Clinical stage drugs targeting inhibitor of apoptosis proteins purge episomal Hepatitis B viral genome in preclinical models

A major unmet clinical need is a therapeutic capable of removing hepatitis B virus (HBV) genome from the liver of infected individuals to reduce their risk of developing liver cancer. A strategy to deliver such a therapy could utilize the ability to target and promote apoptosis of infected hepatocytes. Presently there is no clinically relevant strategy that has been shown to effectively remove persistent episomal covalently closed circular HBV DNA (cccDNA) from the nucleus of hepatocytes. We used linearized single genome length HBV DNA of various genotypes to establish a cccDNA-like reservoir in immunocompetent mice and showed that clinical-stage orally administered drugs that antagonize the function of cellular inhibitor of apoptosis proteins can eliminate HBV replication and episomal HBV genome in the liver. Primary human liver organoid models were used to confirm the clinical relevance of these results. This study underscores a clinically tenable strategy for the potential elimination of chronic HBV reservoirs in patients.Subject terms: Target validation, Hepatitis B, Preclinical research, Translational research     

IgA immunodeficiency leads to inadequate Th cell priming and increased susceptibility to influenza virus infection

IgA is considered to be the principal Ab involved in defense against pathogens in the mucosal compartment. Using mice with a targeted disruption in IgA gene expression (IgA(-/-) mice), we have examined the precise role of IgA in protective anti-influenza responses after intranasal vaccination. IgA(-/-) mice immunized intranasally with soluble hemagglutinin (hemagglutinin subtype 1) and neuraminidase (neuraminidase subtype 1) vaccine in the absence of adjuvant were found to be more susceptible to influenza virus infection than IgA(+/+) mice (13 vs 75% survival after virus challenge). Inclusion of IL-12 during immunization restored the protective efficacy of the vaccine to that seen in IgA(+/+) animals. IgA(-/-) mice had no detectable IgA expression, but displayed enhanced serum and pulmonary IgM and IgG Ab levels after IL-12 treatment. Assessment of T cell function revealed markedly depressed splenic lymphoproliferative responses to PHA in IgA(-/-) animals compared with IgA(+/+) mice. Furthermore, IgA(-/-) animals displayed impaired T cell priming to the H1N1 subunit vaccine, with concomitant reduction in recall memory responses due to a defect in APC function. Collectively, these results provide evidence that a major role of IgA is to facilitate presentation of Ag to mucosal T cells. IL-12 treatment can overcome IgA deficiency by providing adequate T cell priming during vaccination.     

Fc receptor-mediated phagocytosis makes a significant contribution to clearance of influenza virus infections

Fc receptors for IgG expressed on macrophages and NK cells are important mediators of opsonophagocytosis and Ab-dependent cell-mediated cytotoxicity. Phagocyte-mediated opsonophagocytosis is pivotal for protection against bacteria, but its importance in recovery from infection with intracellular pathogens is unclear. We have now investigated the role of opsonophagocytosis in protection against lethal influenza virus infection by using FcR gamma(-/-) mice. Absence of the FcR gamma-chain did not affect the expression of IFN-gamma and IL-10 in the lungs and spleens after intranasal immunization with an influenza subunit vaccine. Titers of serum and respiratory Abs of the IgM, IgG1, IgG2a, and IgA isotypes in FcR gamma(-/-) mice were similar to levels seen in FcR gamma(+/+) mice. Nevertheless, FcR gamma(-/-) mice were highly susceptible to influenza infection, even in the presence of anti-influenza Abs from immune FcR gamma(+/+) mice. NK cells were not necessary for the observed Ab-mediated viral clearance, but macrophages were found to be capable of actively ingesting opsonized virus particles. We conclude that Fc receptor-mediated phagocytosis plays a pivotal role in clearance of respiratory virus infections.     

A fault detection service for wide area distributed computations

The potential for faults in distributed computing systems is a significant complicating factor for application developers. While a variety of techniques exist for detecting and correcting faults, the implementation of these techniques in a particular context can be difficult. Hence, we propose a fault detection service designed to be incorporated, in a modular fashion, into distributed computing systems, tools, or applications. This service uses well-known techniques based on unreliable fault detectors to detect and report component failure, while allowing the user to trade off timeliness of reporting against false positive rates. We describe the architecture of this service, report on experimental results that quantify its cost and accuracy, and describe its use in two applications, monitoring the status of system components of the GUSTO computational grid testbed and as part of the NetSolve network-enabled numerical solver. This revised version was published online in July 2006 with corrections to the Cover Date.     

Suppression of growth hormone by oral glucose in the evaluation of tall stature

Excess secretion of growth hormone is a rare diagnosis in children or adolescents with tall stature. An oral glucose tolerance test (OGT) with determination of growth hormone is generally recommended to exclude this disorder. In order to test the validity of this approach in pediatric subjects, OGT tests were performed in 126 tall subjects (age: 12.4 +/- 1.8 years; height: 3.1 +/- 0.8 SDS). Nonsuppression was present in 39 subjects, however, anthropometric analysis and follow-up excluded the diagnosis of eosinophilic pituitary adenoma in all patients. The lowest GH concentration was reached 90 min after ingestion of oral glucose, GH rose above baseline at 180 min. Plasma concentrations of glucose and insulin did not differ between suppressors and nonsuppressors. In conclusion, absent suppression of growth hormone by oral glucose is common in tall children and adolescents. The test is therefore not recommended as a general screening for excess growth hormone. Prolonging the test beyond 120 min does not increase the diagnostic value.     

Diazaborine treatment of yeast cells inhibits maturation of the 60S ribosomal subunit

Diazaborine treatment of yeast cells was shown previously to cause accumulation of aberrant, 3'-elongated mRNAs. Here we demonstrate that the drug inhibits maturation of rRNAs for the large ribosomal subunit. Pulse-chase analyses showed that the processing of the 27S pre-rRNA to consecutive species was blocked in the drug-treated wild-type strain. The steady-state level of the 7S pre-rRNA was clearly reduced after short-term treatment with the inhibitor. At the same time an increase of the 35S pre-rRNA was observed. Longer incubation with the inhibitor resulted in a decrease of the 27S precursor. Primer extension assays showed that an early step in 27S pre-rRNA processing is inhibited, which results in an accumulation of the 27SA2 pre-rRNA and a strong decrease of the 27SA3, 27SB1L, and 27SB1S precursors. The rRNA processing pattern observed after diazaborine treatment resembles that reported after depletion of the RNA binding protein Nop4p/Nop77p. This protein is essential for correct pre-27S rRNA processing. Using a green fluorescent protein-Nop4 fusion, we found that diazaborine treatment causes, within minutes, a rapid redistribution of the protein from the nucleolus to the periphery of the nucleus, which provides a possible explanation for the effect of diazaborine on rRNA processing.     

Feeling by sight or seeing by touch?

We have addressed the role of occipital and somatosensory cortex in a tactile discrimination task. Sight-ed and congenitally blind subjects rated the roughness and distance spacing for a series of raised dot patterns. When judging roughness, intermediate dot spacings were perceived as being the most rough, while distance judgments generated a linear relation. Low-frequency rTMS applied to somatosensory cortex disrupted roughness without affecting distance judgments, while rTMS to occipital cortex disrupted distance but not roughness judgments. We also tested an early blind patient with bilateral occipital cortex damage. Her performance on the roughness determination task was normal; however, she was greatly impaired with distance judgments. The findings suggest a double-dissociation effect in which roughness and distance are primarily processed in somatosensory and occipital cortex, respectively. The differential effect of rTMS on task performance and corroborative clinical evidence suggest that occipital cortex is engaged in tactile tasks requiring fine spatial discrimination.     

Divergent segmentation mechanism in the short germ insect Tribolium revealed by giant expression and function

Segmentation is well understood in Drosophila, where all segments are determined at the blastoderm stage. In the flour beetle Tribolium castaneum, as in most insects, the posterior segments are added at later stages from a posteriorly located growth zone, suggesting that formation of these segments may rely on a different mechanism. Nevertheless, the expression and function of many segmentation genes seem conserved between Tribolium and Drosophila. We have cloned the Tribolium ortholog of the abdominal gap gene giant. As in Drosophila, Tribolium giant is expressed in two primary domains, one each in the head and trunk. Although the position of the anterior domain is conserved, the posterior domain is located at least four segments anterior to that of Drosophila. Knockdown phenotypes generated with morpholino oligonucleotides, as well as embryonic and parental RNA interference, indicate that giant is required for segment formation and identity also in Tribolium. In giant-depleted embryos, the maxillary and labial segment primordia are normally formed but assume thoracic identity. The segmentation process is disrupted only in postgnathal metamers. Unlike Drosophila, segmentation defects are not restricted to a limited domain but extend to all thoracic and abdominal segments, many of which are specified long after giant expression has ceased. These data show that giant in Tribolium does not function as in Drosophila, and suggest that posterior gap genes underwent major regulatory and functional changes during the evolution from short to long germ embryogenesis.