Prisons and HCV: a review and a report on an experience in New South Wales Australia.

Prison populations in Western countries are characterised by a high hepatitis C prevalence. This reflects a high rate of imprisonment for drug related offences. Prison entrants who are HCV-negative face a significant risk of acquiring hepatitis C. Effective prevention strategies and successful treatment of a significant percentage of hepatitis C-positive inmates could reduce the risk of transmission in the prison context significantly. Several reports of treating hepatitis C in prisoners in major facilities have been published. We report our experience of establishing a liver clinic service in two regional prisons in New South Wales, Australia. Liver biopsy requirements to access treatment in Australia meant that only 46 of 196 reviewed patients were able to commence treatment in our 5-year experience. Treatment completion rate was 61% and end of treatment viral response was 57%. The removal of liver biopsy requirements in Australia in April 2006 has freed up access to treatment and our results encourage further effort to optimise the process of assessment and treatment in this high-risk population.

Targeting XIAP for the treatment of malignancy.

X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis proteins family of caspase inhibitors that selectively binds and inhibits caspases-3, -7 and -9, but not caspase-8. As such, XIAP blocks a substantial portion of the apoptosis pathway and is an attractive target for novel therapeutic agents for the treatment of malignancy. Antisense oligonucleotides directed against XIAP are effective in vitro and are currently being evaluated in clinical trials. Small molecule XIAP inhibitors that target the baculovirus IAP repeat (BIR) 2 or BIR 3 domain are in preclinical development and are advancing toward the clinic. This review will discuss the progress being made in developing antisense and small-molecule XIAP inhibitors.

Structural studies of the purine and SAM binding riboswitches.

Riboswitches are recently discovered genetic regulatory elements found in the 5'-untranslated regions of bacterial mRNAs that act through their ability to specifically bind small-molecule metabolites. Binding of the ligand to the aptamer domain of the riboswitch is communicated to a second domain, the expression platform, which directs transcription or translation of the mRNA. To understand this process on a molecular level, structures of three of these riboswitches bound to their cognate ligands have been solved by X-ray crystallography: the purine, thiamine pyrophosphate (TPP), and S-adenosylmethionine (SAM-I) binding aptamer domains. These studies have uncovered three common themes between the otherwise different molecules. First, the natural RNA aptamers recognize directly or indirectly almost every feature of their ligand to achieve extraordinary specificity. Second, all of these RNAs use a complex tertiary architecture to establish the binding pocket. Finally, in each case, ligand binding serves to stabilize a helix that communicates the binding event to the expression platform. Here, we discuss these properties of riboswitches in the context of the purine and SAM-I riboswitches.

Riboswitches: natural SELEXion.

Advances in our knowledge of the structure and chemistry of RNA have been harnessed in the process known as SELEX to develop artificial RNA-based molecules that can act as enzymes and ligand binders performing a wide variety of functions. The discovery of riboswitches, natural RNA aptamers involved in genetic regulation, offers a basis of comparison between the artificial selection and the natural selection of structured RNAs for small-molecule recognition. The guanine riboswitch structural determination allows us to draw conclusions regarding the apparent increased complexity of the riboswitch aptamers compared to their in-vitro-selected cousins.

Intracerebral haemorrhage and hepatitis C treatment.

We report 3 episodes of intracerebral haemorrhage in a population of 1460 hepatitis C infected patients. We suggest the possibility of a link between HCV and its treatment and the occurrence of this serious complication.

Traditional Chinese medicine prevents inflammation in CCl4-related liver injury in mice.

Alternative medicines are being increasingly used and investigated in the management of a variety of disorders. Hepatitis is a common indication for the use of alternative therapies but evidence for the efficacy of many compounds is lacking. We have utilized a well-defined model of liver injury to study the efficacy of three herbal products designed to assist in the management of liver disease. Mice were exposed to carbon tetrachloride (CCL4) given intragastrically after they had been pretreated for five days with either saline or one of four doses of silymarin extract or CH100 (a Chinese herbal medicine comprising of 19 herbs) or one of two doses of CH101 (a Chinese herbal preparation designed to reduce fibrosis). Animals were sacrificed 24 hours after receiving CCL4. Liver enzymes and hepatic histology formed the basis for evaluating efficacy of the treatments. Each of the alternative medicines reduced the alanine amino transferase (ALT) elevation demonstrated after CCL4 injection. The high dose CH100 regimen was most effective in protecting against injury and this was confirmed with hepatic histology. Other doses of CH100, CH101 and silymarin were not shown to provide protection against the histological damage. In conclusion, Silymarin, CH100 and CH101 are able to reduce ALT elevation in animals exposed to CCL4. High dose CH100 provides protection from hepatocyte necrosis in this model. The data add to our understanding of the capacity some herbal medicines have to modify the reaction of the liver to a variety of insults and suggest the value of studying these agents further in human liver diseases.

Induction interferon and ribavirin for re-treatment of chronic hepatitis C patients unresponsive to interferon alone.

BACKGROUND: The optimal treatment for hepatitis C patients unresponsive to interferon is unclear. High-dose induction interferon may enhance early viral clearance, whilst ribavirin reduces relapse; in combination, they may improve sustained virological response rates. AIM: To compare the efficacy and safety of re-treatment with interferon induction, with or without ribavirin, in interferon non-responders. METHODS: We randomized 218 biochemical interferon non-responders to 10 MU interferon alpha 2b daily for 4 weeks, followed by 5 MU thrice weekly for 48 weeks plus ribavirin (II + R), or to the same interferon regimen plus placebo (II + P). All patients were viraemic at entry. RESULTS: The sustained virological response in the II + R group was 39%[95% confidence interval (CI), 30-48%], compared with 16% (95% CI, 9-23%) in the II + P group (P < 0.002). The study drug was discontinued for intolerable symptoms during induction in 9% of the II + R group and in 5% of the II + P group. By logistic regression, a sustained virological response was more likely following II + R treatment (odds ratio, 4.4; 95% CI, 2.1-9.7) and less likely in patients with genotype 1 or 4 (odds ratio, 0.16; 95% CI, 0.07-0.36). CONCLUSION: High-dose induction interferon plus ribavirin is well tolerated and effective for patients unresponsive to interferon alone.

Interleukin 2 modulates ion secretion and cell proliferation in cultured human small intestinal enterocytes.

AIMS: To determine if interleukin 2 (IL-2) alters epithelial transport and barrier function in cultured human small intestinal enterocytes. METHODS: Confluent monolayers of small intestinal cells derived from duodenal biopsies were treated with IL-2 0.2-50 U/ml for 24 hours prior to study. Transport measurements were performed under short circuited conditions in Ussing chambers, with and without the secretagogues forskolin and 3-isobutyl-1-methyl xanthine (IBMX). Serosal to mucosal flux of 3[H] mannitol (permeability) and 3[H] thymidine uptake (proliferation) were measured. IL-2 receptor and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA were identified using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: IL-2 did not alter baseline electrical parameters but caused a significant increase in cAMP dependent chloride secretion. The effect was mediated by the IL-2 receptor and paralleled a rapid increase in tyrosine phosphorylation, janus kinase 1, and signal transducers and activators of transcription (STATs) 1, 3, and 5. IL-2 significantly increased proliferation but at a lower dose than observed for enhanced secretion but did not alter permeability. IL-2 receptor beta and gammac chains and CFTR mRNA were identified by RT-PCR. CONCLUSIONS: IL-2 treatment enhances cAMP stimulated chloride secretion and cellular proliferation in a human small intestinal cell line expressing a functional IL-2 receptor.

Structural and energetic analysis of RNA recognition by a universally conserved protein from the signal recognition particle.

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the endoplasmic reticulum in eukarya or to the inner membrane in prokarya. The crystal structure of the universally conserved RNA-protein core of the Escherichia coli SRP, refined here to 1.5 A resolution, revealed minor groove recognition of the 4.5 S RNA component by the M domain of the Ffh protein. Within the RNA, nucleotides comprising two phylogenetically conserved internal loops create a unique surface for protein recognition. To determine the energetic importance of conserved nucleotides for SRP assembly, we measured the affinity of the M domain for a series of RNA mutants. This analysis reveals how conserved nucleotides within the two internal loop motifs establish the architecture of the macromolecular interface and position essential functional groups for direct recognition by the protein.

A universal mode of helix packing in RNA.

RNA molecules fold into specific three-dimensional shapes to perform structural and catalytic functions. Large RNAs can form compact globular structures, but the chemical basis for close helical packing within these molecules has been unclear. Analysis of transfer, catalysis, in vitro-selected and ribosomal RNAs reveal that helical packing predominantly involves the interaction of single-stranded adenosines with a helix minor groove. Using the Tetrahymena thermophila group I ribozyme, we show here that the near-perfect shape complementarity between the adenine base and the minor groove allows for optimal van der Waals contacts, extensive hydrogen bonding and hydrophobic surface burial, creating a highly energetically favorable interaction. Adenosine is recognized in a chemically similar fashion by a combination of protein and RNA components in the ribonucleoprotein core of the signal recognition particle. These results provide a thermodynamic explanation for the noted abundance of conserved adenosines within the unpaired regions of RNA secondary structures.

Large-scale purification of a stable form of recombinant tobacco etch virus protease.

Tobacco etch virus NIa proteinase (NIa-Pro) has become the enzyme of choice for removing tags and fusion domains from recombinant proteins in vitro. We have designed a mutant NIa-Pro that resists autoproteolytic inactivation and present an efficient method for producing large amounts of this enzyme that is highly pure, active, and stable over time. Histidine-tagged forms of both wild-type and mutant NIa-Pro were overexpressed in E. coli under conditions in which greater than 95% of the protease was in the insoluble fraction after cell lysis. An inclusion body preparation followed by denaturing purification over a single affinity column and protein renaturation yields greater than 12.5 mg enzyme per liter of bacterial cell culture. NIa-Pro purified according to this protocol has been used for quantitative removal of fusion domains from a variety of proteins prepared for crystallization and biochemical analysis.

Shift of the gastric T-cell response in gastric carcinoma.

BACKGROUND AND AIMS: The etiology and pathophysiology of stomach carcinoma is complex, and the mechanism whereby H. pylori directly or indirectly induces carcinoma remains unclear. In this study, interleukin (IL)-8, IL-4 and interferon (IFN)-gamma were measured in the tissue culture supernatant of gastric organ cultures from subjects with chronic gastritis with or without H. pylori infection, and with or without gastric cancer and gastric dysplasia. RESULTS: Interleukin-8 levels were higher in cancer- and H. pylori-infected gastritis subjects than in H. pylori-negative subjects (12.95 +/- 3.16, 10.48 +/- 1.55 and 4.49 +/- 1.28 ng/mL, respectively). Elevated levels of IFN-gamma were detected in both H. pylori-infected and non-infected subjects with uncomplicated gastritis (72.23 +/- 19.0 and 34.61 +/- 5.30 pg/mL) and in non-infected dysplasia subjects (88 +/- 20.5 pg/mL). Background levels of IL-4 (< or = 9.4 pg/mL) in uncomplicated gastritis subjects and relatively high levels of IL-4 in dysplasia subjects (25.8 +/- 7.3 pg/mL) were detected. In contrast, trace amounts of IFN-gamma (16.01 +/- 0.35 pg/mL) and high levels of IL-4 (42.81 +/- 8.49 pg/mL) in gastric biopsy culture supernatants were found in cancer subjects. Mucosal IL-4 levels (but not IL-8 levels) correlated with infection and mucosal anti-H. pylori immunoglobulin G antibody. CONCLUSIONS: The significant differences between gastritis with and without cancer and dysplasia indicated a shift from a Th1 to a Th2 helper cell pattern of cytokine secretion. This study has identified a local mucosal defect in gastric cancer. The near absence of IFN-gamma production from the mucosa at the margins of the tumor may be a critical factor in promoting growth of neoplastic cells.

Multi-component coupling reactions: synthesis of a guanidine containing analog of the hexahydropyrrolo[3,2-c]quinoline alkaloid martinelline.

A multi-component coupling reaction is used to synthesize a highly functionalized guanidine containing pyrroloquinoline analog of martinelline that displays bradykinin B2 receptor antagonist activity.

Non-urease producing Helicobacter pylori in chronic gastritis.

BACKGROUND: Helicobacter pylori infection is the commonest cause of gastritis. Different patterns of immune response to H. pylori infection and characteristics of bacteria are considered to contribute to clinical outcomes. AIM: To determine characteristics of the host H. pylori relationship in subjects with non-ulcer dyspepsia and a histological diagnosis of gastritis. METHODS: Thirty-five subjects with chronic gastritis undergoing endoscopy (mean age 53 years, range 24-82, 14 male and 21 female) were studied, none of whom was on nonsteroidal anti-inflammatory drugs or antibiotics. H. pylori infection was determined by rapid urease test (CLOtest), culture, antibody and RT-PCR for Ure C, Cag A and 26 kDa gene and histology. Cytokine production of mucosal IL-6 and IL-8 were measured by ELISA. RESULTS: Fifteen subjects were positive by CLOtest and/or bacterial culture. In these subjects histology showed numerous helical forms of H. pylori (Group I). Nine subjects were negative by CLOtest, bacterial culture, and mRNA for urease C fragment, but positive by PCR for the 26 kDa protein encoding gene. Histology in these subjects showed the presence of either coccoid forms (four), or scant helical forms (two), or mixed coccoid/helical forms (three) (Group II). Eleven subjects were negative by all methods of detection (Group III). IgG and IgA antibody levels in serum (p<0.05) and gastric tissue culture supernatant (p<0.001) were significantly higher in Group I than those in Group II or III. There were significant differences in the IgG serum and IgA supernatant antibody levels (p<0.01 and p<0.05) when Group II was compared to Group III. Supernatant IL-6 levels were significantly higher in Group I (p<0.01) than those from Groups II and III. IL-8 levels were higher in Group I (p<0.01) and Group II (p<0.05) when compared to Group III. CONCLUSIONS: 'H. pylori-negative' gastritis can be associated with a non-urease producing form of H. pylori, with a reduction in both local and systemic antibody levels and mucosal pro-inflammatory cytokines.

Circulating T-cell response to Helicobacter pylori infection in chronic gastritis.

BACKGROUND: Helicobacter pylori elicits a specific humoral and cellular immune response. There is increasing evidence that the type of T-cell response contributes to clinical outcome in H. pylori infection. MATERIALS AND METHODS: The host response to H. pylori infection in 34 subjects with chronic gastritis was examined in terms of T-cell proliferation and cytokine production in whole-blood cultures stimulated or unstimulated with H. pylori acid-glycine extract antigens (AGE). RESULTS: The proliferative response in whole-blood cultures was similar for both H. pylori-positive and -negative subjects stimulated with H. pylori AGE. While an increase in interferon-gamma (IFN-gamma) production was observed from both H. pylori-positive and -negative subjects with gastritis, significantly higher levels of IFN-gamma were detected in the former when stimulated with H. pylori AGE. In contrast, interleukin 4 (IL-4) was undetectable regardless of antigen stimulation. However, if an in situ IL-4 antibody capture assay was used, antigen-independent production of IL-4 was detected, but there was no difference between H. pylori-positive and -negative subjects with gastritis. After eradication of H. pylori, antigen-induced production of IL-4 was increased, with no decrease in the levels of secretion of IFN-gamma. IL-4 production was dependent on CD4+ T cells, as addition of anti-CD4 but not anti-CD8 mouse monoclonal antibody or matched IgG isotype to the whole-blood culture inhibited the production of IL-4. CONCLUSION: The results suggest that a shift toward a balanced Th1-Th2 response due to an increase in antigen-induced IL-4 production from CD4+ T cells follows eradication. We suggest that the downregulation of mucosal inflammation consequent on reduction in antigen levels or removal of downregulation after eradication of H. pylori contributes to this shift in cytokine balance.

A general synthetic method for the formation of substituted 5-aminotetrazoles from thioureas: a strategy for diversity amplification.

A general method for the synthesis of 5-aminotetrazoles is outlined using the mercury(II)-promoted attack of azide anion on a thiourea. The reaction proceeds through a guanyl azide intermediate, which undergoes electrocyclization to the tetrazole. The method is high yielding and provides access to mono-, di-, and trisubstituted 5-aminotetrazoles, targets of potential interest for combinatorial library development.