New horizons in translational medicine最新文献

Previous studies have reported that domestic cats can be naturally infected with bovine herpesvirus 4 (BHV4), and experimental inoculations have been linked to feline urolithiasis. It has been difficult to recapitulate initial diagnostic and experimental observations, thus here we have initiated a study to evaluate BHV4 presence in a large cohort of cats at risk for exposure to circulating feline viruses using a sensitive and specific assay. Domestic cat blood DNA samples (n=101) collected from California, Colorado, and Florida were screened for BHV4 using sensitive real time PCR. In contrast to BHV4 containing tissue culture extracts, all domestic cat blood samples were negative for BHV4. Samples were shown to contain intact DNA and to be infected with other horizontally-transmitted feline infections. We conclude that BHV4 is unlikely to be a common pathogen of domestic cats.

Prions are infectious proteins capable of self-propagating and transmitting between organisms. Even though there is no homolog to the mammalian prion protein in yeast, several soluble proteins can form heritable aggregates de novo. These proteins provide a model system to investigate the nucleation, aggregation and propagation steps involved in the formation of a prion fibril. Several prion prediction algorithms have been developed to predict yeast proteins that have the propensity to form prions. One of these algorithms was previously developed in our laboratory (Prion Aggregation Prediction Algorithm, PAPA, Toombs et al., 2012). Therefore, we used PAPA to scan the yeast proteome to extract proteins that contain domains predicted to have prion activity (prion-like domains). These prion-like domains will be tested in four prion activity assays to assess their activity in vivo as well as in vitro. Here we provide preliminary evidence that we are successful at predicting yeast proteins that present prion activity in vivo. Following characterization of these prion-like domains, we will test the respective full-length proteins for prion activity using microscopy as well as developing phenotypic assays. Ultimately, we may identify new prion candidates in yeast, which will contribute information about the parameters necessary for prion formation and insight into the functions prions play in yeast. In addition, by confirming PAPA’s ability to predict prion proteins from the yeast proteome, it allows the possibility to apply this methodology to other proteomes.

Three major vaccine manufacturers in the United States currently sell multivalent vaccines containing modified live bovine herpesvirus 1 (BoHV-1) for use in pregnant cattle. The first of these products entered the US market in 2003. Yet it has been known since the early 1960 s that vaccinal BoHV-1 causes abortion in cattle. The products became popular as they can be used year-round, regardless of pregnancy status in herds. Abortifacient effects have been considered to be minimal, provided initial vaccination is done during the previous 12 months using specific vaccine products and in accordance with label directions. Single nucleotide polymorphisms (SNPs) in BoHV-1 can be used to resolve whether post-vaccination outbreaks of abortion in cattle herds are iatrogenic (Fulton et al.; Vaccine. 2013; 31(11):1471-1479). We tested tissues from 10 abortion episodes (2010–2014) where an apparent association existed between recent use of modified live BoHV-1 and abortion 1–3 months later. Products were used on or off label in individual outbreaks. All 10 episodes had SNP patterns consistent with those of commonly-used modified live BoHV-1 strains (O’Toole et al.; Vet Pathol. 2014, In press). In spite of this, it is likely such products will remain on the market. This is due the absence of meaningful post-marketing surveillance of suspect adverse reactions in animals by the USDA, compounded by the courts’ interpretation of the Virus-Serum-Toxin Act of 1913 [Lynnbrook Farms v. SmithKline Beecham Corp., 79 F.3d 620 (7th Cir.)]. Interesting differences exists between the handling of adverse vaccinal reactions in human patients through the National Vaccine Injury Compensation Program (VICP), and similar reactions in animals following use of federally licensed vaccines.

Alphaviruses are globally distributed, mosquito borne pathogens that cause death and disease in vertebrates, including humans. Therapeutics to combat alphaviral disease are non-existant and only a handful of IND status vaccines are available. Of the available vaccines most are associated with a poor immunological response and a high rate of reactivity, and none protects against more than a single alphavirus species. We designed and tested novel alphavirus vaccines comprised of the E1 glycoproteins of western equine encephalitis virus (WEEV) or Venezuelan equine encephalitis virus (VEEV). Immunization with cationic lipid nucleic acid complexes (CLNCs) and alphavirus E1ecto mixture (lipid-antigen-nucleic acid complexes:LANACs) provided significant protection in mice challenged with either WEEV, VEEV or eastern equine encephalitis virus (EEEV) regardless of challenge route. LANAC immunized mice mount a strong humoral immune response lacking neutralizing antibody. Passive transfer of immune sera from LANAC immunized mice to non-immunized mice confers protection to challenge, indicating that non-neutralizing antibody is sufficient for protection. In summary, our LANAC vaccine has both therapeutic and prophylactic potential and is able to offer protection against distinct alphavirus species irrespective of the route of infection.

Varicella zoster virus (VZV) causes varicella (chickenpox), establishes latency in ganglia and reactivates decades later to produce zoster in the elderly. Clinical, pathological, immunological and virological features of simian varicella virus (SVV) infection of primates parallel human VZV infection. Primary SVV infection of primates, cause varicella, after which virus becomes latent in ganglionic neurons and reactivates upon social and environmental stress. Five rhesus macaques were infected intrabronchially with 4.0x10⁵ pfu of SVV. Two weeks later, the monkeys developed varicella rash. Twenty months later four of the monkeys were treated once with a 50 mg/kg of anti-CD4 antibody. All 5 monkeys developed zoster rash, 7–55 days after the treatment. Punch biopsies of the skin rash were analyzed for the presence of SVV antigens by immunohistochemistry and immunofluorescence. SVV ORF 63 protein and glycoproteins gH and L were detected in sweat glands in skin from all 5 monkeys. Presence of SVV in the β-3-tubilin-positive nerve endings in affected skin suggested possible route of skin infection during zoster.

Poliovirus eradication is one of the most challenging public health endeavors in modern times (www.polioeradication.org). Social, political, economic & scientific factors have made this goal elusive. When eradication goals were first established in 1988, there was little appreciation of viral RNA recombination, enterovirus species groups and their relevance to eradication. Now, it is clear that RNA recombination between live-attenuated vaccine strains of poliovirus and non-polio group C enteroviruses results in circulating vaccine-derived polioviruses (cVDPV) and corresponding outbreaks of paralytic disease, a significant obstacle to eradication. By understanding enterovirus species groups, it becomes clear that poliovirus capsid proteins can be eradicated; however, the remainder of poliovirus RNA genomes will survive indefinitely in other group C enteroviruses. To help address these obstacles to eradication, the Barton lab studies molecular features of 3D^pol involved in viral RNA replication and recombination. A dsRNA clamp of 3D^pol that holds RNA products of replication as they exit the polymerase plays important roles in the polyadenylation of viral RNA, the fidelity of RNA replication, ribavirin sensitivity and viral RNA recombination. In other experiments, we identified a group C enterovirus RNA involved in the inhibition of ribonuclease L, an antiviral endoribonuclease. The RNase L ciRNA plays important but largely unexplored roles in pathogenesis. Using novel deep sequencing methods, we found that RNase L targets viral RNA encoding neutralizing epitopes of capsid proteins, sparing most other regions of viral RNA. These data suggest an important interplay between neutralizing antibodies, neutralization escape mutations and antiviral endoribonucleases. A better understanding of viral RNA recombination, enterovirus species groups and antiviral endoribonucleases should help achieve and maintain poliovirus eradication.

Bardet–Biedl syndrome (BBS) is a rare, multisystemic, genetic disease and member of a group of disorders called ciliopathies. This syndrome provides a mechanistic model for ciliopathies that may also extend to common disorders with complex inheritance patterns, including diabetes mellitus and obesity. Dysregulation of signaling pathways altering the cellular response to the extracellular environment is primary to the ciliopathies and characteristic of BBS. As BBS-centered translational research moves forward, innovative advances provide opportunities to improve the care of individuals with BBS and other rare diseases as well as common related conditions. This review aims to highlight the current understanding of the mechanisms underlying BBS and opportunities for advancing the care of individuals with rare diseases.

Focal points

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  Bedside: understanding the multi-dimensional manifestations of ciliopathies, specifically Bardet–Biedl Syndrome (BBS) as a model ciliopathy, will accelerate research into therapeutic targets for ciliopathies, allowing for improved therapies for individuals with these debilitating disorders.

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  Benchside: elucidating the molecular mechanisms of BBS is likely to increase the chance of discovering novel therapeutic approaches that may be generalizable to other ciliopathies and perhaps to common related disorders, such as obesity and diabetes mellitus.

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  Industry: application of known drugs to new indications, or drug repositioning, and development of novel therapeutics, including gene therapies in BBS, may open new avenues for therapeutic discovery and development.

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  Community: rare diseases affect millions of individuals throughout the world with significant impact on quality of life and longevity. The development of multidisciplinary clinics for BBS and effective implementation of a rare disease registry provides a model for advancing the care of individuals with rare diseases.

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  Government and Regulatory Agencies: the importance of rare disease research and the impact of that research on common disorders should be supported with adequate funding and resources. Understanding the molecular pathways underlying ciliopathies, such as BBS, and advancement of translational medicine in ciliopathies will have far reaching societal benefits

Picornaviruses cause a wide range of ailments, including myocarditis, poliomyelitis, and vesicular lesion type diseases. Excellent vaccines exist for several of them, and the development of the live-attenuated oral polio vaccine (OPV) provided an efficient and cost-effective avenue for successful poliovirus eradication in the majority of the world. However, one hurdle for developing successful live-attenuated vaccines lies with the viral RNA-dependent-RNA-polymerase (RdRP) enzyme whose low replication fidelity allows for reversion of attenuated viruses to disease causing variants. Improving the replication fidelity of RdRPs is an attractive avenue for virus attenuation because it may curtail such reversion issues. We have previously solved the crystal structures of several picornaviral polymerase-RNA complexes that show the structural changes taking place within these polymerases during active site closure and catalysis (Gong et al., 2010, 2013). Based on this, we engineered a panel of fidelity variant coxsackievirus B3 polymerases that caused reduced infectivity and attenuated virus growth in mice (Gnädig et al., PNAS, 2012). We hypothesize that such modulation of polymerase fidelity via structure based protein engineering can provide an effective platform to improve the design of live-attenuated vaccines. To investigate this further we have generated over a dozen mutations in the poliovirus Sabin 2 strain polymerase and carried out in vitro biochemical assays to show that these can either increase or decrease polymerase fidelity while having minor effects on elongation rates and processivity. The fidelity modulation can arise from single point mutations, multi-site mutations that replace entire groups of interacting residues, or from grafting in structurally homologous sequences from related polymerases. The data suggests mutations in the palm domain of the poliovirus RdRP can serve as efficient fidelity modulation sites for protein engineering purposes, and we are now seeking to test these variant polymerases in an infectious virus context.

Approximately 150 million people and almost $40 billion worth of agricultural commodities go through U.S. international ports annually. Ports seize animal and plant products potentially contaminated with high risk diseases that then must be decontaminated before entering the waste stream. Currently, there are only 3 methods of decontamination accepted by the Animal Plant and Health Inspection Service at U.S. ports and borders including incineration, high temperature cooking, and discharge of ground waste as sewage. In this study we assess the efficacy of a relatively new decontamination technology, alkaline digestion, to mitigate infectious agents. Transmissible Spongiform Encephalopathies (TSEs), a member of the protein misfolding diseases (ex: Alzheimer’s and Parkinson’s Diseases), were chosen as the infectious agent for this study because they rank as the hardest to kill microbe/pathogen, affect both human and animal species worldwide and are shed by infected hosts into the environment establishing highly infectious biota. Chronic wasting disease (CWD), an emerging TSE of cervid species (deer, elk, moose) in North America, has recently been spotlighted as a potential concern for European countries, and recapitulates human and animal TSE pathogenesis and shedding. For these reasons CWD is ideal for mitigation studies. We processed CWD positive and negative materials by alkaline digestion under standard temperature and pressure at time intervals of 2, 4, and 6 h. Samples were retrieved after digestion, were neutralized and inoculated intracerebrally into transgenic mice expressing the cervid protein to determine remaining prion infectivity. In addition, the samples (pre and post alkaline digestion) were tested for amplification competent prions by Protein Misfolding Cyclic Amplification (PMCA). Preliminary results suggest a lack of amplification competent prions in samples processed by alkaline digestion at 2, 4, and 6 h cycles as compared to nondigested samples. This work will provide a basis for future studies designed to unravel the mechanisms associated with the ability of prions to bind surfaces enhancing prion mitigation strategies for TSEs and by extension, other protein misfolding diseases.

Progress in the field of translational medicine (TM) within the last decade attests to the importance of the TM initiative in the context of more traditional academic health science centers. In many instances, these advancements have taken place without a clear definition of TM, which signifies the urgent need for a clear, consensus definition that would serve as an integrative blueprint for the various “versions” of TM definition. The various existing definitions are reflecting the diversity of institutional translational research and deployment programs. The European Society for Translational Medicine (EUSTM) is a global non-profit and neutral society whose principal objective is to enhance world-wide healthcare through the specific development and eventual clinical implementation and exploitation of TM-based approaches, resources and expertise. In this position article, the EUSTM defines TM as an interdisciplinary branch of the biomedical field supported by three main pillars: benchside, bedside and community. The goal of TM is to combine disciplines, resources, expertise, and techniques within these pillars to promote enhancements in prevention, diagnosis, and therapies. Accordingly, TM is a highly interdisciplinary field, the primary goal of which is to coalesce assets of various natures within the individual pillars in order to improve the global healthcare system significantly.