New horizons in translational medicine最新文献

Varicella-zoster virus (VZV) is a herpesvirus that causes a characteristic vesicular exanthem in humans with primary infection (varicella) or reactivation (zoster). We have previously observed that vesicular cells are filled with autophagosomes that are easily detectable by confocal microscopy after immunolabeling for the LC3 protein. Through a 3D imaging software program called Imaris we have quantitated autophagosomes as greater than 100 per cell. Similarly, we have assessed autophagy in VZV-infected monolayers after inoculation by the traditional method with infected cells at a ratio of one infected to 8 uninfected cells. Again, autophagosomes are easily detected, but their count is lower than that observed in human skin cells. As an additional control, we enumerated the autophagosomes in the Severe Combined Immuno-Deficient (SCID) Mouse model of VZV infection. In this model, human skin is inserted under the skin of the mouse and subsequently inoculated with VZV-infected cells. Again, autophagy was abundant in the VZV-infected skin and minimal in the mock-infected skin sample. Subsequently, we investigated autophagy following infection with sonically prepared cell free virus in cultured cells. After cell free virus inoculation, autophagy was detected in a majority of infected cells at all time points, but was less than that seen after an infected-cell inoculum. Finally, we investigated VZV-induced autophagic flux by two different methods (radiolabeling proteins and a dual-colored LC3 plasmid); both showed no evidence of a block in autophagy. Overall, therefore, autophagy within a VZV-infected cell was remarkably different from autophagy within an HSV-infected cell, whose genome contains two modifiers of autophagy, ICP34.5 and US11, not present in VZV.

Varicella zoster virus (VZV) is a ubiquitous neurotropic alphaherpesvirus that typically causes childhood varicella (chickenpox) on primary infection and zoster (shingles) after reactivation. During latency most of the ~70 virus genes are transcriptionally silent; however, analysis of latent VZV gene transcription in its natural setting requires analysis of human ganglia removed at autopsy. Recognizing the problems associated with such samples, we have observed that as the post-mortem time interval increases, so do the number of VZV genes transcribed. Based on our data and recent similar findings concerning reactivation of HSV-1, we propose an interesting testable model to describe epigenetic control of neurotropic alphaherpesvirus gene transcription during latency and early reactivation.

In humans only a single prion-forming protein named PrP^c (for “cellular prion protein”) is currently known, yet many more neurodegenerative disorders involve aberrant protein aggregation. The classical model for these diseases has involved cell-autonomous aggregation, assuming that aggregation occurs independently in each cell within a diseased patient. However, more recent models have proposed a non-cell-autonomous progression of disease in which aggregates formed in one cell may be transmitted to neighboring cells. These aggregate seeds then cause aggregation of the soluble protein in the “infected” cells, similar to the prion diseases. Within the past few years, a number of proteins that exhibit prion-like aggregation and spread to neighboring tissues have been discovered in patients with Amyotrophic Lateral Sclerosis (ALS). Although ALS has been studied for a number of decades, these proteins were only recently linked to ALS by chance. This demonstrates a clear need for an accurate method to systematically identify additional proteins that may play a pathological role in neurodegenerative disorders. Taking advantage of the compositional similarity of these proteins to the known yeast prions, I plan to use the prion prediction methodology that our lab has pioneered to develop an entirely new algorithm specifically suited for this class of neuronal proteins.

The long-tailed pygmy rice rat (Oligoryzomys longicaudatus) is the reservoir host of Andes (ANDV) and Oran hantaviruses (Bunyaviridae). To examine transcriptome features of persistently infected rice rats, spleens from ANDV sero-positive wild-caught rice rats were assessed. RNA-seq analysis, de novo reference-independent assembly and stringent orthology assignments produced 17,756 unique coding and non-coding RNAs. Differential expression analysis of persistently-infected seropositive rice rat spleens revealed 18 differentially expressed transcripts from 16 unique genes. A three-pronged effect on the immune response were observed in 1) suppression of the JAK-STAT pathway at Stat5b and Ccnot1, as well as 2) a bias toward a TH2 response in the enrichment of caspase-1 and 3) stimulation of RIG-I pathway factors Ppp1cc and MFF. Two of these differentially expressed transcripts, caspase-1 and STAT5b, code for proteins expected to stimulate T helper follicular (T_FH) cell development, a phenomenon that has also been described for hantavirus-infected P. maniculatus. Differential expression of a single seropositive rice rat with a higher viral load revealed a robust response of 243 differentially expressed transcripts, suggesting an acute infection. Together, these data help define the fundamental features of the immune response in a hantavirus reservoir host.

Members of the genus Phlebovirus (family Bunyaviridae) are new and emerging disease pathogens of humans and animals. Newly identified viruses include Heartland virus (HRTV), Lone Star virus in the USA, and Severe Fever with Thrombocytopenia Syndrome virus in Asia. Assays to support surveillance, epidemiologic studies, and diagnosis of these viruses may also detect related viruses within the genus, confounding interpretation. Rio Grande virus (RGV) was isolated in 1973 from southern plains woodrats (Neotoma micropus) in the United States and has been preliminarily identified as a phlebovirus transmitted by the sand fly Lutzomyia anthophora. RGV is not known to cause disease in humans, but it could be detected by assays designed for HRTV or other phleboviruses. The goal of this study was to determine antigenic cross-reaction between RGV and other phleboviruses. A commercially available ELISA based sand fly fever antigen detection kit was tested for the ability to detect RGV and other New and Old-World phleboviruses, including attenuated Rift valley fever virus (RVFV) strain MP12, Punta Toro virus (PTV), Toscana virus, Aguacate virus, Anhanga virus, Arumowot virus, and Chagres virus. Immunocytochemistry and Western blotting were used to detect cross reactions between RGV, MP12, and PTV using rabbit anti-RVFV nucleocapsid protein and glycoproteins GC and GN, mouse monoclonal anti-PTV, and sheep polyclonal anti-MP12. The ELISA test detected cross reactivity for all phleboviruses excluding RGV, but Western blotting detected the presumed RGV nucleocapsid protein (N) using rabbit anti-RVFV-N serum, RGV-infected cells were also identified when labeled with this antibody. Our findings demonstrate assay specific antigenic cross reactivity between these phleboviruses, thus further characterization of the molecular targets of the cross-reaction is required for proper interpretation of serological assays.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) and is the most common disabling neurological disease of young adults. Although the cause of MS is unknown, genetic and epidemiological studies indicate that MS may be triggered by an environmental agent. The presence of intrathecally produced antibodies, which produce oligoclonal Ig bands in the CNS of MS patients, provides tools for investigating the target of the inflammatory response. In most of the CNS conditions with oligoclonal bands the target is known and the antibody is directed against an infectious, causative agent. For example, in subacute sclerosing panencephalitis, a measles virus (MV) infection of the brain, the oligoclonal bands and intrathecal antibodies are primarily directed against MV. In earlier studies of MS, we demonstrated that the intrathecal antibody response in MS brain does not react to varicella zoster or Epstein-Barr virus. The current study investigates the reactivity of the intrathecal antibody response in MS brain to MV. We isolated individual CD38(+) plasma cells from MS brain to produce recombinant antibodies (rAbs). These rAbs likely represent oligoclonal bands and were used to immunostain MV-infected or uninfected monkey kidney (Vero) cells. Although many of the rAbs from MS brain reacted against auto-antigens in several mouse and human tissues, none of fifteen MS rAbs reacted against MV-infected cells. These results indicate that measles virus is not a disease-relevant antigen in MS.

Presently there are limited options for controlling the transmission of West Nile virus (WNV), including the use of larvicides and adulticides to target the mosquito vector. However, these methods are poorly-targeted, restricted to wealthy semi-urban and urban areas that are able to fund the efforts, and opposed in some communities due to toxicity concerns. This study evaluated the use of endectocide-treated bird feed to control WNV transmission by targeting the primary vector in Colorado, Culex tarsalis. Ivermectin susceptibility in C. tarsalis was first measured through ivermectin-spiked bloodmeals fed using membrane feeders, and the LC₅₀ was determined to be 49.94 ng/ml (39.71-59.93 95% CI, n=988). Chickens were then fed ivermectin-treated feed to examine its safety and palatability, and mosquitoes were blood fed directly on the chickens to assess in vivo effects. Finally, ivermectin pharmokinetics were analyzed using vein blood from chickens as well the C. tarsalis that bloodfed on the chickens. A mixture of 200 mg ivermectin/kg of bird feed was determined to be a palatable and safe dose on which chickens would feed while also being effective in killing C. tarsalis in bioassays. Pharmacokinetic data from the in vivo tests produced conflicting results compared to in vitro blood feeds but drug was detected in chicken blood at concentrations that may be expected to affect C. tarsalis. Dosing, safety, and bioassays are currently being conducted in doves and sparrows. Additional studies are currently determining the effect of ivermectin on mortality in WNV-infected mosquitoes, as well as if ivermectin reduces WNV replication and transmission. Our study indicates that the use of ivermectin-treated bird feed could be a novel method of controlling WNV transmission.

Along with Roche Pharmaceuticals, Roche Diagnostics is an important part of the foundation that modern healthcare builds upon. Our broad range of innovative diagnostic tests and systems play a pivotal role in the groundbreaking area of integrated healthcare solutions and cover the early detection, targeted screening, evaluation and monitoring of disease. Roche Diagnostics is active in all market segments, from scientific research and clinical laboratory systems to patient self-monitoring.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, affecting human and animal species can be transmitted from TSE-infected individuals to naïve susceptible hosts during the long asymptomatic (years to decades) and symptomatic disease stages. The presence of infectious hematogenous prions in asymptomatic TSE-infected hosts demonstrates the highly infectious nature of blood-borne prions in hosts lacking overt clinical symptoms. It is currently unknown when and how infectious prions first enter the blood following initial exposure. We have previously shown that the whole-blood real-time quaking-induced conversion assay (wbRT-QuIC) possesses 100% specificity and >92% sensitivity, making it an ideal tool to address this question. Here, we use wbRT-QuIC to analyze whole blood collected from oral, extranasal or aerosol TSE-exposed hosts for blood-borne prions. Our results demonstrate that conversion competent prions in the inoculum are capable of crossing mucosal surfaces and entering the circulatory system within 30 min—no matter the route of exposure. Detection of the inoculum minutes post exposure is followed by a steady decline in the detection of blood-borne prions up to 3 days which is followed by a progressive increase in the detection of nascent conversion competent prions between 1 and 17 months post exposure. These data provide the first evidence for the facile transport of mucosally acquired prions into the circulatory system, providing evidence for multiple routes of inter- and intra- host prion trafficking and shedding.

Background

Cardiomyocyte enrichment strategies so far have not yielded scalable cardiac specific cell type. More so, the current data is restricted to embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs), wherein the use of viral vectors is fraught with increased risk during clinical use. Herein, we profiled time-dependent gene/protein expression patterns across the cardiac ectoderm, endoderm, and mesoderm for isolating cardiac precursors from human adipose derived stem cells (hADSC).

Methods

Direct cardiac differentiation of hADSCs was carried out with 5-azacytidine and basic fibroblast growth factor (bFGF) in a one month long culture. The cells were periodically harvested, analyzed for unique persistent markers and their inherent regulation using quantitative polymerase chain reaction (qPCR), flow cytometry, immunoblot and immunocytochemistry assays. The identified markers were super paramagnetic iron oxide nanoparticle (SPION) tagged for segregation by magnetic activated cell sorting (MACS) and further evaluated their differentiation potential and checked for the purity by flow cytometry.

Results

The results demonstrated pronounced up-regulation of mesodermal and mature cardiac lineage markers at three weeks, while there was a down-regulation of pluripotent stem cell markers. This perhaps could be attributed to de-differentiation in maintaining the cardiac phenotype. However, signal regulatory protein alpha (SIRPA) and kinase domain receptor (KDR) persisted all through the culture period of one month, making them the most relevant and reliable cardiac specific markers. Dual labeling of these markers to SPION for cardiomyocyte enrichment by MACS column yielded cardiomyogenic-like cells in differentiation cultures with several functional positive markers.

Conclusions

Thus, SIRPA and KDR together provide cues in the enhancement and up-scaling of cardiomyocyte production in the cell replacement therapy.

Focal points

• •

  Benchside

  Identification of specific cell phenotypic markers to identify cardiac precursors in any tissue source with minimal cell manipulation is a novel process development tool in clinical translation.

• •

  Bedside

  A product developed in a closed system would minimize extraneous contaminants in long term cultures and development of such procedures minimizes culture failure rates from bench side.

• •

  Industry

  This unique identification of cell-specific marker would enable a tissue-specific translational plan and immensely help in the cardiac regeneration.

• •

  Government

  Financial inve