High baseline bilirubin and low albumin predict liver decompensation and serious adverse events in HCV-infected patients treated with sofosbuvir-containing regimens.

To conduct surveillance and determine the safety profile of new hepatitis C virus treatments in real-world clinical practice. Hepatic decompensation and other serious adverse events were investigated in an observational cohort study of 511 patients treated with regimens containing sofosbuvir, December 2013-June 2014. Among 499 previously stable patients (no history of hepatic decompensation during the previous 12 months), a nested case-control study was performed to identify predictors of decompensation/serious adverse event. Cases and controls were matched 1:5 based on treatment regimen and duration. Matched conditional logistic regression was used for analysis. Providers scored the likelihood that events were treatment-related (scale = 0-4). The cumulative incidence of decompensation/events was 6.4% for the total cohort. Among 499 previously stable patients, the incidence of decompensation/events was 4.5%; the mortality rate was 0.6%. Sixteen of the 499 experienced one or more serious complications considered to be at least potentially treatment-related, and the sustained virological response rate was 7/16 (44%). Two cases, both on sofosbuvir/simeprevir (without interferon or ribavirin), had complications consistent with autoimmune events (score 3, 'likely treatment-related'), and one experienced a flare of autoimmune hepatitis. Compared to controls, cases had higher baseline median model for end-stage liver disease scores (14 vs 8, P < 0.01). Decompensation/events was independently associated with lower baseline albumin (OR = 0.12/g/dL, P = 0.01) and higher total bilirubin (OR = 4.31/mg/dL, P = 0.01). Reduced hepatic function at baseline increased the risk of liver decompensation/events.

Clinical characteristics of human immunodeficiency virus patients being referred for liver transplant evaluation: a descriptive cohort study.

BACKGROUND: Liver transplantation (LT) is a treatment option for select human immunodeficiency virus (HIV)-infected patients with advanced liver disease. The aim of this study was to describe LT evaluation outcomes in HIV-infected patients. METHODS: All HIV-infected patients referred for their first LT evaluation at the Mount Sinai Medical Center were included in this retrospective, descriptive cohort study. Multivariable logistic regression was used to identify factors independently associated with listing. RESULTS: Between February 2000 and April 2012, 366 patients were evaluated for LT, with 66 (18.0%) listed for LT and 300 (82.0%) not listed. Fifty-one patients (13.9%) died before completing evaluation and 85 (23.2%) were too early for listing. Reasons patients were declined for listing were psychosocial (15.8%), HIV-related (10.4%), loss to follow-up (9.6%), surgical/medical (6.0%), liver-related (4.4%), patient choice (3.4%), and financial (1.6%). Listed patients were more likely to have hepatocellular carcinoma (HCC) (43.1% vs. 17.1%; P < 0.0001) and less likely to have hepatitis B (6.2% vs. 15.7%; P = 0.04) or a psychiatric history (19.7% vs. 35.2%; P = 0.02) than those not listed. In multivariable analysis, HCC (odds ratio [OR] 5.79; 95% confidence interval [95% CI]: 2.97-11.28), model for end-stage liver disease (MELD) score at referral (OR 1.06; 95% CI 1.01-1.11), and hepatitis B (OR 0.26; 95% CI 0.08-0.79) were associated with listing. CONCLUSION: MELD score and HCC were positive predictors of listing in HIV-infected patients referred for LT evaluation and, therefore, timely referrals are vital in these patients. As MELD is a predictor for death while undergoing evaluation, rapid evaluation should be performed in HIV-infected patients with a higher MELD score.

A replication cycle for viroids and other small infectious RNA's.

Experimental data concerning viroid-specific nucleic acids accumulating in tomato plants establish, together with earlier studies, the major features of a replication cycle for viroid RNA in plant cells. Many features of this pathway, which involves multimeric strands of both polarities, may be shared by other small infectious RNA's including certain satellite RNA's and "virusoid" RNA's which replicate in conjunction with conventional plant viruses. The presence, in host plans, of an elaborate machinery for replicating these disease agents suggests a role for endogenous small RNA's in cellular development.

Cell-free circularization of viroid progeny RNA by an RNA ligase from wheat germ.

Linear, potato spindle tuber viroid RNA has been used as a substrate for an RNA ligase purified from wheat germ. Linear viroid molecules are efficiently converted to circular molecules (circles) which are indistinguishable by electrophoretic mobility and two-dimensional oligonucleotide pattern from viroid circles extracted from infected plants. In light of recent evidence for multimeric viroid replication intermediates, cleavage followed by RNA ligation by a cellular enzyme may (i) be a normal step in the viroid life cycle and (ii) may also reflect cellular events.

An ultraviolet-sensitive RNA structural element in a viroid-like domain of the hepatitis delta virus.

The RNA genome of the hepatitis delta virus (HDV) appears to be made up of two parts: a small domain with a high degree of sequence conservation and structural features likely to promote replication; plus a second, larger domain that is less conserved and encodes the delta antigen. This report focuses on one of the several sets of data that have led to the proposal of this model: the existence of a novel structural element in HDV genomic RNA. This structural element lies within the highly conserved domain of HDV RNA and may be related to the local tertiary structure previously mapped to the central conserved region of the plant viroid genome. Both elements occur in regions with no apparent coding capacity and are distinctively responsive to ultraviolet (UV) light. Transcripts containing partial and full-length genomic sequences of HDV readily undergo a UV-induced crosslinking reaction, which establishes a covalent bond between two noncontiguous segments. By locking two segments of the overall structure into place, this crosslink has permitted the unbranched, rodlike model of HDV RNA to be examined and confirmed in the portion of the RNA analyzed. The clustering of the novel tertiary structure and the recently discovered self-cleavage sites into a highly conserved, but apparently noncoding, portion of the genome defines a viroid-like domain in HDV RNA and raises questions about the possible events leading up to the association of free-living RNAs with messenger RNAs and other RNA molecules.

A good antisense molecule is hard to find.

Antisense molecules and ribozymes capture the imagination with their promise of rational drug design and exquisite specificity. However, they are far more difficult to produce than was originally anticipated, and their ability to eliminate the function of a single gene has never been proven. Furthermore, a wide variety of unexpected non-antisense effects have come to light. Although some of these side effects will almost certainly have clinical value, they make it hard to produce drugs that act primarily through true antisense mechanisms and complicate the use of antisense compounds as research reagents. To minimize unwanted non-antisense effects, investigators are searching for antisense compounds and ribozymes whose target sites are particularly vulnerable to attack. This is a challenging quest.

Glucocorticoid regulation of enkephalins in cultured rat adrenal medulla.

The effect of dexamethasone on enkephalin-containing (EC) peptide levels and preproenkephalin mRNA levels was determined in adrenal medullary explants (glands) from sham and hypophysectomized (hypox) rats. Culture for 4 days in serum-free medium without dexamethasone resulted in a 13- and 4-fold increase in EC peptide levels in sham and hypox glands, respectively. The addition of dexamethasone (10(-5) M) produced a 20- to 26-fold increase in EC peptides in sham and hypox glands. In serum free medium, hypox glands showed a concentration dependent increase in EC peptides with the ED50 for dexamethasone equal to 5.7 x 10(-7) M. Since the glucocorticoid antagonist RU486 partially blocked the rise in EC peptides in sham glands, it appears that the increase in EC peptides in sham glands in the absence of dexamethasone is a result of a higher concentration of endogenous corticosterone in sham compared to hypox glands. Dexamethasone resulted in a 6-fold increase in preproenkephalin mRNA in hypox glands cultured for 2 days. This increase was approximately proportional to the increase in EC peptides seen at 4 days. In serum free medium progesterone, testosterone, and deoxycorticosterone failed to increase EC peptides in hypox glands. These results indicate that glucocorticoid treatment is required for maximal proenkephalin gene expression and EC peptide biosynthesis in cultured glands.

Transcripts of the viroid central conserved region contain the local tertiary structural element found in full-length viroid.

The viroid central conserved region (CCR) is highly conserved among different viroids and is thought to be involved in viroid replication. A novel tertiary structure occurs in the CCR of native circular potato spindle tuber RNAs. To permit more detailed studies of this structural element, a small RNA oligonucleotide containing the CCR of the viroid genome was synthesized. The tertiary structure of these CCR transcripts was examined by UV-crosslinking of the RNA, followed by mapping of the crosslink using limited alkaline digestion and classical RNA secondary analysis. The CCR transcript was found to undergo UV-crosslinking between the same two bases as in full-length viroid, indicating that the tertiary structure is the same and that the CCR transcript will be useful for the affinity purification of host components.

Quantitation of preproenkephalin mRNA levels in brain regions from male Fischer rats following chronic cocaine treatment using a recently developed solution hybridization assay.

Quantitative solution hybridization assays were used to determine the picogram amounts of preproenkephalin mRNA (PPenk mRNA) and the microgram quanities of total rat RNA in extracts of eight brain regions from rats which had received three daily intraperitoneal injections of cocaine (10 or 30 mg/kg/day) or saline for 14 days. The young adult male Fischer rats were sacrificed 30 min after the final injection. The highest density of PPenk mRNA (pg PPenk mRNA/micrograms total cellular RNA) was found in extracts of striatum (34.08 +/- 1.79 pg/micrograms for 11 saline-treated rats), followed by extracts of nucleus accumbens (10.08 +/- 0.81 pg/micrograms), and extracts of hypothalamus (2.99 +/- 0.31 pg/micrograms). Extracts of frontal cortex (1.78 +/- 0.24 pg/micrograms), pituitary (1.39 +/- 0.08 pg/micrograms), central grey (1.31 +/- 0.16 pg/micrograms), and cerebellum (1.24 +/- 0.09 pg/micrograms) had intermediate values. Extracts of hippocampus (0.53 +/- 0.03 pg/micrograms) had the lowest density. No significant differences were found among the treatment groups in any brain area investigated. Therefore, chronic cocaine treatment as administered in this protocol did not alter expression of the gene encoding proenkephalin.

Preproenkephalin mRNA and enkephalin in normal and denervated adrenals in the Syrian hamster: comparison with central nervous system tissues.

The distribution and characteristics of preproenkephalin (PPenk) mRNA and enkephalin-containing (EC) peptides are compared in CNS and adrenal tissues from Syrian hamsters and Sprague-Dawley rats. Total cellular RNA extracts from both rat and hamster tissues produce a single hybridization band of PPenk mRNA of approximately 1500 bases when analyzed by Northern blot hybridization. Quantitation by solution hybridization reveals that in the hamster the highest levels of PPenk mRNA are found in adrenal (16.3 +/- 1.4 pg equivalents/micrograms RNA (mean +/- S.E.M.)) and striatum (13.3 +/- 0.7), followed by hypothalamus (0.8 +/- 0.2), and hippocampus (0.4 +/- 0.2). In the rat the highest levels of PPenk mRNA are in the striatum (35 +/- 2 pg/micrograms RNA) followed by the hypothalamus (3.0 +/- 0.5), hippocampus (0.3 +/- 0.1) and adrenal (0.18 +/- 0.04). Thus, the rank order of abundance of PPenk mRNA is similar in these CNS tissues for rat and hamster. The hamster adrenal levels are more than 90-fold greater than those of the rat. The abundance of EC peptides in both hamster and rat tissues mirror the rank order found with PPenk mRNA. Hamster adrenal contains the highest level of EC peptides (441 +/- 37 pmol/mg protein (mean +/- S.E.M.)) which is more than 400-fold greater than that of the rat adrenal and 8- to 12-fold greater than that found in rat and hamster striatum or hypothalamus. Both size exclusion chromatography and Western blot analysis indicate that EC peptides in hamster adrenal are predominantly large proenkephalin-like peptides with approximately 6 copies of Met- and 1 copy of Leu-enkephalin and that included in their number is a prominent EC peptide with a molecular weight of 34 kDa. Unilateral denervation of the hamster adrenal results in a time-dependent ipsilateral decrease in EC peptide and PPenk mRNA levels. Thus, by day 8 postsurgery, PPenk mRNA levels have declined by an average of 80% while EC peptides are reduced by 68% when compared to the innervated contralateral adrenal. These results demonstrate the great abundance of PPenk mRNA and EC peptides in the hamster adrenal. They also demonstrate the apparent need for transsynaptic impulse activity to maintain the high steady-state levels of PPenk and EC peptides. These characteristics of the hamster adrenal system provide opportunities for physiological and pharmacological investigations of the regulation of proenkephalin gene expression.

Time course of enkephalin mRNA and peptides in cultured rat adrenal medulla.

Explantation of rat adrenal medullae to organ culture results in dramatic changes in enkephalins and catecholamines that are similar to the changes seen in vivo in response to denervation, which eliminates transsynaptic impulse activity. We have used rapid and sensitive solution hybridization methods to measure preproenkephalin (PPenk) mRNA and total cellular RNA in samples from rat tissues and adrenal medullary explants. The profiles of adrenal medullary PPenk mRNA, enkephalin-containing (EC) peptides, total cellular RNA and catecholamines [epinephrine (epi) and norepinephrine (norepi)] were measured during 14 days of organ culture. After 8 h in culture, total RNA had declined by 60%, epi and norepi declined 80 to 85% and EC peptides by 50% while the amount of PPenk mRNA per gland increased by 400%. Between 8 h and 14 days total RNA and catecholamine levels remained constant while PPenk mRNA increased to a peak of 85 +/- 10 (S.E.M.) pg/gland at 2-4 days, a value that was 80 times greater than the zero time (preculture) values. EC peptide levels lagged behind the increase in PPenk mRNA and reached a peak of 25 +/- 4 (S.E.M.) pmol Met-enkephalin equivalents/gland at 4 days that was 80 times greater than zero time values. Both PPenk mRNA and EC peptides declined in parallel between 4 and 14 days. The ratio of the copies of proenkephalin (Penk) peptide to PPenk mRNA was estimated to be 25,000 at the time of explantation and after 4 days in culture. From steady-state kinetics half-life estimates of 9.6 h for PPenk mRNA and 14.7 h for Penk peptide were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

The coming impact of gene expression profiling on the diagnosis and treatment of HCV-associated liver disease.

Gene expression profiling allows the level of activity of thousands of genes to be monitored simultaneously. Profiling is often carried out on specialized chips or slides, which have microarrays of gene targets at predetermined addresses. In the immediate future, microarrays promise to yield new insights into hepatitis C virus (HCV) pathogenesis and to produce 'signatures' that can be used in molecular diagnostics. In the longer-term, they may aid the development of serological tests by identifying genes encoding secretory proteins produced by HCV-infected livers, and they may suggest new avenues for disease intervention by detecting genes whose products are retained in the infected liver.

Cloning and characterization of hamster proenkephalin gene.

Our previous studies have shown that the hamster adrenal, like the human, contains high levels of preproenkephalin (PPenk) mRNA and enkephalin peptides, and may serve as a mammalian model for the in vivo study of proenkephalin (Penk) gene expression, peptide biosynthesis, and release. To define further the factors that may regulate hamster Penk gene expression, the hamster Penk gene was isolated from a genomic library prepared from Syrian hamster liver. The hamster Penk gene contains four exons and three introns and encodes 268 amino acids including six copies of Met-enkephalin containing peptides and one copy of Leu-enkephalin. In the 5' upstream region, there are TATA and GC boxes and multiple putative regulatory elements including the cAMP response element, AP-1, AP-2, AP-4, and the glucocorticoid response element (GRE). Possible GREs are also present in the introns. A comparison with the human and the rat Penk genes indicates that both the human and hamster Penk gene contain three introns, while the rat Penk gene has two introns. The intron missing from the rat Penk gene is short and separates the first and second exons of the hamster and human genes. In addition, the hamster and human genes share a region (100 bases) in the 5' upstream sequence that is 98% homologous. It is of interest that Penk gene expression is high in the adrenal medulla of both human and hamster, but is much lower in the rat. These homologous regions and the extra intron may contain regulatory features responsible for a high level of expression in the human and hamster adrenal medulla.

Effect of fibrosis on adverse events in patients with hepatitis C treated with telaprevir.

BACKGROUND: Data about adverse events are needed to optimise telaprevir-based therapy in a broad spectrum of patients. AIM: To investigate adverse events of telaprevir-based therapy in patients with and without advanced fibrosis or cirrhosis in a real-world setting. METHODS: Data on 174 hepatitis C-infected patients initiating telaprevir-based therapy at Mount Sinai and Montefiore medical centres were collected. Biopsy data and FIB-4 scores identified patients with advanced fibrosis. Multivariable fully adjusted models were built to assess the effect of advanced fibrosis on specific adverse events and discontinuation of treatment due to an adverse event. RESULTS: Patients with (n = 71) and without (n = 103) advanced fibrosis were similar in BMI, ribavirin exposure, gender, prior treatment history, haemoglobin and creatinine, but differed in race. Overall, 47% of patients completed treatment and 40% of patients achieved SVR. Treated patients with and without advanced fibrosis or cirrhosis had similar rates of adverse events; advanced fibrosis, however, was independently associated with ano-rectal discomfort (P = 0.03). Three patients decompensated and had advanced fibrosis. The discontinuation of all treatment medications due to an adverse event was significantly associated with older age (P = 0.01), female gender (P = 0.01) and lower platelets (P = 0.03). CONCLUSIONS: Adverse events were common, but were not significantly related to the presence of advanced fibrosis or cirrhosis. More critical monitoring in older and female patients with low platelets throughout treatment may reduce adverse event-related discontinuations.

Antisense drug discovery: can cell-free screens speed the process?

Many conditions must be satisfied for an antisense drug to function. It must colocalize with its target RNA at a sufficient concentration for a bimolecular reaction to occur, and it must have a structure that favors association with its target. In addition, if the antisense compound is to form Watson-Crick bonds with the target RNA, it must be complementary to sites that are amenable to binding. Unfortunately, the peculiarities that cause certain sites to be especially vulnerable to antisense compounds are undefined, as discussed previously (Branch, 1998). Because vulnerable target sites have no common properties allowing them to be identified by sequence analysis, most target sites and their antisense counterparts are found through a trial and error process in which oligomers--each complementary to a different site in the target RNA--are tested individually to find the one with the greatest specificity and lowest inhibitory concentration (IC50). However, testing antisense molecules one at a time can be a taxing process, and there is great interest in developing cell-free screening methods that can reduce the number of compounds that must be tested in cells and in whole animals. These cell-free screens are designed to generate short lists of target sites that include the ideal site--the site most vulnerable to antisense ablation in vivo. They are based on the unproven assumption that ideal sites have distinctive properties, such as susceptibility to RNase H-mediated cleavage, that allow them to be detected in cell-free assays. This is a review of data emerging from studies using RNase H-based screens and a summary of the challenges confronting these and any similar methods that use naked RNAs as surrogates for intracellular RNAs. It is not yet clear if cell-free screening methods will be effective.

A hitchhiker's guide to antisense and nonantisense biochemical pathways.

Antisense pharmaceutical research has sought to provide drugs that would yield effective therapies for diseases resulting from the production of deleterious proteins. The original concept was straightforward: eliminate production of unwanted proteins, such as oncogenic proteins, by blocking the function of their mRNAs; and block their mRNAs by adding "antisense" nucleic acids that bind them through complementary base pairing. However, it has proven difficult to develop clinically useful antisense strategies. Conventional antisense nucleic acids are large, highly charged, complex molecules that interact with a wide variety of unintended cellular and microbial components, often causing "nonantisense effects." It is now clear that a broad knowledge of nucleic acid biochemistry will be needed to optimize antisense molecules for use in patients. The efficacy of naturally occurring antisense molecules and the success of antisense agricultural strategies prove that antisense approaches can be powerful and specific. Pharmaceutical antisense research can be expected to yield many valuable products once sufficient information about antisense mechanisms has been gathered and applied. This article explains the biochemical events that give rise to both antisense and nonantisense effects and provides guidelines for designing and evaluating antisense experiments.

Hepatitis C virus RNA codes for proteins and replicates: does it also trigger the interferon response?

Hepatitis C virus (HCV) is a positive sense virus with a genomic RNA molecule roughly 9,600 nucleotides in length. The single-stranded genomic RNA has a nontranslated region (NTR) at each end and a long open reading frame (coding region) in between. The 5'NTR and portions of the 3'NTR are the most conserved parts of HCV RNA. These conserved regions contain signals for replication and translation. Much of the 5'NTR is folded into a structure that binds ribosomes. This structure, an internal ribosome entry site, promotes the initiation of protein synthesis and is critical for HCV gene expression. The ribosome binding site may extend into the coding region; its exact boundaries are not known. The open reading frame encodes the HCV polyprotein, which is slightly more than 3,000 amino acids in length. The 3'NTR plays a key role in HCV replication and may also influence the rate of HCV protein synthesis. During replication, the genomic RNA is copied by virally encoded enzymes into a complementary antigenomic RNA, which itself is a template for the synthesis of progeny RNAs. At steady state, genomic strands outnumber antigenomic strands about 10 to 1. HCV RNA replication is thought to take place in the cytoplasm and is an error-prone process. It generates a mixed population of RNA sequences (quasispecies), including mutants that may be more fit than the parental type, less fit, or equally fit (but distinct). Natural selection acts upon the progeny RNAs, causing the population to change and drift. Over time, mutation, selection, and population bottlenecks led to the evolution of varied genotypes. The HCV replication complex is a potential source of double-stranded RNA, a powerful inducer of interferon. Thus, HCV-specific double-stranded RNA may trigger the first steps of innate immunity; however, for unknown reasons, the immune system often fails to clear the infection. The plasticity of the HCV genome and the low level of HCV gene expression may counterbalance any immunostimulatory effects of HCV RNA and allow the virus to escape specific immune responses. Antisense drugs and ribozymes directed against HCV RNA are under investigation. Future interventions may include nucleic acid drugs (antisense and ribozymes) and smaller pharmaceuticals that bind to intricate structures in HCV RNA and HCV-specific double-stranded RNA. Infectious clones of HCV RNA are available. These clones and other systems for expressing HCV proteins pave the way for vaccine development.