In-depth characterization of early stages of human induced regulatory T cell differentiation by mass cytometry

Regulatory T cells (Tregs) are responsible for the maintenance of immunological homeostasis and self-tolerance. They are part of the CD4⁺ T helper (Th) cell population, but unlike other Th cells Tregs suppress immune responses by inhibiting other immune system cell responses, and function of antigen presenting cells. Tregs mediate suppression via contact dependent and humoral factor-mediated mechanisms. However, abnormalities in Treg numbers, frequencies, and suppressive function can trigger autoimmune diseases. Several therapies are under investigation to restore and enhance Tregs cell function in autoimmune diseases in vivo. On the other hand, highly activated Tregs can suppress antitumor responses, promoting cancer progression. In cancer, means to inhibit Treg function provide promising targets to control tumor cell growth. The aim of the current study was to perform an in-depth characterization of in vitro generated human induced Treg (iTreg) cell differentiation at early timepoints by high-dimensional single-cell mass cytometry. For this purpose, a panel of 25 markers was designed and validated in iTregs, differentiated in vitro from naïve human umbilical cord blood derived CD4+ T cells. The expression of these markers was further studied in iTregs compared to activated control Th0 cells over time. Additional western blot and flow cytometry analyses were performed to confirm the successful Treg differentiation by determining the Foxp3 expression. The results show an upregulation of key transcription factor Foxp3 and several co-inhibitory molecules including PD-1, CTLA-4, LAG-3 and TIM-3 were expressed and increased with time on iTregs compared to Th0 cells. In addition, surface markers like CD103, CD137, CCR4 and CXCR3, which are interesting targets in context of Treg function and diseases, showed a statistically significant upregulation on iTregs. In conclusion, this study gives insights in the regulation and cell surface marker expression of human Tregs at single cell level and opens new way to study Treg function

Early DNA methylation changes in children developing beta cell autoimmunity at a young age

Aims/hypothesis Type 1 diabetes is a chronic autoimmune disease of complex aetiology, including a potential role for epigenetic regulation. Previous epigenomic studies focused mainly on clinically diagnosed individuals. The aim of the study was to assess early DNA methylation changes associated with type 1 diabetes already before the diagnosis or even before the appearance of autoantibodies. Methods Reduced representation bisulphite sequencing (RRBS) was applied to study DNA methylation in purified CD4(+) T cell, CD8(+) T cell and CD4(-)CD8(-) cell fractions of 226 peripheral blood mononuclear cell samples longitudinally collected from seven type 1 diabetes-specific autoantibody-positive individuals and control individuals matched for age, sex, HLA risk and place of birth. We also explored correlations between DNA methylation and gene expression using RNA sequencing data from the same samples. Technical validation of RRBS results was performed using pyrosequencing. Results We identified 79, 56 and 45 differentially methylated regions in CD4(+) T cells, CD8(+) T cells and CD4-CD8- cell fractions, respectively, between type 1 diabetes-specific autoantibody-positive individuals and control participants. The analysis of pre-seroconversion samples identified DNA methylation signatures at the very early stage of disease, including differential methylation at the promoter of IRF5 in CD4(+) T cells. Further, we validated RRBS results using pyrosequencing at the following CpG sites: chr19:18118304 in the promoter of ARRDC2; chr21:47307815 in the intron of PCBP3; and chr14:81128398 in the intergenic region near TRAF3 in CD4(+) T cells. Conclusions/interpretation These preliminary results provide novel insights into cell type-specific differential epigenetic regulation of genes, which may contribute to type 1 diabetes pathogenesis at the very early stage of disease development. Should these findings be validated, they may serve as a potential signature useful for disease prediction and management.Peer reviewe

Early DNA methylation changes in children developing beta cell autoimmunity at a young age

Aims/hypothesis Type 1 diabetes is a chronic autoimmune disease of complex aetiology, including a potential role for epigenetic regulation. Previous epigenomic studies focused mainly on clinically diagnosed individuals. The aim of the study was to assess early DNA methylation changes associated with type 1 diabetes already before the diagnosis or even before the appearance of autoantibodies.Methods Reduced representation bisulphite sequencing (RRBS) was applied to study DNA methylation in purified CD4(+) T cell, CD8(+) T cell and CD4(-)CD8(-) cell fractions of 226 peripheral blood mononuclear cell samples longitudinally collected from seven type 1 diabetes-specific autoantibody-positive individuals and control individuals matched for age, sex, HLA risk and place of birth. We also explored correlations between DNA methylation and gene expression using RNA sequencing data from the same samples. Technical validation of RRBS results was performed using pyrosequencing.Results We identified 79, 56 and 45 differentially methylated regions in CD4(+) T cells, CD8(+) T cells and CD4-CD8- cell fractions, respectively, between type 1 diabetes-specific autoantibody-positive individuals and control participants. The analysis of pre-seroconversion samples identified DNA methylation signatures at the very early stage of disease, including differential methylation at the promoter of IRF5 in CD4(+) T cells. Further, we validated RRBS results using pyrosequencing at the following CpG sites: chr19:18118304 in the promoter of ARRDC2; chr21:47307815 in the intron of PCBP3; and chr14:81128398 in the intergenic region near TRAF3 in CD4(+) T cells.Conclusions/interpretation These preliminary results provide novel insights into cell type-specific differential epigenetic regulation of genes, which may contribute to type 1 diabetes pathogenesis at the very early stage of disease development. Should these findings be validated, they may serve as a potential signature useful for disease prediction and management.</p

Umbilical cord blood DNA methylation in children who later develop type 1 diabetes

Aims/hypothesis Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes. Methods Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis. Results No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate Conclusions/interpretation Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.Peer reviewe

Umbilical cord blood DNA methylation in children who later develop type 1 diabetes

Aims/hypothesis: Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes.Methods: Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis.Results: No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate Conclusions/interpretation: Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.</p

Chillaa-mobiilisovellus yläkouluikäisten positiivisen mielenterveyden edistämisessä

Nuorten mielenterveysongelmat nousevat esille yhteiskunnassa yhä enemmän. Moni nuori kärsii sosiaalisesta jännittämisestä, mikä voi häiritä nuoren arkea, opiskelua ja sosiaalisia suhteita. Mielen hyvinvointiin eli positiiviseen mielenterveyteen tulisi kiinnittää huomiota jo ennen kuin syntyy mielenterveysongelmia. Mielenterveyden edistämiseen on kehitelty paljon erilaisia digitaalisia terveyssovelluksia, ja niiden vaikuttavuudesta on saatu jo jonkin verran hyvää tutkimusnäyttöä. Digitaalisten sovellusten yleistyminen on auttanut nuoria löytämään apua helpommin, ja siten ne ovat auttaneet nuoria saamaan tukea mielen hyvinvointiin. Opinnäytetyön tarkoituksena oli kokeilla Chillaa-mobiilisovellusta kahden kahdeksannen luokan nuorten kanssa noin kuukauden kestävän kokeilujakson ajan. Tavoitteena oli saada sovellus laajempaan käyttöön, jotta nuoret saavat enemmän tietoa siitä, kuinka edistää omaa positiivista mielenterveyttään. Tavoitteena oli myös, että kouluterveydenhoitajat voisivat hyödyntää sovellusta enemmän suosittelemalla sitä vastaanotoillaan koululaisille. Opinnäytetyön menetelmäksi valittiin toiminnallinen opinnäytetyö. Toiminnallisessa osuudessa Chillaa-mobiilisovellusta käytiin esittelemässä Lapuan yläkoululla kahden kahdeksannen luokan nuorille, jotka kokeilivat sovellusta sen jälkeen noin kuukauden ajan. Kokeiluun osallistui noin 25 henkilöä. Nuorten kokemuksia Chillaa-sovelluksesta kysyttiin kyselylomakkeilla, ja kyselyiden tuloksien avulla arvioitiin sitä, voisiko Chillaa-sovellusta hyödyntää kouluterveydenhoitajan vastaanotolla. Kyselyn tulosten perusteella suurin osa nuorista ei kokenut sovellusta itselleen tarpeellisena, mutta enemmistö heistä oli kuitenkin sitä mieltä, että kouluterveydenhoitaja voisi suositella Chillaa-mobiilisovellusta vastaanotollaan. Ne nuoret, jotka käyttivät sovellusta vielä kokeilun jälkeenkin, käyttävät sovellusta eniten paniikin, ahdistuksen ja stressin lieventämiseen sekä unen saamiseen. Hyötyjä sovelluksesta saivat pääosin ne nuoret, jotka pitivät Chillaa-mobiilisovelluksesta. Nuorten mielestä mielenterveyssovellukset ovat tarpeellisia, koska niistä voi olla apua joillekin ihmisille.The mental health problems of adolescents are becoming more and more prominent in society. Many young people suffer from social anxiety, which can disrupt a young person's everyday life, studies, and social relationships. Attention should be paid to mental well-being, i.e., positive mental health, even before mental health problems arise. Many different digital health applications have been developed to promote mental health, and some good research evidence has already been obtained about their effectiveness. The proliferation of digital applications has helped young people find help more easily and thus they have helped young people get support for mental well-being. The purpose of the thesis was to test the Chillaa mobile application with two eighth grade groups during a trial period lasting approximately one month. The goal was to make the application more widely available so that young people get more information about how to promote their positive mental health. The goal was also that the school nurses could make more use of the application by recommending it to schoolchildren at their receptions. The methodology of the thesis was functional. In the functional part, the Chillaa mobile application was presented at the middle school in Lapua to the pupils of two eighth grade groups, who then tried the application for about a month. About 25 people participated in the trial. Young people's experiences with the Chillaa application were asked using questionnaires, and the results of the surveys were used to evaluate whether the Chillaa application could be used at the school health nurse's reception. The results of the survey showed that the majority of young people did not feel that the application was necessary for them, but the majority of them thought that the school nurse could recommend the Chillaa mobile application at their reception. Those adolescents who used the app even after the trial use it mostly to relieve panic, anxiety, and stress, and to get sleep. Those young people who liked the Chillaa mobile application benefited most from the application. Adolescents think mental health apps are necessary because they can help some people

Umbilical cord blood DNA methylation in children who later develop type 1 diabetes

Abstract Aims/hypothesis: Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes. Methods: Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis. Results: No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate &lt;0.05. Conclusions/interpretation: Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset