Characterisation of fasted state gastric and intestinal fluids collected from children.

Fundamental knowledge about the composition of intestinal fluids in paediatric populations is currently unavailable. This study aimed to characterise gastric and intestinal fluid from paediatric populations. Gastric and intestinal fluid samples were obtained during routine clinical endoscopy from paediatric patients at a large teaching hospital. These fluids were characterised to measure the pH; buffer capacity; osmolality; bile acid concentration and composition. A total of 55 children were recruited to the study aged from 11 months to 15 years of age where 53 gastric fluid samples and 40 intestinal fluid samples were obtained. pH values recorded ranged from pH 0.57 to 11.05 (median: 2.50) in gastric fluids and from 0.89 to 8.97 (median: 3.27) in intestinal fluids. The buffer capacity did not change significantly between gastric and intestinal fluids with median values of 12 mM/L/ΔpH for both fluids. Gastric fluid osmolality values ranged from 1 to 615 mOsm/kg, while intestinal fluid values ranged from 35 to 631 mOsm/kg. Gastric fluid bile acid concentrations ranged from 0.002 to 2.3 mM with a median value of 0.017 mM whilst intestinal fluid bile acid concentrations ranged from 0.0008 to 3.3 mM with a median value of 0.178 mM. Glycocholate; taurocholic acid; glycochenodeoxycholate and taurochenodeoxycholate were the most commonly identified bile acids within paediatric intestinal fluids. All compositional components were associated with large inter-individual variability. Further work is required to develop simulated paediatric media and to explore the impact of these media on drug solubility and dissolution.

Duplication of the A17L locus of vaccinia virus provides an alternate route to rifampin resistance.

UNLABELLED: Specific gene duplications can enable double-stranded DNA viruses to adapt rapidly to environmental pressures despite the low mutation rate of their high-fidelity DNA polymerases. We report on the rapid positive selection of a novel vaccinia virus genomic duplication mutant in the presence of the assembly inhibitor rifampin. Until now, all known rifampin-resistant vaccinia virus isolates have contained missense mutations in the D13L gene, which encodes a capsid-like scaffold protein required for stabilizing membrane curvature during the early stage of virion assembly. Here we describe a second pathway to rifampin resistance involving A17, a membrane protein that binds and anchors D13 to the immature virion. After one round of selection, a rifampin-resistant virus that contained a genomic duplication in the A17L-A21L region was recovered. The mutant had both C-terminally truncated and full-length A17L open reading frames. Expression of the truncated A17 protein was retained when the virus was passaged in the presence of rifampin but was lost in the absence of the drug, suggesting that the duplication decreased general fitness. Both forms of A17 were bound to the virion membrane and associated with D13. Moreover, insertion of an additional truncated or inducible full-length A17L open reading frame into the genome of the wild-type virus was sufficient to confer rifampin resistance. In summary, this report contains the first evidence of an alternate mechanism for resistance of poxviruses to rifampin, indicates a direct relationship between A17 levels and the resistance phenotype, and provides further evidence of the ability of double-stranded DNA viruses to acquire drug resistance through gene duplication. IMPORTANCE: The present study provides the first evidence of a new mechanism of resistance of a poxvirus to the antiviral drug rifampin. In addition, it affirms the importance of the interaction between the D13 scaffold protein and the A17 membrane protein for assembly of virus particles. Resistance to rifampin was linked to a partial duplication of the gene encoding the A17 protein, similar to the resistance to hydroxyurea enabled by duplication of the gene encoding the small subunit of ribonucleotide reductase and of the K3L gene to allow adaptation to the antiviral action of protein kinase R. Gene duplication may provide a way for poxviruses and other DNA viruses with high-fidelity DNA polymerases to adjust rapidly to changes in the environment.

Reverse genetics analysis of poxvirus intermediate transcription factors.

Vaccinia virus transcription is regulated in three stages. An intermediate transcription factor, comprised of virus-encoded polypeptides A8 and A23, was previously identified by in vitro analyses. To investigate its role, we engineered cells that stably expressed both subunits and complemented the replication of A8 and A23 deletion mutant viruses. Without A8 or A23, viral early gene expression and DNA replication occurred but intermediate and late gene expression and resolution of genome concatemers were not detected.

Duration of etanercept treatment and reasons for discontinuation in a cohort of juvenile idiopathic arthritis patients.

OBJECTIVE: Since 2004, juvenile idiopathic arthritis (JIA) patients treated with etanercept and/or MTX have been monitored in the British Society for Paediatric and Adolescent Rheumatology Biologics and New Drug Register. Here, we report the duration of etanercept use for the first 5 years of the register and reasons for discontinuation. METHODS: Disease subtype and activity, comorbidity, treatment efficacy and safety data were recorded. Etanercept discontinuation was defined as stopping the drug because of disease remission or treatment failure. Time to discontinuation was explored using Kaplan-Meier survival analysis with remaining patients censored at 5-year follow-up. RESULTS: A total of 483 etanercept-treated JIA patients were enrolled from 30 UK centres, representing 941 patient-years of follow-up. A total of 100 (20.7%) patients discontinued etanercept; 9 due to disease control, 88 because of treatment failure, 2 for unknown reasons and 1 because of a change in diagnosis. Of the 53 patients in whom etanercept was perceived to be ineffective at controlling the inflammation, 48 were prescribed other biologic drugs [26/48 (54%) infliximab]. In 21 patients with intolerance, infections, CNS events and a few isolated events were associated with discontinuation. Using Kaplan-Meier analysis, at 5 years 69% (95% CI 61, 77%) had not experienced treatment failure. Discontinuation of etanercept for inefficacy was associated with systemic arthritis subtype [odds ratio (OR) 2.55, 95% CI 1.27, 5.14], chronic anterior uveitis (OR 2.39, 95% CI 1.06, 5.35) and inefficacy of MTX before starting etanercept (OR 8.3, 95% CI 1.14, 60.58). CONCLUSIONS: In a cohort of JIA patients treated with etanercept and followed for a median of 2 years (maximum 5 years), the majority (69%) remain on the drug.

Elucidating and minimizing the loss by recombinant vaccinia virus of human immunodeficiency virus gene expression resulting from spontaneous mutations and positive selection.

While characterizing modified vaccinia virus recombinants (rMVAs) containing human immunodeficiency virus env and gag-pol genes, we detected nonexpressing mutants by immunostaining individual plaques. In many cases, the numbers of mutants increased during successive passages, indicating strong selection pressure. This phenomenon provided an opportunity to investigate the formation of spontaneous mutations in vaccinia virus, which encodes its own cytoplasmic replication system, and a challenge to reduce the occurrence of mutations for vaccine production. Analysis of virus from individual plaques indicated that loss of expression was due to frameshift mutations, mostly by addition or deletion of a single nucleotide in runs of four to six Gs or Cs, and large deletions that included MVA DNA flanking the recombinant gene. Interruption of the runs of Gs and Cs by silent codon alterations and moving the recombinant gene to a site between essential, highly conserved MVA genes eliminated or reduced frameshifts and viable deletion mutants, respectively. The rapidity at which nonexpressing mutants accumulated depended on the individual env and gag-pol genes and their suppressive effects on virus replication. Both the extracellular and transmembrane domains contributed to the selection of nonexpressing Env mutants. Stability of an unstable Env was improved by swapping external or transmembrane domains with a more stable Env. Most dramatically, removal of the transmembrane and cytoplasmic domains stabilized even the most highly unstable Env. Understanding the causes of instability and taking preemptive actions will facilitate the development of rMVA and other poxviruses as human and veterinary recombinant vaccines.