New horizons in translational medicine最新文献

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus. During primary infection, VZV causes varicella (chicken pox), after which the virus go latent in ganglionic neurons along the entire neuraxis before reactivating decades later to cause zoster (shingles). Interferon gamma (IFNγ), produced during viral infection, stimulates transcription of genes that mediate antiviral responses. Herein, it was tested whether IFNγ treatment of human neurons inhibits VZV infection of human neurons in vitro. Infected neurons not treated with IFNγ developed a cytopathic effect in 4 weeks, during which time VZV DNA increased 7-fold and viral RNA accumulated. Infected neurons cultured in the presence of IFNγ for 8 weeks or infected neurons cultured in IFNγ for 4 weeks followed by cytokine removal for an additional 4 weeks had only a 2.8- and 3.6-fold increase of viral DNA, respectively in the 8 weeks post-infection. Furthermore, levels of VZV transcripts did not increase between 4 and 8 weeks post-infection when IFNγ was removed at 4 weeks post-infection, and even began to decrease when the cultures were maintained in IFNγ for the entire 8 weeks. In accordance with reduced DNA accumulation and mRNA levels when infected neurons were maintained in IFNγ, less CPE was evident at 8 weeks post-infection compared to cultures which had IFNγ removed at 4 weeks post-infection. Replication of VZV DNA and transcription of viral genes was inhibited by IFNγ, and the extent of virus gene expression in IFNγ-treated neurons compared to VZV expression in latently infected human ganglia remains to be determined.

Biomarkers have shown to improve success rates in the development of novel drugs, providing essential information in the early phases of clinical development for decision-making. Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is pursued as a drug target for a number of inflammatory diseases. CRTH2 antagonists block the activation and migration of key inflammatory cells such as eosinophils, basophils, and Th2 cells. The mechanism of action of CRTH2 antagonists was established in cells isolated from human blood. Biomarkers derived from these experiments were included in clinical studies to investigate the mechanism of action and potency of CRTH2 antagonists in human. For clinical phase I studies with the CRTH2 antagonist ACT-453859, a follow-up molecule of setipiprant, inclusion of the most precise and robust pharmacodynamic (PD) biomarker with a clinically relevant target effect was desired to aid phase II dose selection.

Candidate biomarkers such as IL-13 secretion from Th2 cells and CRTH2, CD11b and CD203 modulation on basophils and eosinophils in whole blood were compared in terms of signal intensity and variability. Blockade of CRTH2 receptor internalization was finally chosen as PD biomarker and rigorously tested in a feasibility study. The assay showed excellent robustness, an intra-assay precision of 5% and inter-subject variability smaller than 15%. Based on phase II clinical study results with setipiprant, 90% CRTH2 receptor blockade was defined as clinically relevant PD effect. This target PD effect provides the means to take decisions based on the data generated in the phase I clinical studies with ACT-453859.

Focal points

• •

  Bedside

  Biomarkers offer a great potential to influence decisions taken during early clinical development. For clinical phase I studies with the CRTH2 antagonist ACT-453859, a follow-up molecule of setipiprant, inclusion of a biomarker was desired to aid phase II dose selection. In order to facilitate decision-making, we developed a biomarker that delivers high quality data under clinical circumstance and defined a relevant target biomarker effect.

• •

  Benchside

  In-vitro experiments with human whole blood identified CRTH2 receptor internalization on basophils and eosinophils as the most precise and robust biomarker. Clinical results obtained with setipiprant in a seasonal allergic rhinitis study were used to define the clinically relevant target biomarker effect of 90% CRTH2 receptor blockade. Proof for the chosen target biomarker effect remains to be demonstrated in phase II clinical studies with ACT-453859.

Chronic wasting disease (CWD) is the transmissible spongiform encephalopathy (TSE), or prion disease, of free-ranging and captive cervids (deer, elk and moose). The presence of sufficient infectious prions in the tissues, bodily fluids (urine, saliva, and blood) and environments of clinical and preclinical CWD-infected animals is thought to account for its high transmission efficiency. Recently it has been recognized that transmission from mother to offspring may contribute to the facile transmission of some TSEs. Although the mechanism of maternal transmission has yet to be elucidated, the extended asymptomatic TSE carrier phase, lasting years to decades, suggests that maternal transmission may have implications in the spread of prions.

Placental trafficking and/or secretion in milk are two means by which maternal prion transmission may occur. In these studies we explore CWD maternal transmission during early and late CWD infection using a transgenic mouse model (TgCerPRP) expressing cervid prion protein. Naïve and CWD-infected dams were bred during early (45 dpi) and late (120 dpi) infection and were allowed to bear and raise their offspring. Milk was collected from the dams for prion analysis, and the offspring were observed for TSE disease progression. Terminal tissues harvested from these dams and offspring were analyzed for prions.

We have demonstrated: 1) that CWD-infected TgCerPRP females successfully breed and bear offspring, 2) the presence of PrP^CWD in reproductive and mammary tissue harvested from CWD-infected dams, and 3) clinical disease progression in offspring born to CWD-infected dams. We are currently analyzing terminal tissue harvested from offspring born to CWD-infected dams for the detection of PrP^CWD and amplification competent prions. These studies will provide insight into the potential mechanisms and biological significance associated with mother to offspring transmission of TSEs.

Alphaviruses most often associated with neuroinvasive disease are limited to the Americas and include strains of EEEV, VEEV, and WEEV. The process of alphavirus entry into the CNS of infected vertebrates following challenge is not well-understood. It is thought that virus entry into the CNS depends on the inoculation route. It is well-established that olfactory sensory neurons provide access to the CNS following challenge with airborne virus. However, less knowledge is available regarding virus entry into the CNS following peripheral, non-olfactory infection, which appears to rely on some form of hematogenous spread. We sought to determine the precise route of CNS entry following footpad inoculation by using a combination of in vivo/ex vivo bioluminescence imaging and traditional histological examination methods. We found a consistent pattern in the spatiotemporal distribution of virus among the imaged brains, none of which involved the olfactory bulb. Extending these studies by performing histological analysis on the imaged tissues, led to the finding that CNS entry by WEEV likely occurs in areas of the CNS where the blood-brain barrier is naturally absent. These areas include the hypothalamus, the subfornical organ, the pineal gland, and the area postrema. Importantly, these results reveal a previously unrecognized method of alphavirus entry into the CNS.

Tacaribe virus (TCRV) is a bisegmented, ambisense, RNA virus within the genus Arenavirus. Arenaviruses are grouped into Old World lymphocytic choriomeningitis-Lassa virus complex and the New World Tacaribe complex viruses. TCRV is placed within the Tacaribe complex along with the South American hemorrhagic fever viruses: Chapare, Guanarito, Junin, Machupo, and Sabia viruses. The only isolates of TCRV were from 11 artibeus bats collected by investigators at the Trinidad Regional Virology Laboratory in the Republic of Trinidad in the 1950 s. TCRV has not been isolated since, although serological data from the 1970 s suggested it may circulate among Caribbean bats. Only one isolate remains, TRVL-11573, and it has been passaged in suckling mice and Vero cells. We sought to determine if TCRV is still circulating in bat populations in Trinidad through serological investigation. We developed an ELISA and western blot assay using His-tagged recombinant TCRV nucleocapsid antigen. Serum from Artibeus jamaicensis that had been experimentally infected with TCRV was used as a positive control, and serum collected from an uninfected A. jamaicensis used as a negative control. ELISA screen of bloods from 84 bats of various species captured in Trinidad identified several, mostly artibeus bats, as seropositive for antibodies to TCRV. Some of these were tested by western blot. Four were negative, eight were weakly positive, and five were strongly positive. These results suggest that TCRV or other arenaviruses continue to circulate among bats in Trinidad.

Dengue virus (DENV) infection is a significant global health concern with over 40% of the world’s population at risk and currently no therapeutics or vaccines available. Understanding host viral interactions is key to developing novel therapeutic options. Dengue virus is a positive sense RNA virus that induces the formation of invaginations in the endoplasmic reticulum to replicate its genome. Increased phospholipid biosynthesis is key to the formation of these replication compartments as well as viral maturation and release. It is now evident that viral proteins mediate this change in the cellular phospholipid repertoire, but the precise mechanisms are unknown. We have shown that siRNA mediated knockdown as well as pharmacological inhibition of key enzymes in the unsaturated phospholipid biosynthesis pathway reduces DENV replication. Unsaturated fatty acids, when incorporated into membrane phospholipids are a key mechanism for providing fluidity and curvature of membranes enhancing the assembly and function of membrane bound enzymes. Several of the enzymes are conserved from bacteria to mammals and are high profile therapeutic targets for obesity, hepatic steatosis and metabolic disease. This indicates a novel pathway for drug discovery and exploration of viral host interactions. We will discuss mechanistic details of how this pathway influences DENV replication.

The availability of broad epigenomic profiles of human tissues provides an opportunity to uncover viral sequences and their corresponding functional regulatory elements in otherwise overlooked datasets. We developed Viralign, a throughput screening method to discover and interpret viral functional information in existing short read archive data. Using a comprehensive reference database, Viralign scans sequence data for known viral sequences and generates an alignment report with read information and genome coverage. Viralign analyzes functional datasets for regulatory elements and provides coordinate and visualization files that can be viewed in a genome browser. Additionally, this method searches for potential integration sites and variants by genome assembly. In a pilot study, we performed H3K27me3 ChIP-seq in monocytes of an HHV6 infected individual and compared this to U2OS cells infected with HHV6A and HHV6B and use Viralign to detect HHV6 insertion loci and H3K27me3 enriched regions. The source code as well as additional data for Viralign will be made publicly available.

Aedes aegypti mosquitoes are the primary vectors transmitting dengue virus (DENV), one of the most aggressive re-emerging pathogens worldwide causing more than 390 million infections per year. The spread of the virus is greatly dependent upon successful replication within both the human host and mosquito vector. Much effort has been placed in understanding the dynamics of virus transmission and replication in both organisms, but little is known about the global impact of DENV on metabolic pathways. Previous studies have demonstrated perturbations in human and Aedes albopictus cellular metabolic environments during DENV infection. Some of these perturbations include increasing the production of membranous lipids that had the capability to induce membrane curvature and permeability, as well as visibly altering both human and mosquito intracellular membrane architecture to support DENV replication. In this study, we have explored metabolic changes in Aedes aegypti midgut and salivary glands upon DENV (serotype 2) infection. We have found several significant fluctuations in the lipid and metabolite repertoire from infected tissues compared to uninfected controls, including differential expression of molecules that function as membrane building blocks, bioactive messengers, energy storage and intermediates in lipid biosynthesis and lipolysis pathways. These results and their relevance to dengue virus infection of its mosquito vector will be discussed.