genotypes, and the cases were stratified for etiology. The number of IFNL4 transcripts within nonmalignant and malignant tissues was found to be more abundant in patients with HCV or HCV/HBV infections when compared to other risk factors. Moreover, in patients with HCV infection as a risk factor, a close, positive relationship was found between ISG activation and the number of functional IFNL4 transcripts. Data on this independent TCGA sample support the concept of an IFNL4-dependent HCV-driven activation of hepatic ISGs. In addition to that, they add to the understanding of etiology-related host immunological phenotypes in HCC.

Interferon-lambda (IFNL) germline variations and their significance for HCC and PDAC progression: an analysis of The Cancer Genome Atlas (TCGA) data.

BACKGROUND: Hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC) are malignancies with a leading lethality. With reference to interferons (IFNs) known to mediate antitumor activities, this study investigated the relationship between germline genetic variations in type III IFN genes and cancer disease progression from The Cancer Genome Atlas (TCGA) data. The genetic variations under study tag a gain-or-loss-of-function dinucleotide polymorphism within the IFNL4 gene, rs368234815 [TT/ΔG]. METHODS: The entirety of the TCGA sequencing data was used to assess genotypes of 187 patients with HCC and of 162 patients with PDAC matched for ethnicity. Stratified for IFNL genotypes, both cohorts were subjected to time-to-event analyses according to Kaplan-Meier with regard to the length of the specific progression free interval (PFI) and the overall survival (OS) time as two clinical endpoints for disease progression. RESULTS: Logrank analysis revealed a significant relationship between IFNL genotypes and disease outcome for PDAC. This relationship was not found for HCC. A multiple Cox regression analysis employing patients' age, tumor grade and tumor stage as further covariates proved IFNL genotypes to be independent predictors for PDAC disease outcome. CONCLUSION: This repository-based approach unveiled clinical evidence suggestive for an impact of IFNL germline variations for PDAC progression with an IFNL haplotype predisposing for IFNL4 expression being favorable.

Danger-Associated Molecular Patterns (DAMPs): Molecular Triggers for Sterile Inflammation in the Liver.

Inflammatory liver diseases in the absence of pathogens such as intoxication by xenobiotics, cholestatic liver injury, hepatic ischemia-reperfusion injury (I/R), non-alcoholic steatohepatitis (NASH), or alcoholic liver disease (ALD) remain threatening conditions demanding specific therapeutic options. Caused by various different noxae, all these conditions have been recognized to be triggered by danger- or death-associated molecular patterns (DAMPs), discompartmentalized self-structures released by dying cells. These endogenous, ectopic molecules comprise proteins, nucleic acids, adenosine triphosphate (ATP), or mitochondrial compounds, among others. This review resumes the respective modes of their release-passively by necrotic hepatocytes or actively by viable or apoptotic parenchymal cells-and their particular roles in sterile liver pathology. It addresses their sensors and the initial inflammatory responses they provoke. It further addresses a resulting second wave of parenchymal death that might be of different mode, boosting the release of additional, second-line DAMPs. Thus, triggering a more complex and pronounced response. Initial and secondary inflammatory responses comprise the activation of Kupffer cells (KCs), the attraction and activation of monocytes and neutrophil granulocytes, and the induction of type I interferons (IFNs) and their effectors. A thorough understanding of pathophysiology is a prerequisite for identifying rational therapeutic targets.

Hepatic Interferon-λ3 (IFNL3) Gene Expression Reveals Not to Be Attenuated in Non-Favorable IFNL3 rs4803217 or IFNL4 rs368234815 Minor Allele Carriers in Chronic Hepatitis C.

Genetic polymorphisms in the region of the interferon-λ genes (IFNL) associate with clearance of hepatitis C virus (HCV) infection. One of these polymorphisms, IFNL4 rs368234815, determines loss or gain of function of the IFNL4 gene by frameshift variation. The very same and a second one, IFNL3 rs4803217, are supposed to impact the expression of IFNL3: while IFNL4 rs368234815 is suggested to modulate IFNL3 transcription, IFNL3 rs4803217 is thought to alter IFNL3 mRNA stability. The latter process is believed to be partially driven by an HCV-induced ectopic expression of myosin heavy chain genes 7B and 7 and their co-expressed microRNAs mir499 and mir208B. These ideas are evidenced by functional investigations on peripheral blood mononuclear and hepatoma cells in culture. Our study aimed at exploring IFNL3 gene expression in clinical samples, i.e., in ex vivo derived liver tissue from patients with chronic hepatitis C (n = 57) and various other diseases (n = 56). By applying an assay designed to specifically quantify IFNL3 and discriminating paralogous IFNL2 transcripts, IFNL3 mRNA expression was not found to differ significantly between chronic hepatitis C and control samples. Among patients with chronic HCV infection, moreover, IFNL3 rs4803217 or IFNL4 rs368234815 minor alleles did not associate with reduced IFNL3 gene expression. Finally, myosin heavy chain genes 7B and 7 and corresponding microRNAs mir499 and mir208B were not found activated in liver in chronic HCV infection. Of note, detectability of MYH7 mRNA related to the procedure of liver biopsy sampling, as tissue obtained by direct punctation of the liver during laparoscopic inspection was less likely to contain MYH7 transcripts than samples acquired by percutaneous punctation. In conclusion, data on ex vivo derived liver tissue samples argue against an attenuating impact of IFNL3 rs4803217 or IFNL4 rs368234815 minor alleles on hepatic IFNL3 gene expression in vivo.

Association of serum vitamin B12 levels with stage of liver fibrosis and treatment outcome in patients with chronic hepatitis C virus genotype 1 infection: a retrospective study.

BACKGROUND: Chronic hepatitis C (CHC) is a global health challenge. New therapeutic agents with excellent sustained virological response (SVR) rates are available mainly in developed countries, while the majority of CHC patients live in countries with low health budget. Predictors of therapeutic response are therefore necessary. Vitamin B12 appears to be involved in hepatitis C virus replication. METHODS: We therefore studied retrospectively the relationship between baseline serum vitamin B12 levels and clinical features in 116 CHC genotype 1 infected patients. Logistic regression models with univariate and multivariate analysis were used in the statistical analysis. RESULTS: Baseline serum vitamin B12 levels were found to be positively associated with serum transaminase activities (AST, p = 0.002, ALT, p = 0.04), baseline viral load (p < 0.0001), stage of fibrosis (p = 0.0001) and favorable interferon-λ3/4 (IFNL3/IFNL4) rs12979860 genotypes (p = 0.04), and inversely with SVR (p < 0.001) as well as with rapid virological response (p = 0.001). Patients with baseline serum vitamin B12 levels below a cut-off value of 570 ng/L achieved a SVR rate of 59% with an odds ratio (OR) of 13.4 [confidence interval (CI) 4.3-41.9, p < 0.0001] compared to patients above the cut-off value. By combining serum vitamin B12 levels and IFNL3/IFNL4 rs12979860 genotypes, patients with baseline serum vitamin B12 levels below the cut-off value of 570 ng/L and IFNL3/IFNL4 rs12979860 CC genotype achieved a SVR rate of even 80% with an OR of 54 (CI 9.9-293, p < 0.0001) compared to patients above the cut-off value and non-CC-genotypes. CONCLUSION: Our data suggest baseline serum vitamin B12 levels as useful noninvasive marker for characterizing CHC patients. They might further help to identify responders to a standard treatment.

Activation of Type I and Type III Interferons in Chronic Hepatitis C.

Infection with hepatitis C virus (HCV) results in chronic and progressive liver disease. Persistency rates add up to 85%. Despite recognition of the virus by the human host in peripheral blood and in the liver, immune response appears to be ineffective in clearing infection. The ability to spontaneously eradicate the virus as well as the outcome of infection upon therapy with human recombinant interferon-α (IFN-α) was found to correlate most closely with genetic variations within the region encoding the IFN-λ genes, as revealed by genome-wide association studies on main ethnic populations in 2009. This review summarizes the induction of type I and type III IFN genes and their effectors, the IFN-stimulated genes. It focusses on the in vivo situation in chronic HCV infection in man both in the peripheral blood compartment and in the liver. It also addresses the impact of genetic polymorphisms in the region of type III IFN genes on their activation. Finally, it discusses how antiviral drugs (i.e. IFN-α, ribavirin and the direct-acting antivirals) may complementarily control the activation of endogenous IFNs and succeed in combatting infections.

Erythropoietin rs1617640 G allele associates with an attenuated rise of serum erythropoietin and a marked decline of hemoglobin in hepatitis C patients undergoing antiviral therapy.

BACKGROUND: A decline in hemoglobin (Hb) concentration during antiviral therapy in chronic hepatitis C (CHC) is a serious side effect. It may compel to dose reduction or even termination of antiviral treatment. The activation of erythropoietin (EPO) synthesis as a physiological response to anemia and its relation to a genetic variation within the EPO gene has not been evaluated yet. METHODS: Data of 348 CHC patients were reviewed retrospectively. Samples were genotyped for EPO rs1617640 and inosine triphosphatase (ITPA) rs1127354. Serum EPO concentrations were determined before and during therapy. Primary endpoints were set as Hb decline >3 g/dl at weeks 4 and 12. RESULTS: EPO rs1617640 G homozygotes showed a significantly lower rise of serum EPO level over time than T allele carriers (p < 0.001). The cumulative frequency of a significant Hb reduction added up to 40%. Multivariate analysis revealed that besides age, ribavirin starting dose and baseline Hb also EPO rs1617640 G homozygosity associates with Hb reduction at week 4 (p = 0.025) and 12 (p = 0.029), while ITPA C homozygotes are at risk for Hb decline particularly early during treatment. Furthermore, EPO rs1617640 G homozygotes were more frequently in need for blood transfusion, epoetin-α supplementation, or ribavirin dose reduction (p < 0.001). CONCLUSIONS: Our data suggest that EPO rs1617640 genotype, the rise of serum EPO concentration as well as ITPA rs1127354 genotype are promising parameters to evaluate the Hb decline during antiviral therapy. A rational adjustment of therapy with epoetin-α supplementation might prevent serious adverse events or the need to terminate treatment.

Combination of alcohol and fructose exacerbates metabolic imbalance in terms of hepatic damage, dyslipidemia, and insulin resistance in rats.

Although both alcohol and fructose are particularly steatogenic, their long-term effect in the development of a metabolic syndrome has not been studied in vivo. Consumption of fructose generally leads to obesity, whereas ethanol can induce liver damage in the absence of overweight. Here, Sprague-Dawley rats were fed ad libitum for 28 days on five diets: chow (control), liquid Lieber-DeCarli (LDC) diet, LDC +30%J of ethanol (L-Et) or fructose (L-Fr), and LDC combined with 30%J ethanol and 30%J fructose (L-EF). Body weight (BW) and liver weight (LW) were measured. Blood and liver samples were harvested and subjected to biochemical tests, histopathological examinations, and RT-PCR. Alcohol-containing diets substantially reduced the food intake and BW (≤3rd week), whereas fructose-fed animals had higher LW than controls (P<0.05). Additionally, leukocytes, plasma AST and leptin levels were the highest in the fructose-administered rats. Compared to the chow and LDC diets, the L-EF diet significantly elevated blood glucose, insulin, and total-cholesterol levels (also vs. the L-Et group). The albumin and Quick-test levels were the lowest, whereas ALT activity was the highest in the L-EF group. Moreover, the L-EF diet aggravated plasma triglyceride and reduced HDL-cholesterol levels more than 2.7-fold compared to the sum of the effects of the L-Et and L-Fr diets. The decreased hepatic insulin clearance in the L-EF group vs. control and LDC groups was reflected by a significantly decreased C-peptide:insulin ratio. All diets except the control caused hepatosteatosis, as evidenced by Nile red and H&E staining. Hepatic transcription of insulin receptor substrate-1/2 was mainly suppressed by the L-Fr and L-EF diets. The L-EF diet did not enhance the mitochondrial ß-oxidation of fatty acids (Cpt1α and Ppar-α expressions) compared to the L-Et or L-Fr diet. Together, our data provide evidence for the coaction of ethanol and fructose with a high-fat-diet on dyslipidemia and insulin resistance-accompanied liver damage.

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 Sprague-Dawley 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 fat degeneration 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-α 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, phospho-MAPK, caspase-9, cytochrome c and 4-hydroxynonenal was increased in the L-HFr group. Conversely, the hepatic expression of PGC-1α (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.

Ninety-day survival rate of patients with sepsis relates to programmed cell death 1 genetic polymorphism rs11568821.

BACKGROUND: Sepsis is a life-threatening condition. Programmed cell death 1 protein (PD-1), a negative costimulatory molecule, is suggested to be involved in pathogenesis as mortality is associated with high expression and as neutralizing antibodies improve survival in a mouse model. The PD-1 gene harbors an intronic single-nucleotide polymorphism, rs11568821, which is located in a transcription factor-binding site and supposed to affect PD-1 transcription. OBJECTIVE: This study aimed at investigating whether mortality (90-day) among patients with sepsis associates with PD-1 rs11568821 genotypes. METHODS: Adult white patients with sepsis from the surgical intensive care units of a university medical center were followed up for 90 days, and mortality was recorded as primary outcome variable. Blood samples were taken for PD-1 rs11568821 genotyping. Sequential Organ Failure Assessment scores increased at enrollment and during the observation period to monitor morbidity. RESULTS: Two hundred nineteen critically ill patients with sepsis were enrolled in this investigation. Ninety-day mortality was significantly higher among G homozygotes than among A allele carriers (P = 0.0032). During intensive care unit stay, G homozygotes experienced higher Sequential Organ Failure Assessment scores (P < 0.001) and a higher demand of vasopressor therapy (P = 0.0107). CONCLUSIONS: Data provide first associative evidence for PD-1 rs11568821 as a prognostic indicator in patients with sepsis.

Interferon-λ4 (IFNL4) transcript expression in human liver tissue samples.

Eradication of hepatitis C virus (HCV) infection, both spontaneous and treatment-induced, is marked by the wildtype allele C of a single nucleotide polymorphism upstream of the IL28B gene, rs12979860. This favorable allele was recently described to be in linkage disequilibrium with the wildtype allele TT of a dinucleotide polymorphism, ss469415590, located within a new protein-coding gene. While the TT allele introduces a frame-shift and disrupts the open reading frame, only the variant allele, ΔG, creates a novel type III interferon (IFN) protein, IFN-λ4/IFNL4. Absence of IFNL4 is thus supposed to favor resolution of HCV infection. As to date IFNL4 mRNA transcription has only been investigated in polyI:C-stimulated primary human hepatocytes and not yet in HCV infection in vivo, this study analyzed IFNL4 mRNA expression in human liver biopsy specimens. Samples were obtained from patients with a broad panel of disorders including no liver disease, liver diseases of non-viral etiology, chronic hepatitis B and chronic hepatitis C. Hepatic IFNL4 transcripts were detectable exclusively in a subgroup of chronic hepatitis C patients (24/45). Their amounts were positively related to liver HCV RNA copy numbers (p = 0.0023, r = 0.56) suggesting that the hepatic viral load influences IFNL4 transcription irrespective of IFNL4 governing genotype. Both, the IFNL4 creating allele ΔG (p<0.0001) and actual IFNL4 transcription (p = 0.0015) were found to be correlated to the activation of IFN stimulatory genes (ISGs). By contrast, IFNL4 ss469415590 genotypes were not found to be related to IFN-λ2/3/IL28 or IFN-λ1/IL29 gene expression. In conclusion, this study is the first report on intrahepatic transcript levels of the recently discovered IFNL4 gene. Data indicate that HCV infection in particular might activate IFNL4 transcription in the liver. It provides a possible explanation as to why hepatitis C patients show ISG stimulation in their livers in the apparent absence of an induction of other IFN subtypes.

Vitamin D status and serum ferritin concentration in chronic hepatitis C virus type 1 infection.

The circulating 25-hydroxylated form of vitamin D(3), 25(OH)D, and serum ferritin concentrations have been described to be associated with disease progression in chronic hepatitis C. Both parameters also have been assessed with regard to treatment outcome, however, with divergent results. This study examined both the pre- and posttreatment serum concentrations of 25(OH)D and ferritin in 191 patients infected chronically with hepatitis C virus (HCV) type 1 with regard to liver inflammatory activity (grading), disease progression in terms of fibrosis (staging) and an antiviral treatment outcome. Mean pretreatment serum 25(OH)D and ferritin concentrations were 18 ± 10 ng/ml and 280 ± 225 µg/L, respectively. Multivariate analysis revealed lower pretreatment serum 25(OH)D and higher ferritin concentrations to be significantly related to both severity of inflammatory activity and of fibrotic alterations. Pretreatment serum ferritin concentration, furthermore, unlike 25(OH)D concentration, was found to be associated with a sustained virological response by uni- and multivariate analyses. A sustained virological response was featured by a significant increase in serum 25(OH)D levels (18 ± 10 ng/ml vs. 22 ± 11 ng/ml; P < 0.01), a reduction of serum ferritin concentration (191 ± 156 µg/L vs. 103 ± 63 µg/L; P < 0.001) and a normalization of serum alanine aminotransferase (ALT) and γ-glutamyl-transferase (γ-GT) activities. Taken together, decreased 25(OH)D and increased ferritin serum levels indicate the severity of hepatic inflammation and fibrosis in patients infected chronically with HCV type 1. Elevated ferritin, furthermore, was found to be an independent predictor for standard IFN-based therapy responsiveness.

Ferritin L is the sole serum ferritin constituent and a positive hepatic acute-phase protein.

Ferritin L (FTL) and ferritin H (FTH) subunits are responsible for intracellular iron storage. Serum ferritin levels are not only dependant on body iron stores. Aims of the present study are to demonstrate nature, source, and major regulatory mediators of serum ferritin in an animal model of acute-phase (AP) response. Animals (rats, wild-type [WT] mice, and interleukin [IL]-6ko mice) were injected with turpentine oil (TO) intra-muscularity to induce a sterile abscess and sacrificed at different time points afterward. Rat hepatocytes were isolated for cell culture and, after reaching confluence, stimulated with major AP cytokines to induce AP conditions. We found a significantly increased expression of both ferritin subunits in liver at mRNA and protein levels during AP response. In the serum of both control and TO-injected rats, only FTL was detectable by Western blotting, whereas no increase in serum FTL was measured by Western blot or enzyme-linked immunosorbent assay. An increase in protein expression of FTL and FTH was observed in lysates of rat hepatocytes after treatment with IL-6, IL-1ß, and tumor necrosis factor-α; however, only FTL was increasingly released into supernatant. In both TO-injected rats and WT mice, a dramatic increase in serum IL-6 levels was observed, along with an increased amount of hepatic ferritin subunits. However, an increase of hepatic FTL but not of FTH protein expression was observed in IL-6ko mice after TO injection. Our data demonstrate that FTL is the only rat serum ferritin whose release into circulation from the hepatocytes is increased by the effect of AP cytokines (e.g., IL-6). In contrast, FTH expression is intracellular in both under physiological and AP conditions.

Early anemia and rapid virological response improve the predictive efficiency of IL28B-genotype for treatment outcome to antiviral combination therapy in patients infected with chronic HCV genotype 1.

IL28B genotypes and virological response within 4 weeks are predictors of sustained virological response in patients infected with chronic hepatitis C virus (HCV) genotype 1 treated with antiviral dual combination therapy. The predictive value of "early" anemia (within 4 weeks) alone or in combination with the two other predictors has not been studied yet. A total of 305 pegylated interferon-α and ribavirin-treated patients with HCV genotype 1 were included in this study. Hemoglobin values at week 0, 4, 8, and 12 as well as the predictive efficiency of early anemia (hemoglobin value below the gender-specific lower limit: female < 11.5; male < 13.5 g/dl) during therapy were assessed with IL28B genotypes and rapid virological response. Forty-eight percent of treated patients developed early anemia. In both females and males (64%), a decrease of hemoglobin concentration of 3 g/dl (female: 14.7 ± 1.1 to 11.4 ± 1.3; male: 15.2 ± 1.2 to 12.2 ± 1.5) significantly correlated with sustained virological response. 64% of IL28B-CC patients showed a sustained virological response. Seventy-eight percent of patients with rapid virological response definitively eliminated the virus. Early anemia (81:48:41%) and rapid virological response (83:91:92%) increased the predictive efficiency of IL28B rs12979860 genotype distribution (CC:CT:TT). IL28B-CC and early anemia as well as IL28B-CC and rapid virological response had an Odds ratio of 42.4 or 75 to achieve a sustained virological response compared to TT without early anemia or rapid virological response. This finding may help to early identify responders to standard PEG-IFN-α and ribavirin treatment even within those with unfavorable IL28B genotype.

Ultra-rapid virological response, young age, low γ-GT/ALT-ratio, and absence of steatosis identify a subgroup of HCV Genotype 3 patients who achieve SVR with IFN-α(2a) monotherapy.

BACKGROUND AND AIMS: The standard treatment regimen for chronic HCV genotype 3 (HCV-G3) hepatitis consists of PEGylated interferon-α (IFN-α) and ribavirin at varying doses ranging from 400 to 1,200 mg and results in response rates of 80%. However, this therapy has substantial side-effects including anemia, is teratogenic, and costly. To reduce the side-effects of therapy, the role of monotherapy consisting of only IFN-α was investigated. METHODS: A retrospective analysis of individual therapy courses of HCV-G3-infected patients who were treated with IFN-α(2a) monotherapy or a combination therapy with attention to the treatment outcome and the presence of IL28B rs12979860 and IL28B rs8099917 single-nucleotide polymorphism genotypes was performed. Conventional prognostic features in each case were assessed as well. RESULTS: In the study, 15/30 (50%) of patients treated with IFN-α(2a) monotherapy and 32/36 (89%) treated with combination therapy achieved a sustained virological response (SVR). In addition, 7/11 (64%) of those treated initially with monotherapy and subsequently with combination therapy achieved an SVR. An "ultra-rapid" virological response occurring within 2 weeks of initiation of therapy (p = 0.005), young age (<40; p < 0.001) and low initial γ-GT/ALT-ratio (p = 0.03) were associated with a SVR to IFN-α(2a) monotherapy. An SVR in those treated with combination therapy was found to be associated with a rapid virological response (RVR) (p = 0.03). The absence of histologic steatosis was associated with SVR in all patient groups (p = 0.01). Therapy duration (24 vs. 48 weeks) did not affect the SVR in either group. As expected, combination therapy resulted in more hematological side-effects than did monotherapy. CONCLUSIONS: An "ultra-rapid" virological response, young age, low initial γ-GT/ALT-ratio and absence of steatosis were each associated with an SVR in those receiving IFN-α(2a) monotherapy. Therefore, monotherapy in these patients should still be discussed independently of the existence of the IL28B polymorphisms.

Functional relevance of the IRF-1 promoter polymorphism rs2549009 on transcriptional activity in a native genomic environment.

Interferon regulatory factor-1 (IRF-1), a transcription regulator involved both in inducing and in mediating the effects of interferon, is encoded by a highly polymorphic gene in different ethnic populations. Some of these genetic variations have been described to be associated to disease traits in hepatitis C virus and in human immunodeficiency virus infection, including one single-nucleotide polymorphism rs2549009 within the promoter region. This study aimed at investigating the functional relevance of rs2549009 on IRF-1 transcriptional activity in peripheral blood mononuclear cells in its natural genomic environment. Haplotype-specific chromatin immunoprecipitation using antibodies directed against both the transcriptionally inactive and active RNA polymerase II (RNAPII) and allele-specific transcript quantification techniques were applied to ex vivo-derived samples from healthy heterozygous donors. Inactive serine 5 phosphorylated RNAPII was found to be preferentially bound to the rs2549009 A allele in all donors investigated. Active serine 2 phosphorylated (ser2-P) RNAPII, in contrast, was found to be precipitable, depending on the donor, preferentially either with the A or the G promoter variants or without any preference. The ratio of rs2549009 A/G promoter variants engaged by ser2-P RNAPII was closely related to the relative frequency of the respective IRF-1 transcripts, and relative allelic expression was found to be associated to total IRF-1 gene expression. These results provide evidence for a bidirectional IRF-1 gene expression imbalance that appears not to be solely controlled by rs2549009 in cis and may rely on a yet unidentified variant or haplotype or on environmental control in trans.

Toll-like receptor 7 rs179008/Gln11Leu gene variants in chronic hepatitis C virus infection.

Hepatitis C virus (HCV) infection affects an estimated 3% of the world's population. The natural outcome of infection and the natural course of disease are highly variable. Sensing of viral single-stranded RNA (ssRNA) by Toll-like receptor 7 (TLR7) is likely involved in early pathogen detection and host response to viral infections. This study analyzed epidemiological and clinical data from 136 patients with HCV infection with regard to rs179008/Gln11Leu, a non-synonymous polymorphism within exon 3 of the X-linked TLR7 gene, the variant allele of which is suggested to code for a functionally impaired protein. Allele-specific transcript quantification (ASTQ) analyses in heterozygous females revealed individual skewed mosaicism in peripheral blood mononuclear cells (PBMCs). Thus, analyses were restricted to homo- and hemizygous individuals. Among the clinical and histological parameters studied, the variant allele T was found to be solely associated with the presence of portal lymphoid aggregates. Whereas hepatic viral load and expression of genes known to be induced in chronic HCV infection were not found to differ in patients with wild-type or variant TLR7 rs179008 genotype, significant lower gene expression of interleukin-29 (IL-29)/lambda(1) interferon (IFN-λ(1)) and both of its receptor subunits was found for T homo- and hemizygous patients. Irrespective of the minor differences in disease phenotype including hepatic viral load, natural, and alpha interferon (IFN-α)-mediated outcome of infection, and disease activity and progression, the significant differences in hepatic IL-29/IFN-λ(1) and IFN-λ receptor gene expression between TLR7 rs179008 T and A allele patients might have implications for responsiveness to future IFN-λ-based approaches.