Thyroid and Virus Detection and Intervention

Autoimmune thyroid diseases Virus Detection and Intervention StudyThere is growing evidence that viral infection is a trigger for development of autoimmune thyroid disease. In this study, we are pursuing detection of virus and viral tissue markers. Furthermore, we have developed a protocol for an intervention study with antiviral drugs to test if this treatment reverse or stop the progression of the disease.Autoimmune thyroid diseases (AITD), mainly consisting of Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). The ethiopathogenesis of the disease is not fully elucidated. However, it is generally accepted that genetic factors together with environmental trigger factors leading to a break of tolerance. Viral infection is considered as an important factor, shown by our groups and others. This study is based on our findings in type 1 diabetes and autoimmune thyroid diseases. We have developed a protocol for an intervention study with antiviral drug. While applying extra funding for the intervention study, we are continuing the search for virus and viral tissue markers in already collected thyroid tissue. Coxsackie and adenovirus receptor (CAR), enteroviral capsid 1 (VP1) and tissue markers for viral infection (HLA class I, signal transducer and activation of transcription 1 (STAT1), and protein kinase R (PKR)) were assessed by IHC in thyroid tissue from patients and controls collected in our previous cohort. The coxsackie and adenovirus receptor (CAR) was found in thyroid cells, proving that thyroid cells are susceptible to enteroviral infection. There is also upregulation of antiviral tissue markers. HLA class I expression was increased both GD and HT compared to the controls. STAT1 was found in a higher proportion of HT and GD samples. STAT1 was found, and it was co-expressed with HLA class I. Similar results were found for PKR which was co-localized with VP1. Furthermore, in collaboration with prof. Toniolo in Italy, we analyzed thyroid tissue using a specific permissive cell line. The results showed that virus is often present in thyroid tissue. Most common viruses were enetrovirus and herpes virus simplex. Taken together with a previously reported increase in the downstream interferon type 1 response protein, myxovirus resistance protein 1, plasmacytoid dendritic cells and CD8+ T cells in the same cohort, our results support the hypothesis of an association between enteroviral infections and AITD. We are continuously applying for funding for the intervention study. Furthermore, we are collaborating with Professor Hyöty who is working on development of enterovirus vaccine. Maybe in a long run we can develop new treatment strategies to prevent or to stop the progression of the disease. We are continuing the analysis of collected samples.

Thyroid and Virus Detection and InterventionThere is growing evidence that viral infection may trigger development of autoimmune thyroid disease. In this study, we are pursuing detection of virus and viral tissue markers. Furthermore, we plan to start an intervention study with antiviral drugs to test if this treatment reverse or stop the progression of the disease.Autoimmune thyroid diseases (AITD), mainly consisting of Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). The ethiopathogenesis of the disease is not fully elucidated. However, it is generally accepted that genetic factors together with environmental trigger factors leading to a break of tolerance. Viral infection is considered as an important factor, shown by our groups and others. This study is based on our findings in type 1 diabetes and autoimmune thyroid diseases. Coxsackie and adenovirus receptor (CAR), enteroviral capsid 1 (VP1) and tissue markers for viral infection (HLA class I, signal transducer and activation of transcription 1 (STAT1), and protein kinase R (PKR)) were assessed by IHC in thyroid tissue from patients and controls collected in our previous cohort. The coxsackie and adenovirus receptor (CAR) was found in thyroid cells, proving that thyroid cells are susceptible to enteroviral infection. There is also upregulation of antiviral tissue markers. HLA class I expression was increased both GD and HT compared to the controls. STAT1 was found in a higher proportion of HT and GD samples. STAT1 was found, and it was co-expressed with HLA class I. Similar results were found for PKR which was co-localized with VP1. Furthermore, in collaboartion with Prof. Toniolo in Italy, we tested the presence of 8 different virus genome in thyroid tissue using a specific permissive cell line. Our results showed that virus are often present in thyroid, however we did not find any differences between normal thyroid and those with AITD. Taken together with a previously reported increase in the downstream interferon type 1 response protein, myxovirus resistance protein 1, plasmacytoid dendritic cells and CD8+ T cells in the same cohort, our results support the hypothesis of an association between enteroviral infections and AITD. We have developed a protocol for an intervention study with antiviral drug. This study is approved by REK. While applying extra funding for the intervention study, we are continuing the search for virus and viral tissue markers in already collected thyroid tissue. We are further continuing the analysis of collected samp les. Due to Covid19 pandemic, there has been a delay in analysis of the biological material in collaboration with our international partners.  HLA analysis of DNA will be performed with our collaborator Professor Tomer, The head of department of Medicine and Thyroid Albert Einstein college of medicine in NY;  In-situ production of cytokines and chemokines by Luminex and mRNA expression determined by rt-qPCR in fresh frozen tissue or in FFPE tissue and assessment of CD8 T cell presence, specificity and activation in tissues. Probing for CD8 T cells reactive against viral antigens (Tetramer protocols),  Professor Von-Herath the head of Immunology center at La Jolla Institute, USA.; and gene expression with highthroughput RNA/DNA seq. In collaboration with Riitta Lahesmaa (Director of Turku Bioscience Center Finland).

Thyroid Virus Detection and InterventionThe results of this study demonstrate a strong link between viral infection and autoimmune thyroid diseases. Our results highlight the potential benefit of interventions in the early phases of AITD such as vaccines or antiviral treatment to prevent or cure the disease.Direct examination of a large number thyroid tissue from patients with autoimmune thyroid diseases (Graves disease, Hashimoto thyroiditis) and controls indicates that viral infection may be involved in development of thyroid autoimmunity and thyroid dysfunction. Detection of tissue markers for viral infection in collected thyroid samples is performed by immunohistochemistry in thyroid tissue. In collaboration with Professor Morgan in Exeter. We detected increased expression of HLA class I in thyroid tissue from patients with both Graves' disease and Hashimoto thyroiditis. Furthermore, the expression of STAT1 and PKR (other intracellular molecules produced in response to viral infection) were increased. The results are recently published in "The journal of Endocrinology and Metabolism". In collaboration with Prof Toniolo in Varese, we investigated the presence of several common viruses directly in thyroid tissue samples from patients with autoimmune thyroid diseases and controls. Using both conventional methods such as PCR and immunofluorescence, and a novel method of pre-enrichment of virus in cell cultures, we searched for enterovirus, parvovirus B19, human herpesvirus 6, Epstein-Barr virus, cytomegalovirus and hepatitis C virus in addition to five gastroenteric viruses. Surprisingly, several viruses were frequently found in the thyroid tissue and specimens were rarely devoid of virus. Interestingly, 76% of the investigated cases carried at least one virus, suggesting that unapparent viral infection of the thyroid is commonplace (manuscript under preparation). The viral detection rate between patients and controls did not differ significantly. However, the high detection rate of several viruses is novel knowledge and needs to be addressed in the future, as well as the pathogenic role of these infections remains to be established. All taken together, the results of this study shows that viral infection of thyroid tissue is common, which may explain the high prevalence of autoimmune thyroid diseases. The potential effect of antiviral therapy in early stages will be tested in the intervention study. The protocol for the intervention study is ready and the project is approved by REK. We are continuously working to obtain funding to pursue our intervention study ThyrVInt.

Autoimmune thyroid diseases Virus Detection and Intervention Study ThyrVIntThere is growing evidence that viral infection is a trigger for development of autoimmune thyroid disease. In this study, we are pursuing detection of virus and viral tissue markers. Furthermore, we plan to start an intervention study with antiviral drugs to test if this treatment reverse or stop the progression of the disease.Autoimmune thyroid diseases (AITD), mainly consisting of Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). The ethiopathogenesis of the disease is not fully elucidated. However, it is generally accepted that genetic factors together with environmental trigger factors leading to a break of tolerance. Viral infection is considered as an important factor, shown by our groups and others. This study is based on our findings in type 1 diabetes and autoimmune thyroid diseases. We have developed a protocol for an intervention study with antiviral drug. This study is approved by REK. While applying extra funding for the intervention study, we are continuing the search for virus and viral tissue markers in already collected thyroid tissue. Coxsackie and adenovirus receptor (CAR), enteroviral capsid 1 (VP1) and tissue markers for viral infection (HLA class I, signal transducer and activation of transcription 1 (STAT1), and protein kinase R (PKR)) were assessed by IHC in thyroid tissue from patients and controls collected in our previous cohort. The coxsackie and adenovirus receptor (CAR) was found in thyroid cells, proving that thyroid cells are susceptible to enteroviral infection. There is also upregulation of antiviral tissue markers. HLA class I expression was increased both GD and HT compared to the controls. STAT1 was found in a higher proportion of HT and GD samples. STAT1 was found, and it was co-expressed with HLA class I. Similar results were found for PKR which was co-localized with VP1. Furthermore, we are working on detection of a range of virus in thyroid tissue using a specific permissive cell line. Taken together with a previously reported increase in the downstream interferon type 1 response protein, myxovirus resistance protein 1, plasmacytoid dendritic cells and CD8+ T cells in the same cohort, our results support the hypothesis of an association between enteroviral infections and AITD.

Autoimmune thyroid diseases Virus Detection and Intervention Study ThyrVIntThere is growing evidence that viral infection play an important role as a trigger for development of autoimmune thyroid disease. In this study, we are pursuing detection of virus and viral tissue markers. Furthermore, we will start an intervention study with antiviral drugs to test if this treatment reverse or stop the progression of the disease.Autoimmune thyroid diseases (AITDs), mainly consisting of Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). The ethiopathogenesis of the disease is not fully elucidated. However, it is generally accepted that genetic factors together with invorenmental trigger factors leading to a break of tolerance. Viral infection is considered as an important factor, shown by our groups and others. This study is based on our findings in type 1 diabetes and autoimmune thyroid diseases. We have developed a protocol for an intervention study with antiviral drug. This study is approved by REK. While applying extra funding for the intervention study, we are continuing the search for virus and viral tissue markers in already collected thyroid tissue. Coxsackie and adenovirus receptor (CAR), enteroviral capsid 1 (VP1) and tissue markers for viral infection (HLA class I, signal transducer and activation of transcription 1 (STAT1), and protein kinase R (PKR)) were assessed by IHC in thyroid tissue from patients and controls collected in our previous cohort. The coxsackie and adenovirus receptor (CAR) was found in a distinct granular expression in thyroid cells in all samples, proving that thyroid cells are susceptible to enterovirus infection. There was a clear upregulation of HLA class I in both GD and HT compared to the controls. Assessed with a semi-quantitative immunoreactivity score taking intensity and proportion into account, both patient groups had a significantly higher score than controls. The mean number of VP1 positive thyroid cells was significantly higher in the GD group (30.3%) than in controls (14.9%). In the HT group, there were more, albeit not significantly, VP1 positive thyroid cells (20.1%) than in the controls (14.9%). STAT1 was found in a higher proportion og HT and GD samples. Both cytosolic and nuclear STAT1 was found, and it was co-expressed with HLA class I. Similar results were found for PKR which was co-localized with VP1. Taken together with a previously reported increase in the downstream interferon type 1 response protein, myxovirus resistance protein 1, plasmacytoid dendritic cells and CD8+ T cells in the same cohort, our results support the hypothesis of an association between enteroviral infections and AITD.

Thyroid and Virus Detection and InterventionI denne studien ser vi på sammenhengen mellom virusinfeksjon og utvikling av autoimmun stoffskiftesykdom. Vi har undersøkt thyreoideavev fra pasienter med autoimmun stoffskiftesykdom. Vi finner oppregulering av HLA klasse I i både Graves sykdom og Hashimotos thyreoiditt. I tillegg fant vi andre markører på virusrespons som PKR, CAR og STAT1.Prosjektet bygger på et tidligere prosjekt gjort av Dr. Sara Hammerstad, som er prosjektleder for prosjektet. Alle prøver og analyser som er gjort kommer fra biologisk materiale fra pasienter med autoimmun stoffskiftesykdom. Prosjektet omfatter en vitenskapelig stilling; PhD-kandidat Therese Weider. I 2018 har vi gått videre med direkte analyser av skjoldbruskkjertelvev fra pasienter med autoimmun stoffskiftesykdom. I samarbeid med Universitetet i Exeter, England, har vi ved hjelp av immunhistokjemi og immunfluorescens funnet flere proteiner som indikerer gjennomgått virusinfeksjon. Vi har undersøkt human leukocytt antigen klasse I (HLA I), signal transducer and activator of transcription 1 (STAT1) coxsackie og adenovirusreseptor (CAR), enteroviruskapsid 1 (VP1) og CD20 i thyreoideasnitt fra 118 pasienter og kontroller. Alle snittene ble analysert for HLA klasse I og VP1, med en standard immunmhistokjemiprotokoll. PKR/VP1, CAR/VP1 og STAT1/HLAI ble analysert med immunfluorescens hos en gruppe pasienter. I mange snitt fant vi både PKR og CAR uttrykt i samme området som VP1. STAT1 fant vi i både i cellekjerner og cytosol i de samme cellene hvor det også fantes HLA I. Første utkast til den første artikkelen er klart, og tittelen er Upregulation of HLA class I in Hashimoto’s Thyroiditis. I samarbeid med Dr. Toniolo i Varese, Italia, har vi undersøkt frosset thyreoideavev fra en gruppe av pasienter og kontroller fra samme kohort med en ny metode for viruspåvisning. Metoden går ut på at man dyrker opp virus i cellekultur før man leter etter viralt genom og antigen. Ca. halvparten av prøvene er analysert, og man fant enterovirus, rhinovirus og coxsackievirus. Vi har planlagt å analysere DNA fra samme kohort for å se hvor stor andel av pasientene med autoimmun thyreoideasykdom som har en kjent HLA-variant som vi vet gir økt risiko for autoimmun thyreoideasykdom ( samarbeid med Professor Benedicte Lie og Marte Viken-OUS). Vi planlegger oppstart av intervensjonsstudiet, ThyrVInt, i løpet av sommer 2019. Protokollen er klargjort og første søknad er sendt til REK. Studien er en randomisert, placebo-kontrollert, dobbelt-blindet studie med tre-armet design. Vi skal gi antiviral behandling til pasienter med autoimmun stoffskiftesykdom, og se om dette gir raskere normalisering av stoffskiftet. Vi har planlagt samarbeid med Universitetssykehuset i Bergen, Trondheim og Tromsø. Flere kilder er søkt for finansiering av prosjektet.

Thyroid and Virus detection and Intervention (ThyrVINT)Our previous study showed the presence of low grad EV infection and/or increased viral footprint in throid tissue in Graves’ disease and Hashimoto thyroiditis. Other than preparing an RTC with antiviral treatment, we are continuing the search for viral and immune response markers in thyroid tissue.Autoimmune Thyroid diseases (AITD) is a common condition affecting up to 6% of the population and especially women. The etio-pathology of the disease is still unclear. Our previous study together med result in T1D showed that EV infection may be a trigger factor for development of autoimmunity. Dr. Therese Weider is the PhD candidate working on this project. This study consists of 2 parts: 1. ThyrVInt RCT: The primary aim of this study is to perform an innovative randomized controlled intervention trial (RCT) to retard or stop the progression of AITD, and the proof of concept that virus may trigger autoimmune thyroid diseases. Our hypothesis is that a low grade persistent viral infection might be a trigger factor for initiation of AITD in genetically susceptible individuals. Patients with newly diagnosed Graves’ disease and Hashimoto thyroiditis will be recruited. Candidates will be treated with anti-viral medication (Ribavirin and Pleconaril for 26 weeks) or placebo. The protocol for this study is ready. We are currently in negotiation regarded the study medication/placebo. The applications to the Regional Ethical Committee and SLV will be soon submitted. 2. We are continuing the search for virus and viral footprint in already collected thyroid tissue in patients with Graves’ Disease and Hashimoto thyroiditis. In collaboration Exeter Medical School (Dr. Sarah Richardson, and Prof. Noel Morgan), we are currently working on detection of EV capsid protein. Furthermore, we are analyzing the expression of HLA class I, STATA 1, and PKR. Increased expression of these markers shown to be associated with IFNα expression, possibly in response to viral infection.