Detection and localization of viral infection in the pancreas of patients with type 1 diabetes using short fluorescently-labelled oligonucleotide probes
Busse, Niels; Paroni, Federico; Richardson, Sarah J.; Laiho, Jutta E.; Oikarinen, Maarit; Frisk, Gun; Hyöty, Heikki; de Koning, Eelco; Morgan, Noel G.; Maedler, Kathrin
(2017) Oncotarget, volume 8, issue 8, pp. 12620 - 12636
(Article)
Abstract
Enteroviruses, specifically of the Coxsackie B virus family, have been implicated in triggering islet autoimmunity and type 1 diabetes, but their presence in pancreata of patients with diabetes has not been fully confirmed. To detect the presence of very low copies of the virus genome in tissue samples from T1D
... read more
patients, we designed a panel of fluorescently labeled oligonucleotide probes, each of 17-22 nucleotides in length with a unique sequence to specifically bind to the enteroviral genome of the picornaviridae family. With these probes enteroviral RNA was detected with high sensitivity and specificity in infected cells and tissues, including in FFPE pancreas sections from patients with T1D. Detection was not impeded by variations in sample processing and storage thereby overcoming the potential limitations of fragmented RNA. Co-staining of small RNA probes in parallel with classical immunstaining enabled virus detection in a cell-specific manner and more sensitively than by viral protein.
show less
Download/Full Text
Keywords: Enteroviruses, Islets, Oligonucleotide probes, Pancreas, Pathology Section, Type 1 diabetes, Oncology, Journal Article
ISSN: 1949-2553
Publisher: Impact Journals
Note: Funding Information: We would like to thank Katrischa Hennekens (Uni Bremen) for excellent technical assistance. Human islets were provided through the JDRF award 31-2008-416 (ECIT Islet for Basic Research program). We are grateful to our collaborators for providing the cell lines and viruses for this study; HEK293 and FHRK-4 cells and all viruses (cytomegalovirus (CMV), hepatitis-A-Virus (HAV), coxsackievirus B3 (Nancy) and B2 were kindly provided by Dr. Andreas Dotzauer, Institute for Virology, University of Bremen, Germany. CM9 cells were kindly provided by Dr. Paolo Pozzilli, Barts and the London School of Medicine, Queen Mary, University of London, UK. INS-1E cells were kindly provided by Dr. Claes Wollheim, Universities of Geneva, Switzerland and Lund, Sweden. This research was performed with the support of the Network for Pancreatic Organ Donors with Diabetes (nPOD), a collaborative type 1 diabetes research project sponsored by JDRF. Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at http://www.jdrfnpod.org/forpartners/npod-partners/. This study was also supported by JDRF grant 25-2012-516 to the nPOD-Virus Group (Alberto Pugliese). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
(Peer reviewed)
