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Our aim was to assess whether, in the United States, with the predominant hepatitis C viral (HCV) genotypes 1a/I and 1b/II, hepatic interferon-alpha receptor (IFNAR) mRNA expression correlated with response to IFN therapy, levels of HCV RNA, or histologic activity index (HAI). Nine of 24 patients (38%) had an initial response to IFN treatment, 5 of whom (21%) had a sustained response. The corrected hepatic IFNAR mRNA expression (measured by RT-PCR) for the sustained responder group (mean +/- SE, 0.16 +/- 0.06, n = 5) was significantly higher than for the nonresponding group (0.059 +/- 0.01, n = 15) (p0.02). Patients who relapsed had an intermediate value (0.092 +/- 0.029, n = 4). Higher IFNAR expression was inversely correlated with a lower serum HCV RNA titer (p0.01), and responders to IFN treatment tended to have a lower titer of HCV RNA (p = 0.056). We found no significant correlation between the amounts of IFNAR with (1) the total HAI (low HAIor = 7, IFNAR 0.076 +/- 0.013, n = 10; high HAIor = 8, IFNAR 0.092 +/- 0.027, n = 14, ns) or (2) individual inflammation, necrosis, or fibrosis components of the HAI. As with Japanese HCV patients with genotypes 1b/II-2b/IV, higher hepatic IFNAR mRNA expression in the United States with predominant genotypes 1a/I and 1b/II appears to correlate with response to IFN therapy and a low HCV RNA titer but not with the total HAI or its components.

Heme oxygenase (HO) is responsible for the physiological breakdown of heme into equimolar amounts of biliverdin, carbon monoxide, and iron. Three isoforms (HO-1, HO-2, and HO-3) have been identified. HO-1 is ubiquitous and its mRNA and activity can be increased several-fold by heme, other metalloporphyrins, transition metals, and stimuli that induce cellular stress. HO-1 is recognized as a major heat shock/stress response protein. Recent work from our laboratory has demonstrated several potential consensus regulatory elements in the 5'-untranslated region (UTR) of HO-1, including activator protein 1 (AP-1), metal responsive element (MRE), oncogene c-myc/max heterodimer binding site (Myc/Max), antioxidant response element (ARE), and GC box binding (Sp1) sites. Using deletion-reporter gene constructs, we have mapped sites that mediate the arsenite-dependent induction of HO-1, and we have shown that components of the extracellular signal-regulated kinase (ERK) and p38 (a homologue of the yeast HOG1 kinase), but not c-jun N-terminal kinase (JNK), mitogen-activated protein (MAP) kinase pathways are involved in arsenite-dependent upregulation. In contrast, HO-2 is present chiefly in the brain and testes and is virtually uninducible. HO-3 has very low activity; its physiological function probably involves heme binding. Products of the HO reaction have important effects: carbon monoxide is a potent vasodilator, which is thought to play a key role in the modulation of vascular tone, especially in the liver under physiological conditions, and in many organs under "stressful" conditions associated with HO-1 induction. Biliverdin and its product bilirubin, formed in most mammals, are potent antioxidants. In contrast, "free" iron increases oxidative stress and regulates the expression of many mRNAs (e.g., DCT-1, ferritin, and transferrin receptor) by affecting the conformation of iron regulatory protein (IRP)-1 and its binding to iron regulatory elements (IREs) in the 5'- or 3'-UTRs of the mRNAs.

Non-alcoholic steatohepatitis (NASH) is increasingly recognized, and its pathogenesis is believed to involve increased oxidative stress. Elevated levels of serum ferritin and positive liver iron stains are often observed in patients with NASH, and the pathogenesis of liver injury due to iron is also thought to involve oxidative stress. The aim of this study was to determine whether there is an association of NASH and mutations in the HFE gene associated with hereditary hemochromatosis (HHC).Clinical, laboratory, and histopathological data on all 57 subjects with a final diagnosis of NASH seen between August 1990 and August 1997 at our Liver Center were analyzed. Thirty-six Caucasian subjects (23 men) with NASH underwent mutational analyses of HFE gene mutations performed. The prevalence of HFE gene mutations was compared to that in 348 Caucasian normal controls. Data were analyzed by both parameteric and non-parametric methods with similar results.One subject (2.8%) with NASH was homozygous for the C282Y mutation and six (16.7%) were heterozygous, compared with 0%, and 11.2%, respectively, of controls. Two (5.6%) subjects with NASH were homozygous for the H63D mutation and 16 (44.4%) were heterozygous, whereas 2.9% and 26.4%, respectively, of controls had these genotypes. The prevalence of heterozygosity (61.1%) for either mutation was significantly higher in subjects with NASH than in controls (38%) (p = 0.008), and the prevalence of homozygosity or heterozygosity combined in NASH subjects (69.4%) was significantly higher than for controls (40.5%, p = 0.001). Sex (63-67% male) and age at diagnosis of NASH did not differ between those with or without HFE mutations, but men with NASH were significantly more likely than women to have the H63D mutation (15/23 vs. 3/13, p0.05) Levels of serum ferritin, iron, transferrin saturation levels, and the degree of hepatic iron staining were significantly higher (p0.05) in subjects with NASH who carried an HFE mutation than in those without. Differences in hepatic iron concentrations or hepatic iron indices between NASH subjects with and without HFE mutations were not significant. Those with C282Y mutations had significantly more hepatic fibrosis than those without (p0.05). Those with HFE mutations had significantly higher levels of serum ALT (90+/-11 [mean +/- SE]) than those without (55+/-6; p = 0.02).The prevalences of the HFE gene mutations associated with hereditary hemochromatosis are increased among North American subjects with NASH.

When patients with acute porphyrias are treated with antihypertensives and analgesics, they could be placed at increased risk of developing porphyric attacks, since little is known about the potential for many of these drugs to induce these attacks. We used primary chick embryo liver cells, which maintain intact heme synthesis and regulation, to study the effects of antihypertensives and analgesics on porphyrin accumulation. Cells were treated with desferrioxamine to block heme synthesis partially, simulating conditions encountered in porphyric patients. Typically, cells were treated for 20 hr with the test drugs (3.16 to 1000 μM), along with desferrioxamine. Porphyrins were measured spectrofluorometrically, as uro-, copro,- and protoporphyrin. The evaluated drugs included six antihypertensives (two calcium channel blockers, an angiotensin receptor antagonist, and three inhibitors of angiotensin converting enzyme) and eight analgesics. Of the calcium channel blockers tested, nifedipine greatly increased porphyrin accumulation, whereas diltiazem caused only a slight increase. Losartan (an angiotensin receptor antagonist), captopril, or lisinopril (two angiotensin converting enzyme inhibitors) produced only small increases in porphyrin accumulation. In contrast, enalapril (another angiotensin converting enzyme inhibitor) substantially increased porphyrin accumulation when given in high concentrations. Among the analgesics tested, fentanyl and tramadol produced the highest porphyrin accumulations. Nalbuphine, hydrocodone, oxycodone, and dezocine were moderately or weakly porphyrogenic, whereas buprenorphine and morphine did not increase porphyrin accumulation. These studies suggest that patients with acute porphyrias may be at greater risk for developing porphyric attacks when treated with nifedipine (compared with diltiazem), enalapril (compared with captopril or lisinopril), and tramadol (compared with the other analgesics).

Patients with chronic hepatitis C who have not had a sustained hepatitis C virus (HCV)-RNA response or serum alanine transaminase (ALT) response to a 6-month course of interferon (IFN) may respond to higher dose retreatment with consensus interferon (CIFN). Some nonresponders to initial IFN treatment have a transient response defined as undetectable HCV RNA or normalization of ALT during treatment, but subsequently have a "breakthrough" while still on treatment. The aim of this study was to determine if nonresponders who had breakthroughs responded differently to CIFN retreatment than nonresponders without breakthroughs using data from a large, multicenter trial. ALT and HCV RNA were monitored frequently during initial IFN therapy (either 9 mcg CIFN or 3 MU IFN-alpha2b 3 times per week). HCV-RNA breakthroughs were observed in 86 of 467 (18%) of all treated patients, and ALT breakthroughs were observed in 90 of 467 (19%) of all treated patients. There was no association between breakthroughs and the presence of either binding or neutralizing anti-IFN antibodies. When the patients who were nonresponders to initial IFN treatment were retreated with CIFN (15 mcg) for 12 months, 27% of those with viral breakthroughs had a sustained viral response compared with 8% in prior nonresponders without breakthroughs (P =.102). Sustained ALT responses were observed in 39% with breakthroughs compared with 10% in those without breakthroughs (P =.014). The data suggest that prior nonresponders with breakthroughs have a greater chance of responding to retreatment than do nonresponders without breakthroughs. However, most breakthrough patients would be missed unless repeated HCV-RNA testing were conducted during therapy.

To identify a magnetic resonance (MR) imaging method sufficiently sensitive and specific in the estimation of hepatic iron content to obviate liver biopsy.Thirty-eight patients underwent percutaneous needle biopsy of the liver with chemical measurement of the hepatic iron concentration and hepatic MR imaging with several spin-echo and gradient-recalled-echo (GRE) techniques. Correlations between MR imaging parameters and the hepatic iron concentration were determined.Inverse curvilinear relationships were noted between several MR parameters and hepatic iron concentrations. GRE sequences with short repetition and echo times were more accurate and precise than spin-echo sequences for the estimation of hepatic iron concentration. A GRE sequence with a repetition time of 18 msec, an echo time of 5 msec, and a flip angle of 10 degrees showed close correlation between the hepatic iron concentration and the natural logarithm of the ratio of the signal intensity of liver to the SD of background noise (r = -0.94) and low coefficient of variation (12%).MR imaging with these parameters is a rapid, noninvasive, and accurate modality for estimation of hepatic iron concentration; it is sufficiently accurate and precise to obviate liver biopsy for the purpose of measuring hepatic iron concentration.

Heme oxygenase (HO) catalyzes the conversion of heme into biliverdin, iron, and carbon monoxide (CO). Two isoforms of HO have been identified: the inducible HO-1 and the constitutive HO-2. CO, like nitric oxide, is an endogenous vasodilator that could contribute to modulation of systemic and local vascular tone. The aim of the present study was to determine the expression of HO isoforms in liver cells and splanchnic organs from portal hypertensive (PH) and sham-operated (SO) rats. Liver cells (hepatocytes, Kupffer and stellate cells), and splanchnic organs (liver, mesentery, intestine, colon, and spleen) were isolated from PH and SO rats. Expression of HO mRNA and protein was assessed by reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. In SO rats, HO-1 mRNA expression was only detected in spleen. In contrast, in PH rats, HO-1 mRNA was expressed in hepatocytes, Kupffer cells, and in all the splanchnic organs studied. Moreover, levels of HO-1 protein in splanchnic organs were significantly higher in PH rats than in SO animals. In addition, HO-2 expression was observed in all liver cell types and splanchnic organs studied from both PH and SO rats. These results indicate that HO-2 is expressed in parenchymal and nonparenchymal liver cells, as well as splanchnic organs, of both PH and SO rats. In addition, HO-1 is up-regulated in hepatocytes and splanchnic organs of PH rats, compared with SO animals, suggesting a possible pathophysiological role of HO-1 in chronic portal hypertension.

Erythropoietic protoporphyria (EPP) is marked by a deficiency of ferrochelatase, which occurs in all cells and tissues, preventing effective conversion of proto porphyrin IX to heme and thereby blocking effective feedback inhibition of heme synthesis. The major source of the excess protoporphyrin is the bone marrow. Protoporphyrin IX may accumulate, with resultant toxicity chiefly of the marrow, skin, nervous system, and liver. Orthotopic liver transplantation (OLT) is, at present, the only adequate intervention for severe liver compromise secondary to protoporphyrin deposition, but it has been complicated by severe photosensitivity and polyneuropathy. Intravenous heme and plasmapheresis have been proposed but not previously reported as means to reduce the protoporphyrin burden before liver transplantation. We report a man with EPP who underwent preoperative heme-albumin administration and plasmaphereses that led to marked reductions in plasma and erythrocyte protoporphyrin levels. His OLT was uneventful, and he developed neither polyneuropathy nor exacerbation of photosensitivity.

Heavy iron overload, as occurs in primary and secondary hemochromatosis, may cause fibrosis of parenchymal organs, including the heart, liver, and pancreas, and it is a risk factor for the development of hepatocellular carcinoma. Recent evidence indicates that lesser degrees of hepatic iron deposition are also risk factors for nonhemochromatotic liver disease. For example, several recent studies showed extraordinarily high prevalences (about 60% to 75%) of HFE mutations in patients with porphyria cutanea tarda and significantly increased prevalences of these mutations in patients with nonalcoholic steatohepatitis from Australia and the United States. It is less well established that the prevalence of the HFE mutations is increased in alcoholic liver disease and in chronic viral hepatitis, but in both conditions, patients harboring one of these mutations, especially C282Y, are more likely to have advanced hepatic fibrosis or cirrhosis. Thus, these mutations both incite and exacerbate nonhemochromatotic liver disease. In this review, we summarize current knowledge of these associations and emphasize important unresolved questions that require further study.

The natural history, prognosis, and clinical significance of chronic hepatitis C are highly variable and somewhat controversial. The purpose of this study was to evaluate the effect of chronic hepatitis C infection on patients' perceptions of health-related quality of life (HRQOL) and to evaluate whether treatment with interferon improves HRQOL. A total of 642 patients with compensated liver disease who were enrolled in a multicenter trial of interferon therapy for chronic hepatitis C had evaluation of HRQOL using the SF-36 and other instruments derived from the Medical Outcomes Study (MOS). These instruments were self-administered by patients at baseline and at the end of a 24-week post-treatment observation period after 24 weeks of interferon treatment. Patients with chronic hepatitis C were compared with healthy controls (n = 750) selected from a representative sample of adults in the United States. Unadjusted and age/gender-adjusted results were similar, as were analyses using parametric or nonparametric methods. Compared with healthy controls, patients with chronic hepatitis C at baseline had lower HRQOL on all eight scales of the SF-36 (P

Cytotoxic T lymphocytes (CTL) capable of recognizing prototype hepatitis C virus (HCV) sequences have been shown to localize to the liver in chronically infected individuals, where they are thought to influence hepatic inflammation and viral replication. We isolated three intrahepatic CD8 + CTL clones from two individuals with chronic HCV infection and compared the recognition of prototype and autologous HCV sequences. These CTL recognized epitopes within the NS2 (amino acids 957–964) or NS3 (amino acids 1402–1410 and 1406–1415) proteins in the context of HLA B37, B8, or A2.1, respectively. The corresponding predominant autologous HCV sequences (SDWAANGL, ELAAKLVGL, and ALRGMGVNAV, respectively) differed from the HCV-1 sequences used for screening (RDWAHNGL, ELAAKLVAL, and KLVALGINAV, respectively) at one to five residues. For each CTL clone, recognition of the autologous HCV sequence required significantly higher peptide concentrations than did recognition of the HCV-1 sequence; for two of the clones, recognition was minimal or absent at peptide concentrations as high as 25 μM. These data show that intrahepatic HCV-specific CD8 + CTL clones can be relatively inefficient at recognizing autologous viral epitopes. Inefficient recognition of autologous HCV sequences should influence the interpretation of data generated using prototype HCV sequences and might have implications in vivo .

Heme oxygenase-1 is an inducible enzyme that catalyzes heme degradation and has been proposed to play a role in protecting cells against oxidative stress-related injury. We investigated the induction of heme oxygenase-1 by the tumor promoter arsenite in a chicken hepatoma cell line, LMH. We identified a heme oxygenase-1 promoter-driven luciferase reporter construct that was highly and reproducibly expressed in response to sodium arsenite treatment. This construct was used to investigate the role of mitogen-activated protein (MAP) kinases in arsenite-mediated heme oxygenase-1 gene expression. In LMH cells, sodium arsenite, cadmium, and heat shock, but not heme, induced activity of the MAP kinases extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. To examine whether these MAP kinases were involved in mediating heme oxygenase-1 gene expression, we utilized constitutively activated and dominant negative components of the ERK, JNK, and p38 MAP kinase signaling pathways. Involvement of an AP-1 site in arsenite induction of heme oxygenase-1 gene expression was studied. We conclude that the MAP kinases ERK and p38 are involved in the induction of heme oxygenase-1, and that at least one AP-1 element (located −1576 base pairs upstream of the transcription start site) is involved in this response.

To test the hypothesis that inflammation in hepatitis C follows mechanisms common to immune-activated pathways, the distributions of T and B cells, adhesion molecules and transforming growth factor-beta (TGF-beta) were assessed in liver biopsies with chronic inflammation due to hepatitis C (HCV, n = 8) and other causes (non-HCV, n = 10). Frozen sections were immunostained using primary antibodies to CD2, CD20, CD4, CD8, intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM)-1, HLA-DR, lymphocyte function-associated antigen (LFA)-1, and TGF-beta. Inflammatory cells positive for each immunophenotypic marker were counted, and positive staining for adhesion molecules, HLA-DR and TGF beta was graded in triads and lobules and compared in HCV and non-HCV biopsies. In all biopsies, T cells were more frequent than B cells, both in triads and lobules. CD20+, CD4+, CD8+ and LFA-1+ cells were increased in HCV compared to non-HCV biopsies. Portal lymphoid aggregates were present in 6 of 8 HCV biopsies and 3 of 10 non-HCV biopsies. Aggregates consisted of CD20+, CD4+, CD8+ and LFA-1+ cells, and ICAM-1 and VCAM-1 were increased. Sinusoidal lining cells in HCV biopsies and non-HCV biopsies with inflammation expressed HLA-DR, ICAM-1, and CD4. TGF-beta was increased in foci of necrosis. Inflammation in chronic HCV involves common immune-mediated cellular effector pathways and the inflammation in the portal triads represents aggregation of both T and B cells, mediated in part by upregulation of adhesion molecules on portal stromal cells; this is possibly in response to antigens draining from necroinflammatory foci in the lobules. TGF-beta is increased in active necroinflammatory foci, but not in portal lymphoid aggregates.

Some patients with acute hereditary porphyrias have seizures and require anticonvulsant therapy, but many anticonvulsants induce exacerbations of the hepatic porphyrias. Recently, several new anticonvulsants have become available. Among these are gabapentin, vigabatrin, felbamate, lamotrigine, and tiagabine. Little is known about their potential for induction of porphyric attacks. We used a cell culture model of primary chicken embryo liver cells, which maintain intact heme synthesis and regulation, to study the effects of these new anticonvulsants on porphyrin accumulation. Treatment of the cells with deferoxamine (250 microM) led to a partial block in heme synthesis, simulating the conditions encountered in human beings with porphyria. Concomitant exposure of these cells to phenobarbital (2 mM) strongly induced accumulation of porphyrins, serving as a positive control in this model. Cells were treated for 20 hours with increasing doses (3.2 to 1,000 microM) of the newer anticonvulsants, with or without deferoxamine. For most of these anticonvulsants 5 to 100 microM is representative of the concentrations achieved in humans with therapeutic doses. Porphyrins were measured spectrofluorometrically as uro-, copro-, and protoporphyrins. Results were confirmed by high-pressure liquid chromatography. Neither vigabatrin nor gabapentin treatment, with or without deferoxamine, led to any increase in porphyrin accumulation. Similar doses of felbamate (with deferoxamine) led to a marked increase in (mainly proto-) porphyrin levels, qualitatively and quantitatively almost identical to the accumulation produced by phenobarbital. Lamotrigine or tiagabine (with deferoxamine) caused similar porphyrin accumulation. Tiagabine treatment up to 100 microM (with deferoxamine) also resulted in very high levels of predominantly proto-porphyrin. In contrast to the other anticonvulsants tested, tiagabine without deferoxamine led to mild porphyrin accumulation. In the presence of deferoxamine, phenobarbital, felbamate, lamotrigine, or tiagabine, but not gabapentin or vigabatrin, increased levels of the mRNA of ALA synthase, the first and rate-controlling enzyme of porphyrin synthesis. Such enzyme induction is a sine qua non for acute porphyric attacks. We conclude that neither vigabatrin nor gabapentin is porphyrogenic, whereas felbamate, lamotrigine, and, especially, tiagabine lead to much accumulation of porphyrins. The latter three anticonvulsants, therefore, may precipitate or exacerbate acute porphyric attacks in humans. We recommend use of vigabatrin or gabapentin, but not felbamate, lamotrigine, or tiagabine, in patients with acute porphyria and seizures.

Induction of heme oxygenase (HO) has been proposed as a protective cellular mechanism against oxidative damage. In previous work (Tyrrell et al., Carcinogenesis [1993] 14, 761-765), portions of the 5' promoter region of the human HO-1 gene linked to the reporter gene chloramphenicol acetyl transferase (CAT), had been transiently expressed in HeLa cells. To extend the study of human HO gene expression into primary liver cells, these reporter gene fusion constructs, containing 121 or 1416 base pairs of the untranscribed 5'-upstream sequences of the human HO-1 gene, were used along with pSV beta-Gal plasmid to dually transfect primary cultures of chick embryo liver cells (CELC). The transfected cells were treated with selected metals, heme, phorbol ester, and chemical agents that produce oxidative stress (H2O2 or sodium arsenite). Reporter gene activities were measured 18-20 h later. Our major findings are: (1) these HO-CAT constructs were expressed in CELC; (2) unlike HeLa cells, the expression of CAT was detected in CELC without the need for the SV40 enhancer; (3) sodium arsenite and cobalt chloride induced the expression of the HO-CAT constructs whereas heme had no effect on or decreased CAT expression for all of the transfected constructs; (4) study of endogenous chick HO-1 gene expression in CELC showed that HO-1 responded to sodium arsenite treatment in a dose-dependent fashion, and the response was rapid and transient. We conclude that, in chick liver cell cultures, induction of the HO-1 gene by heme is fundamentally different from that produced by transition metals or sodium arsenite. Furthermore, the results suggest that expression of the HO-1 gene is highly conserved across species.

Heme oxygenase catalyzes the first and rate-controlling step in heme catabolism. One of the two forms of heme oxygenase (heme oxygenase-1) has been shown to be increased by heme, metals, and in some systems, by certain environmental stresses. However, it remains uncertain whether heme induces hepatic heme oxygenase-1 by a general stress response, or a specific heme-dependent cellular response. The work communicated here explores this issue by examining possible mechanisms whereby heme and other metalloporphyrins induce heme oxygenase-1 in normal liver cells. Primary cultures of chick embryo liver cells were tested for their ability to increase heme oxygenase mRNA after exposure to selected metalloporphyrins (heme, chromium mesoporphyrin, cobalt protoporphyrin and manganese protoporphyrin). The ability of antioxidants to decrease metalloporphyrin-mediated induction of heme oxygenase-1 mRNA was also tested. Our results indicate that: 1) the increase in heme oxygenase-1 mRNA mediated by heme or other metalloporphyrins may involve a short-lived protein(s) since the increase was prevented by several inhibitors of protein synthesis; and 2) in normal liver cells, heme-dependent oxidative stress does not play a key role in the heme-mediated induction of heme oxygenase-1. We conclude that heme and other non-heme metalloporphyrins induce heme oxygenase-1 through a mechanism requiring protein synthesis, not because metalloporphyrins increase cellular oxidative or other stress.

In this study, the frequencies of the common hemochromatosis gene mutations were assessed in 75 Iranian subjects with chronic hepatitis B infection. We found that the major C282Y mutation was significantly more frequent in subjects infected with hepatitis B virus (4%) than in 194 control subjects (0%, ; Fisher’s exact test). P p .02 Hepatitis B virus (HBV) infection can cause a broad spectrum of disease, ranging from asymptomatic inactive HBV carriage to acute hepatitis, chronic hepatitis, cirrhosis, and primary hepatocellular carcinoma. The reasons for this variability in the pattern and clinical outcome of HBV infection are still poorly understood. The role of iron in modulating the course of viral hepatitis was first highlighted by Blumberg et al. [1]. They found that patients with higher levels of serum iron or ferritin were less likely to achieve spontaneous recovery after acute HBV infection. Elevations in serum ferritin level and serum iron saturation percentage were also noted in patients with chronic hepatitis C [2‐4]. Recent studies have indicated that, among patients with chronic hepatitis C, there is an increased prevalence of the C2824 mutation, the major mutation of the hemochromatosis gene (HFE) associated with hereditary hemo

Hepatitis C virus (HCV) infection is one of main causes of hepatocellular carcinoma (HCC) and the prevalence of HCV-associated HCC is on the rise worldwide. It is particularly important and helpful to identify potential markers for screening and early diagnosis of HCC among high-risk individuals with chronic hepatitis C, and to identify target molecules for the prevention and treatment of HCV-associated-HCC. Small non-coding RNAs, mainly microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) with size greater than 200 nucleotides, are likely to play important roles in a variety of biological processes, including development and progression of HCC. For the most part their underlying mechanisms of action remain largely unknown. In recent years, with the advance of high-resolution of microarray and application of next generation sequencing techniques, a significant number of non-coding RNAs (ncRNAs) associated with HCC, particularly caused by HCV infection, have been found to be differentially expressed and to be involved in pathogenesis of HCV-associated HCC. In this review, we focus on recent studies of ncRNAs, especially miRNAs and lncRNAs related to HCV-induced HCC. We summarize those ncRNAs aberrantly expressed in HCV-associated HCC and highlight the potential uses of ncRNAs in early detection, diagnosis and therapy of HCV-associated HCC. We also discuss the limitations of recent studies, and suggest future directions for research in the field. miRNAs, lncRNAs and their target genes may represent new candidate molecules for the prevention, diagnosis and treatment of HCC in patients with HCV infection. Studies of the potential uses of miRNAs and lncRNAs as diagnostic tools or therapies are still in their infancy.

Porphyrins and metalloporphyrins are the key pigments of life on earth as we know it, because they include chlorophyll (a magnesium-containing metalloporphyrin) and heme (iron protoporphyrin). In eukaryotes, porphyrins and heme are synthesized by a multistep pathway that involves eight enzymes. The first and rate-controlling step is the formation of delta-aminolevulinic acid (ALA) from glycine plus succinyl CoA, catalyzed by ALA synthase. Intermediate steps occur in the cytoplasm, with formation of the monopyrrole porphobilinogen and the tetrapyrroles hydroxymethylbilane and a series of porphyrinogens, which are serially decarboxylated. Heme is utilized chiefly for the formation of hemoglobin in erythrocytes, myoglobin in muscle cells, cytochromes P-450 and mitochondrial cytochromes, and other hemoproteins in hepatocytes. The rate-controlling step of heme breakdown is catalyzed by heme oxygenase (HMOX), of which there are two isoforms, called HMOX1 and HMOX2. HMOX breaks down heme to form biliverdin, carbon monoxide, and iron. The porphyrias are a group of disorders, mainly inherited, in which there are defects in normal porphyrin and heme synthesis. The cardinal clinical features are cutaneous (due to the skin-damaging effects of excess deposited porphyrins) or neurovisceral attacks of pain, sometimes with weakness, delirium, seizures, and the like (probably due mainly to neurotoxic effects of ALA). The treatment of choice for the acute hepatic porphyrias is intravenous heme therapy, which repletes a critical regulatory heme pool in hepatocytes and leads to downregulation of hepatic ALA synthase, which is a biochemical hallmark of all forms of acute porphyria in relapse.

Acute intermittent porphyria (AIP) is an inherited disorder of haem synthesis wherein a partial deficiency of porphobilinogen (PBG) deaminase (PBGD) with other factors may give rise to biochemical and clinical manifestations of disease. The biochemical hallmarks of active AIP are relative hepatic haem deficiency and uncontrolled up-regulation of hepatic 5-aminolevulinic acid (ALA) synthase-1 (ALAS1) with over-production of ALA and PBG. The treatment of choice is intravenous haem, which restores the deficient regulatory haem pool of the liver and represses ALAS1. Recently, haem has been shown to influence circadian rhythms by controlling their negative feedback loops. We evaluated whether subjects with AIP exhibited an altered circadian profile.Over a 21-h period, we measured levels of serum cortisol, melatonin, ALA, PBG and mRNA levels (in peripheral blood mononuclear cells) of selected clock-controlled genes and genes involved in haem synthesis in 10 Caucasian (European-American) women who were either postmenopausal or had been receiving female hormone therapy, six of whom have AIP and four do not and are considered controls.Four AIP subjects with biochemical activity exhibited higher levels of PBG and lower levels and dampened oscillation of serum cortisol, and a trend for lower levels of serum melatonin, than controls or AIP subjects without biochemical activity. Levels of clock-controlled gene mRNAs showed significant increases over baseline in all subjects at 5 a.m. and 11 p.m., whereas mRNA levels of ALAS1, ALAS2 and PBGD were increased only at 11 p.m. in subjects with active AIP.This pilot study provides evidence for disturbances of circadian markers in women with active AIP that may trigger or sustain some common clinical features of AIP.

Many herbal dietary supplements (HDS) contain green tea extract (GTE) and its component catechins, although their presence may not always be indicated on the product label. Because GTE and catechins have been implicated in human hepatotoxicity in several case reports, our objective was to determine whether catechins were present in HDS that were implicated in hepatotoxicity, even if not identified among the labeled ingredients, and whether these compounds could be associated with liver injury. We assayed 97 HDS implicated in human hepatotoxicity for catechins. We found that 29 of 73 HDS (39.7 %) that did not identify GTE or any of its component catechins on their label contained catechins. Among patients with confirmed hepatotoxicity, there was no statistically significant association between the presence of catechin or the dose consumed and liver injury causality score, severity, or pattern of liver injury. Catechin levels tended to be highest in products used for weight loss, although catechin concentrations were low in most products. Many HDS commonly contain catechins that are implicated in hepatotoxicity, although their presence may not be indicated on the product label. Although our results did not establish an association between GTE or catechins with hepatotoxicity, they highlight some of the many complexities and uncertainties that surround the attribution of drug-induced liver injury (DILI) to HDS.

Post-transplant hepatitis C is a major challenge after liver transplantation (LT). Antiviral therapy is associated with lower efficacy in the post-transplant setting.The purpose of this study was to determine the safety and effect of intravenous interferon (IFN) during the anhepatic phase of LT on hepatitis C viral load.Fifteen consecutive subjects undergoing liver transplant for hepatitis C cirrhosis were enrolled in the study, ten of which received study drug and five subjects served as controls. Cases received weight-based ribavirin and subcutaneous IFN at time of incision followed by intravenous IFN at the start of the anhepatic phase. Adverse events and viral levels were recorded. Repeated measures ANOVA was employed to test for differences over time, between the groups, and time by group interaction.All subjects had genotype 1 virus. Hepatitis C viral load was lower at week 4 in cases compared to controls (769,004 ± 924,082 IU/ml and 2,329,896 ± 3,731,749 IU/ml, respectively), but did not reach statistical significance (p = 0.50). Three subjects developed adverse events related to IFN including pulmonary edema, rejection, and neutropenia.Intravenous IFN administered during the anhepatic phase of liver transplant did not prevent graft reinfection and was associated with manageable adverse events. This regimen could be further studied if direct acting antiviral agents alone are insufficient for treating post-transplant hepatitis C.

AIM: To assess effects of heme on messenger RNA (mRNA) and microRNA (miRNA) profiles of liver cells derived from humans. METHODS: We exposed human hepatoma cell line Huh-7 cells to excess iron protoporphyrin (heme) (10 μmol/L) or induced heme deficiency by addition of 4, 6-dioxoheptanoic acid (500 μmol/L), a potent inhibitor of aminolevulinic acid dehydratase, for 6 h or 24 h. We harvested total RNA from the cells and performed both mRNA and miRNA array analyses, with use of Affymetrix chips, reagents, and instruments (human genome U133 plus 2.0 and miRNA 2.0 arrays). We assessed changes and their significance and interrelationships with Target Scan, Pathway Studios, and Ingenuity software. RESULTS: Changes in mRNA levels were most numerous and striking at 6 h after heme treatment but were similar and still numerous at 24 h. After 6 h of heme exposure, the increase in heme oxygenase 1 gene expression was 60-fold by mRNA and 88-fold by quantitative reverse transcription-polymerase chain reaction. We found striking changes, especially up-regulation by heme of nuclear erythroid-2 related factor-mediated oxidative stress responses, protein ubiquitination, glucocorticoid signaling, P53 signaling, and changes in RNAs that regulate intermediary metabolism. Fewer mRNAs were down-regulated by heme, and the fold decreases were less exuberant than were the increases. Notable decreases after 24 h of heme exposure were patatin-like phospholipase domain-containing protein 3 (-6.5-fold), neuronal PAS domain protein 2 (-1.93-fold), and protoporphyrinogen oxidase (-1.7-fold). CONCLUSION: Heme excess exhibits several toxic effects on liver and kidney, which deserve study in humans and in animal models of the human porphyrias or other disorders.

Hepatitis C virus (HCV) infection, which results in chronic hepatitis C (CHC) in most patients (70-85%), is a major cause of liver disease and remains a major therapeutic challenge. The mechanisms determining liver damage and the key factors that lead to a high rate of CHC remain imperfectly understood. The precise role of cytoskeletal (CS) proteins in HCV infection remains to be determined. Some studies including our recent study have demonstrated that changes occur in the expression of CS proteins in HCV-infected hepatocytes. A variety of host proteins interact with HCV proteins. Association between CS and HCV proteins may have implications in future design of CS protein-targeted therapy for the treatment for HCV infection. This chapter will focus on the interaction between host CS and viral proteins to signify the importance of this event in HCV entry, replication and transportation.

Erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are inborn errors of heme biosynthesis with the same phenotype but resulting from autosomal recessive loss-of-function mutations in the ferrochelatase (FECH) gene and gain-of-function mutations in the X-linked erythroid-specific 5-aminolevulinate synthase (ALAS2) gene, respectively. The EPP phenotype is characterized by acute, painful, cutaneous photosensitivity and elevated erythrocyte protoporphyrin levels. We report the FECH and ALAS2 mutations in 155 unrelated North American patients with the EPP phenotype. FECH sequencing and dosage analyses identified 140 patients with EPP: 134 with one loss-of-function allele and the common IVS3-48T>C low expression allele, three with two loss-of-function mutations and three with one loss-of-function mutation and two low expression alleles. There were 48 previously reported and 23 novel FECH mutations. The remaining 15 probands had ALAS2 gain-of-function mutations causing XLP: 13 with the previously reported deletion, c.1706_1709delAGTG, and two with novel mutations, c.1734delG and c.1642C>T(p.Q548X). Notably, XLP represented ~10% of EPP phenotype patients in North America, two to five times more than in Western Europe. XLP males had twofold higher erythrocyte protoporphyrin levels than EPP patients, predisposing to more severe photosensitivity and liver disease. Identification of XLP patients permits accurate diagnosis and counseling of at-risk relatives and asymptomatic heterozygotes.

Drug-induced liver injury (DILI) is the most common cause of acute liver failure in the United-States. The aim of the study was to describe serum immune profiles associated with acute DILI, to investigate whether there are profiles associated with clinical features or types of DILI and/or with prognosis, and to assess temporal changes in levels. Twenty-seven immune analytes were measured in the sera of 78 DILI subjects in the Drug-Induced Liver Injury Network (DILIN) and compared with 40 healthy controls. Immune analytes (14 cytokines, 7 chemokines and 6 growth factors) were measured by BioPlex multiplex ELISA at DILI onset and after 6 months. A modeling process utilizing immune principles was used to select a final set of variables among 27 immune analytes and several additional clinical lab values for prediction of early death (within 6 months of DILI onset). Nineteen of the 27 immune analytes were differentially expressed among healthy control, DILI onset and 6-month cohorts. Disparate patterns of immune responses, especially innate and adaptive cellular (mostly TH17) immunity were evident. Low values of four immune analytes (IL-9, IL-17, PDGF-bb and RANTES) and serum albumin are predictive of early death [PPV = 88% (95% CI, 65%-100%), NPV = 97% (95% CI, 93%-100%), accuracy = 96% (95% CI, 92%-100%)]. Conclusions Acute DILI is associated with robust and varying immune responses. High levels of expression of cytokines associated with innate immunity are associated with a poor prognosis, whereas high levels of expression of adaptive cytokines are associated with good long-term prognosis and eventual recovery. Serum immune analyte profiles at DILI onset appear to be of prognostic, and perhaps, diagnostic significance.

Hepatitis C virus (HCV) infection, which results in chronic hepatitis C (CHC) in most patients (70–85%), is a major cause of liver disease and remains a major therapeutic challenge. The mechanisms determining liver damage and the key factors that lead to a high rate of CHC remain imperfectly understood. The precise role of cytoskeletal (CS) proteins in HCV infection remains to be determined. Some studies including our recent study have demonstrated that changes occur in the expression of CS proteins in HCV-infected hepatocytes. A variety of host proteins interact with HCV proteins. Association between CS and HCV proteins may have implications in future design of CS protein-targeted therapy for the treatment for HCV infection. This chapter will focus on the interaction between host CS and viral proteins to signify the importance of this event in HCV entry, replication and transportation.

HHC is the most common inherited metabolic disease among the white population worldwide, with a gene frequency of about 10% and a frequency of homozygosity of about 1 of 250. Many patients harbor a common haplotype of informative markers on chromosome 6p2l.23, suggesting a strong founder effect exerted by a common Celtic ancestor. With the advent of screening tests (serum Tf saturation, fe), many subjects with HHC are being identified before development of cirrhosis or diabetes mellitus, and early detection is important because prompt and vigorous iron reduction prevents development of such complications and assures normal life expectancy. The HIC can be estimated as accurately by specialized magnetic resonance imaging or susceptometric measurements as by chemical measurements on liver biopsy specimens. However, biopsy specimens retain value for showing fibrosis/cirrhosis and dysplastic hepatocytes, both of which increase risks of HCC development. There is growing evidence that iron in the liver plays an important role in non-HHC diseases, such as alcoholic liver disease, chronic viral hepatitis, and porphyria cutanea tarda. The complicated, manifold roles of iron in pathogenesis of the latter disorder include enhancement of production and irreversible oxidation of uroporphyrinogen, as well as formation of an inhibitor targeted specifically at hepatic uroporphyrinogen decarboxylase. The nature of the gene and gene product that are abnormal in HHC remain elusive, despite the intense efforts of several investigative groups. The search has been hampered by a dearth of informative markers in HHC patients in the relevant region of chromosome 6p. Note added in proof: The cloning of a candidate gene, the mutation of which may perhaps cause HLA-linked hemochromatosis, has just been reported (Feder et al: A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nature (Genetics) 1996;399-408). These workers identified a 250-kb region move than three megabases telomeric of the MHC that was identical in 85% of chromosomes of HHC patients. Within this region, they identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations one of which is predicted to inactivate this class of proteins. 83% of 178 patients were homozygous for this mutation (Cys 282Tyr). This variant was also found on 3.2% of control chromosomes, as would be expected for such a common disorder. Functional studies are awaited with great interest.

Heme oxygenase catalyzes the degradation of heme into biliverdin, carbon monoxide, and iron. Two forms of this enzyme, heme oxygenase-1 and -2, have been identified; only heme oxygenase-1 is subject to induction by heme, metal ions, and other chemical and physical perturbations (e.g. drugs, oxidants, and heat shock). Primary chick embryo liver cells are widely used for the study of heme metabolism because of their ease of preparation, low cost, and high degree of similarity to human heme metabolism. Nonetheless, this system has some limitations: new cultures must be prepared every week; the resulting cell populations are non-homogeneous; and cells are short-lived, limiting the feasible duration of time course and transfection studies. LMH cells are the first chicken hepatoma cell line to be established. The aim of this study was to characterize the regulation of heme oxygenase-1 in LMH cells, and to compare this regulation to that previously described in primary chick embryo liver cells. The induction of heme oxygenase-1 was assessed by measuring changes in mRNA levels or enzyme activities in response to several treatments, including heme, heavy metals, sodium arsenite, and heat shock, which have been shown to increase the expression of heme oxygenase. Similarities were observed with respect to regulation of heme oxygenase-1 expression in primary hepatocytes and LMH cells. We report the first measurable heat shock response of heme oxygenase-1 in CELC or LMH cells; and show that LMH cells are a useful model for the study of heme oxygenase-1 regulation.

There is growing evidence that normal or only mildly increased amounts of iron in the liver can be damaging, particularly when they are combined with other hepatotoxic factors such as alcohol, porphyrogenic drugs, or chronic viral hepatitis. Iron enhances the pathogenicity of microorganisms, adversely affects the function of macrophages and lymphocytes, and enhances fibrogenic pathways, all of which may increase hepatic injury due to iron itself or to iron and other factors. Iron may also be a co-carcinogen or promoter of hepatocellular carcinoma, even in patients without HC or cirrhosis. Based on this and other evidence, we hope that the era of indiscriminate iron supplementation will come to an end. Bloodletting, a therapy much in vogue 2 centuries ago, is deservedly enjoying a renaissance, based on our current understanding of the toxic effects of iron and the benefits of its depletion.

To study the regulation of expression of the metallothionein gene in normal liver cells, we transfected chick embryo liver cells in primary cultures with constructs containing luciferase or chloramphenicol acetyl transferase (as reporter genes) under the control of differing lengths of the 5'-promoter region of the chick metallothionein gene (containing 30, 122, 190, or 623 base pairs upstream of the transcriptional start site). We controlled for efficiency of transfection by co-transfections with a plasmid containing a bacterial beta-galactosidase gene under the control of the SV 40 promoter and enhancer. Treatment of the transfected cells with transition metallic ions (cadmium, cobalt, and zinc) or sodium arsenite produced increases in activities of luciferase or chloramphenicol acetyl transferase, relative to beta-galactosidase, and this activity mapped to the first 122 base pairs of the promoter. Although heme has recently been reported to induce the endogenous metallothionein gene in chick embryo liver cells, 10-50 microM heme did not increase reporter gene activities in transfected cells. Nevertheless, the heme-dependent induction of endogenous heme oxygenase-1 in these cells was normal. We conclude that the heme-dependent induction of the liver metallothionein gene depends upon DNA region(s) outside the regulatory region of the chick metallothionein gene studied here and that elements within the first 122 base pairs of the metallothionein promoter are sufficient to confer responsiveness to transition metals or sodium arsenite.

Relatively few patients with chronic hepatitis C treated with standard doses of interferon-alpha 2b (3 million units per week for 24 weeks) have a sustained response. Our aim was to evaluate whether higher doses of interferon would improve this rate of response. Twenty-four patients with chronic hepatitis C who had failed to respond to (N = 21) or had relapsed after (N = 3) an initial course of standard interferon therapy were randomized to 15 million units (N = 13) or 22.5-30 million units per week (N = 11) for 24 weeks. Five of 13 subjects given 15 million units per week and 3/11 of subjects given 22.5-30 million units per week had complete normalization of serum alanine aminotransferase levels during therapy. Five patients (24% who had not responded to standard interferon had a complete response to high-dose interferon during therapy. Only one patient had a sustained response, with normal serum alanine aminotransferase 24 weeks after stopping interferon. Six patients were withdrawn before completing treatment, five in the 22.5-30 million unit per week group. We conclude that higher doses of interferon ameliorate the severity of hepatitis in patients who failed to respond to or relapsed after standard interferon therapy, but are unlikely to produce a sustained response. High-dose therapy is associated with an increase in side effects.

Recent evidence suggests that patients with chronic hepatitis C virus (CHCV) who respond to interferon-alpha (IFN) therapy have a lower hepatic iron concentration than those who do not. The object of this study was to assess the concentration and distribution of iron in liver biopsies from 15 patients with CHCV seen at the authors' medical center between June 1992 and March 1993. Patients with complete response to IFN were compared to those with non-complete response with respect to quantitative hepatic iron concentration, serum iron indices, and a detailed analysis of histologic features of hematoxylin-and-eosin and iron-stained pre-IFN biopsies. Patients with non-complete response had significantly higher scores for stainable iron in sinusoidal cells (P = .02) and portal tracts (P = .05) than did patients with complete response. Total hepatic iron scores, mean quantitative hepatic iron, and mean serum ferritin were higher in patients with noncomplete response, but the differences were not significant. In conclusion, iron deposition in sinusoidal cells and portal tracts is significantly less frequent in patients with complete response to IFN than in those with poor or no response, and may be a useful, objective predictor of response to IFN therapy.

The advent of specific antiviral therapy for chronic hepatitis C has increased the importance of establishing the correct etiology of chronic hepatitis in patients, especially because interferon alfa (IFN-alpha) has been reported to exacerbate autoimmune hepatitis (AIH), whereas corticosteroids increase viral replication in chronic hepatitis C. In our medical center, we have treated many patients with apparent chronic hepatitis C and serological or clinical evidence of autoimmunity. Our aim was to estimate the prevalence of this association and to learn whether demographic or clinical features distinguished between patients with or without autoimmune markers. We performed a retrospective review of the records of 244 unselected patients seen at the Clinics and Hospital of the University of Massachusetts between May 1991 and November 1993, who had elevated serum aminotransferases. One hundred seventeen patients had chronic hepatitis C defined by elevations of serum alanine transaminase (ALT) for at least 6 months, positive serum antibodies to hepatitis C virus (HCV; second-generation enzyme immunoassay [EIA2] or recombinant immunoblot assay [RIBA]), and absence of hepatitis B surface antigen in the serum. Records were reviewed for results of autoimmune markers in sera, including anti-nuclear antibodies (ANAs), anti-smooth muscle antibodies (SMAs), rheumatoid factor (RF), antimitochondrial antibodies (AMAs), anti-liver and kidney microsomal (LKM) antibodies, and cryoglobulins. We found a high prevalence of positivity, particularly for anti-SMAs (66%) and RF (76%) in both men and women. Forty of 41 patients tested negative for anti-LKM antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Background. Essential mixed cryoglobulinemia is frequently associated with hepatitis C virus (HCV) infection. A beneficial effect of interferon alfa therapy has been reported, but we do not know whether the antiviral activity of the drug affects the clinical and biochemical manifestations of disease. Methods. In a prospective randomized, controlled trial, we studied 53 patients with HCV-associated type II cryoglobulinemia. A group of 27 patients received recombinant interferon alfa-2a thrice weekly at a dose of 1.5 million units for a week and then 3 million units thrice weekly for the following 23 weeks. The 26 control patients did not receive anything apart from previously prescribed treatments. All patients were then followed for an additional 24 to 28 weeks. Results. Interferon was usually well tolerated, but it was permanently discontinued in two patients because of atrial fibrillation and depression. Two of the 26 patients in the control group were lost to follow-up. After the treatment period, serum HCV RNA was undetectable in 15 of the remaining 25 patients who received interferon alfa-2a, but in none of the controls. In comparison with the control group, the 15 patients with undetectable levels of HCV RNA in serum had significant improvement in cutaneous vasculitis (P = 0.04) and significant decreases in serum levels of anti-HCV-antibody activity (P = 0.007), cryoglobulins (P = 0.002), IgM (P = 0.002), rheumatoid factor (P = 0.001), and creatinine (P = 0.006). After treatment with interferon alfa-2a was discontinued, viremia and cryoglobulinemia recurred in all 15 HCV RNA-negative patients. On resumption of treatment, three of four patients had virologic, clinical, and biochemical response. Conclusions. The therapeutic efficacy of interferon alfa-2a in HCV-associated cryoglobulinemia is closely related to its antiviral activity, thus supporting the idea that HCV infection may be a cause of this disease. (N Engl J Med 1994;330:751–6.) Background/Aims: Mixed cryoglobulinemia is frequently associated with liver diseases. The respective role of hepatitis C virus (HCV) and liver damage in the pathogenesis of cryoglobulinemia is investigated in this study. Methods: The prevalence of cryoglobulinemia in 226 consecutive patients with chronic liver diseases (hepatitis C, 127; hepatitis B, 40; other diseases 59) was studied, and the epidemiological, biological, histological, and virological features in these three groups were analyzed. Anti-HCV antibodies, HCV proteins, and HCV RNA were searched in the cryoprecipitates. Results: The prevalence of cryoglobulinemia was high (41.5%) in patients with liver diseases and higher in patients with hepatitis C (54.3%) than in patients with hepatitis B (15%) or other causes of liver disease (32%). Patients with cryoglobulinemia had cirrhosis more frequently and had a longer history of hepatitis. In patients with hepatitis C, HCV RNA sequences and HCV proteins were detected in the cryoprecipitate. Cryoglobulins became undetectable in 21 of 43 patients treated with interferon. Conclusions: These findings suggest that HCV is a major cause of cryoglobulinemia. Besides viral infection itself, multiple factors appear to be responsible for the production of cryoglobulins, including cirrhosis and duration of liver disease.

Iron is an essential element for cell survival; it serves as a cofactor for essential enzymes in oxidative metabolism and (in the form of heme) as the major oxygen transporter in most forms of life on earth. Both deficiency and excess of iron often lead to disease. Iron is necessary for the proliferation of microorganisms and neoplastic cells. The presence of iron overload facilitates infection, as evidenced by the increased risk of persons with hemochromatosis to certain infections and by the fact that patients with lesser amounts of hepatic iron appear to respond better to interferon therapy for chronic viral hepatitis than those with larger amounts of hepatic iron. Viral hepatitis is a common cause of morbidity and mortality. Recent studies suggest that there is a key link between iron metabolism and the pathophysiology of viral hepatitis. The lobular and cellular distribution of iron in the liver may be as important as the total quantity of iron present. Whether iron removal will prove useful in the long-term management of chronic viral hepatitis is an issue in need of further well-designed, randomized, controlled trials.

AIM: To investigate roles of genetic polymorphisms in non-alcoholic fatty liver disease (NAFLD) onset, severity, and outcome through systematic literature review. METHODS: The authors conducted both systematic and specific searches of PubMed through December 2015 with special emphasis on more recent data (from 2012 onward) while still drawing from more historical data for background. We identified several specific genetic polymorphisms that have been most researched and, at this time, appear to have the greatest clinical significance on NAFLD and similar hepatic diseases. These were further investigated to assess their specific effects on disease onset and progression and the mechanisms by which these effects occur. RESULTS: We focus particularly on genetic polymorphisms of the following genes: PNPLA3, particularly the p. I148M variant, TM6SF2, particularly the p. E167K variant, and on variants in FTO, LIPA, IFNλ4, and iron metabolism, specifically focusing on HFE, and HMOX-1. We discuss the effect of these genetic variations and their resultant protein variants on the onset of fatty liver disease and its severity, including the effect on likelihood of progression to cirrhosis and hepatocellular carcinoma. While our principal focus is on NAFLD, we also discuss briefly effects of some of the variants on development and severity of other hepatic diseases, including hepatitis C and alcoholic liver disease. These results are briefly discussed in terms of clinical application and future potential for personalized medicine. CONCLUSION: Polymorphisms and genetic factors of several genes contribute to NAFLD and its end results. These genes hold keys to future improvements in diagnosis and management.

Pancreatic cancer is an aggressive disease with nearly equal yearly rates of diagnosis and death. Current therapies have failed to improve outcomes due to rapid disease progression and late stage at presentation. Recently, pathways involved in progression and metastasis have been elucidated; however, new knowledge has not generated more effective therapies. We report on the use of subcellular fractionation and liquid chromatography (LC)-mass spectrometry to identify 3,907 proteins in four pancreatic cancer cell lines, 540 of which are unique to primary cancer cells, and 487 unique to cells derived from metastatic sites. Statistical analysis identified 134 proteins significantly differentially expressed between the two populations. The subcellular localization of these proteins was determined and expression levels for four targets were validated using western blot techniques. These identified proteins can be further investigated to determine their roles in progression and metastasis and may serve as therapeutic targets in the development of more effective treatments for pancreatic cancer.

Background Flavocoxid is a prescription medical food that is used to treat osteoarthritis. It is a proprietary blend of 2 flavonoids, baicalin and catechins, which are derived from the botanicals Scutellaria baicalensis and Acacia catechu, respectively. Objective To describe characteristics of patients with acute liver injury suspected of being caused by flavocoxid. Design Case series. Setting Drug-Induced Liver Injury Network Prospective Study ongoing at multiple academic medical centers since 2004. Patients Four adults with liver injury. Measurements Clinical characteristics, liver biochemistry values, and outcomes. Results Among 877 patients enrolled in the prospective study, 4 had liver injury suspected to have been caused by flavocoxid. All were women; ages ranged from 57 to 68 years. All developed symptoms and signs of liver injury within 1 to 3 months after initiating flavocoxid. Liver injury was characterized by marked elevations in levels of alanine aminotransferase (mean peak, 1268 U/L; range, 741 to 1540 U/L), alkaline phosphatase (mean peak, 510 U/L; range, 286 to 770 U/L), and serum bilirubin (mean peak, 160.7 µmol/L [9.4 mg/dL]; range, 34.2 to 356 µmol/L [2.0 to 20.8 mg/dL]). Liver biochemistry values decreased to the normal range within 3 to 12 weeks after flavocoxid was stopped, and all patients recovered without experiencing acute liver failure or chronic liver injury. Causality was adjudicated as highly likely in 3 patients and as possible in 1 patient. Limitation The frequency and mechanism of liver injury could not be assessed. Conclusion Flavocoxid can cause clinically significant liver injury, which seems to resolve within weeks after cessation.

Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGFβ). microRNAs (miRs), small non-coding RNAs modulating mRNA and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGFβ signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Since miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGFβ signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGFβ signaling components demonstrated by decreased TGFβ receptor II (TGFβRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3’UTR of TGFβRII was validated by luciferase reporter assay. Inhibition of TGFβ signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGFβ-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased αSMA expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared to normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared to normal human livers. CONCLUSIONS miR 19b is a novel regulator of TGFβ signaling in HSCs suggesting a potential therapeutic approach for hepatic fibrosis.

The aim of this study was to describe the presenting features and outcomes of consecutive patients with liver injury attributed to interferon beta.The presenting features of eight subjects with clinically apparent liver injury attributed to interferon beta enrolled in the U.S. Drug-Induced Liver Injury Network (DILIN) prospective registry between 2004 and 2010 were reviewed and compared to 11 published reports of symptomatic hepatotoxicity.All eight of the DILIN patients were women, 75 % were Caucasian and the mean age was 49 years. Most subjects presented with an acute hepatocellular injury pattern and mean serum alanine aminotransferase (ALT) levels were 725 ± 593 U/L. The median duration of interferon beta use before injury onset was 462 days, and four patients had been treated for more than a year. No patient had detectable antinuclear or smooth muscle antibodies. One patient died of acute liver failure and the remaining patients usually recovered within 2-3 months. Causality assessment scored three cases as definite, three highly likely, one probable and one possible. Eleven additional published cases were all women, mean age was 40 years, mean ALT at onset 840 U/L, and 7 (63 %) had autoantibodies. Liver histology in three cases from DILIN and nine from the literature commented upon centrilobular (zone 3) necrosis and infiltrates with lymphocytes and plasma cells.Interferon beta hepatotoxicity occurs mostly in women and has a variable, but often prolonged time to onset. Most patients have self-limited acute hepatocellular liver injury but several have required liver transplantation or died of acute liver failure. Liver histology available in three cases demonstrated zone 3 necrosis and autoimmune features suggestive of an immunologic basis to this adverse drug reaction.

Idiosyncratic drug-induced liver injury (DILI) is a complex disorder that is difficult to predict, diagnose and treat.To describe the global serum proteome of patients with DILI and controls.A label-free, mass spectrometry-based quantitative proteomic approach was used to explore protein expression in serum samples from 74 DILI patients (collected within 14 days of DILI onset) and 40 controls. A longitudinal analysis was conducted in a subset of 21 DILI patients with available 6-month follow-up serum samples.Comparison of DILI patients based on pattern, severity and causality assessment of liver injury revealed many differentially expressed priority 1 proteins among groups. Expression of fumarylacetoacetase was correlated with alanine aminotransferase (ALT; r = 0.237; P = 0.047), aspartate aminotransferase (AST; r = 0.389; P = 0.001) and alkaline phosphatase (r = -0.240; P = 0.043), and this was the only protein with significant differential expression when comparing patients with hepatocellular vs. cholestatic or mixed injury. In the longitudinal analysis, expression of 53 priority 1 proteins changed significantly from onset of DILI to 6-month follow-up, and nearly all proteins returned to expression levels comparable to control subjects. Ninety-two serum priority 1 proteins with significant differential expression were identified when comparing the DILI and control groups. Pattern analysis revealed proteins that are components of inflammation, immune system activation and several hepatotoxicity-specific pathways. Apolipoprotein E expression had the greatest power to differentiate DILI patients from controls (89% correct classification; AUROC = 0.97).This proteomic analysis identified differentially expressed proteins that are components of pathways previously implicated in the pathogenesis of idiosyncratic drug-induced liver injury.

Contemporary Western medicines approved by the U.S. Food and Drug Administration (FDA) for the treatment of chronic hepatitis B (CHB), although available in China, have high costs, or major side effects and limited effectiveness. Research efforts have focused on looking for natural products as alternative medicines with low cost and good safety for CHB treatment. Chinese medicine (CM) has ancient, time-honored theories about methods of diagnosis and treatment for liver diseases. In recent decades, a large number of clinical trials and pre-clinical studies, which were performed in China and other countries, indicated that CM has potential benefit in several aspects of the treatment of CHB, e.g., anti-inflammatory, anticancer, antioxidant, immunomodulating, antifibrosis, and antiviral. However, there are many concerns regarding the study design and the quality of clinical trials. Further larger, stringently designed, double-blind, placebo control, randomized clinical trials and long-term follow-up are needed to provide conclusive evidence of their efficacy and safety. Components of CM deserve further study in pre-clinical models of HBV infection and in clinical trials world-wide.

Amyotrophic lateral sclerosis (ALS) is one of the group of diseases of the central nerve system (CNS), which are characterized by progressive loss of structure and function of neurons in different regions of brain or spinal cord. Therefore, these diseases are collectively designated as “Neurodegenerative Diseases” (NDDs). Usually the loss of specific functions precedes the death of affected neurons, and the related clinical features depend on localization and degree of neurodegeneration. NDDs include such diseases as Alzheimer’s, Parkinson’s, Huntington’s, spinocerebellar ataxias, and ALS. In spite of differences in predominant localization of neurodegeneration and clinical features, there are many parallels among different neurodegenerative disorders. These include involvement of mitochondrial dysfunctions, increased oxidative stress, and atypical protein assemblies (Backman et al., 2006). Amyotrophic lateral sclerosis (ALS) refers to several adult-onset conditions characterized by progressive degeneration of motor neurons. “Amyotrophic” refers to the muscle atrophy, weakness, and fasciculation (spontaneous contraction affecting a small number of muscle fibers) that signify disease of the lower motor neurons. There are two forms of this fatal disease: sporadic, with no known genetic component, and familial, which make up about 10% of all ALS cases (Rowland & Schneider, 2001; Martin et al., 2009). Among the familial cases, approximately 20% are caused by dominantly inherited mutations in the Cu/Zn superoxide dismutase (SOD1) gene, with more than 100 known mutations (reviewed in Bruijn et al., 2004). So far, there is very little information that links familial and sporadic cases of the disease. One established fact, based on studies of patients and transgenic animals, is that mitochondria dysfunction is an early manifestation. However, it is unclear whether mitochondrial dysfunctions are the primary pathogenic mechanism, or the result of some other proximate pathogenic mechanism. Although the cases associated with mutations in the SOD1 gene comprise only about 2% of all ALS cases, understandably, transgenic animals bearing mutated SOD1 gene

The let-7 microRNA (miRNA) plays important roles in human liver development and diseases such as hepatocellular carcinoma, liver fibrosis and hepatitis wherein oxidative stress accelerates the progression of these diseases. To date, the role of the let-7 miRNA family in modulation of heme oxygenase 1 (HMOX1), a key cytoprotective enzyme, remains unknown. Our aims were to determine whether let-7 miRNA directly regulates Bach1, a transcriptional repressor of the HMOX1 gene, and whether indirect up-regulation of HMOX1 by let-7 miRNA attenuates oxidant injury in human hepatocytes. The effects of let-7 miRNA on Bach1 and HMOX1 gene expression in Huh-7 and HepG2 cells were determined by real-time qRT-PCR, Western blot, and luciferase reporter assays. Dual luciferase reporter assays revealed that let-7b, let-7c, or miR-98 significantly decreased Bach1 3'-untranslated region (3'-UTR)-dependent luciferase activity but not mutant Bach1 3'-UTR-dependent luciferase activity, whereas mutant let-7 miRNA containing base complementarity with mutant Bach1 3'-UTR restored its effect on mutant reporter activity. let-7b, let-7c, or miR-98 down-regulated Bach1 protein levels by 50-70%, and subsequently up-regulated HMOX1 gene expression by 3-4 fold, compared with non-specific controls. Furthermore, Huh-7 cells transfected with let-7b, let-7c or miR-98 mimic showed increased resistance against oxidant injury induced by tert-butyl-hydroperoxide (tBuOOH), whereas the protection was abrogated by over-expression of Bach1. In conclusion, let-7 miRNA directly acts on the 3'-UTR of Bach1 and negatively regulates expression of this protein, and thereby up-regulates HMOX1 gene expression. Over-expression of the let-7 miRNA family members may represent a novel approach to protecting human hepatocytes from oxidant injury.

Mifepristone (RU-486), a potent progesterone receptor antagonist and inducer of cytochromes P-450, is currently in use in Europe, particularly as a post-coital oral contraceptive. Soon it will be available in the United States, as well. Since progesterone has been implicated in the pathogenesis of acute attacks of porphyria, the use of RU-486 or related compounds might be considered in porphyric patients. However, as with other cytochrome P-450 inducers, RU-486 may have the ability to precipitate or exacerbate attacks of acute porphyria. The acute porphyrias in relapse are associated with an increase in activity of delta-aminolevulinic acid synthase, the first and normally rate-controlling enzyme in heme biosynthesis. We have used primary cultures of chick embryo liver cells to test the ability of RU-486 to induce delta-aminolevulinic acid synthase activity and mRNA, cytochromes P-450, porphyrin accumulation, and heme oxygenase. We found that RU-486, at concentrations observed in human plasma after a single oral dose, induced the mRNA and activity of delta-aminolevulinic acid synthase, both by itself and in the presence of deferoxamine, a potent iron chelator that inhibits ferrochelatase. RU-486 and deferoxamine together also produced significant accumulations of protoporphyrin. These results indicate that RU-486 may pose a risk in patients with known acute porphyria and should be used with caution. RU-486 increased the concentration of total cytochrome P-450, and the activity of erythromycin demethylase, an activity specifically catalyzed by cytochrome P-450 3A. Unlike several other porphyrogens (e.g. hydantoins, barbiturates), RU-486 does not produce accumulation of uroporphyrin or induction of heme oxygenase in chick embryo liver cells.

The serum protein hemopexin is considered to have a major role in the mechanism of the uptake of heme by hepatocytes by means of a heme-hemopexin receptor. Therefore, we examined in primary cultures of adult rat and embryonic chick hepatocytes whether the presence of hemopexin would affect the heme-mediated repression of 5-aminolevulinate synthase activity (the rate-limiting enzyme of heme biosynthesis) and the heme-induced increase of heme oxygenase activity (the rate-limiting step of heme degradation). Both of these heme-mediated effects were partly or entirely prevented by the presence of hemopexin. We conclude that homologous hemopexin, at molar concentrations exceeding that of heme, inhibited the uptake of heme into hepatocytes. These results suggest that heme, in amounts sufficient to affect the rate-limiting steps of heme synthesis and degradation, can only enter hepatocytes in primary culture when the binding capacity of hemopexin for heme has been exceeded or altered.