Anticossos contra antígens de membrana neuronal en encefalitis paraneoplàsica i idiopàtica

Inicialment s’havia descrit el perfil clínic d’un grup concret de malalts amb encefalitis límbica paraneoplàsica i anticossos (Acs) contra diferents proteïnes onconeuronals, en el que gairebé la meitat d’aquests pacients tenien càncer de pulmó de cèl•lula petita i anticossos associats principalment a l’antigen (Ag) Hu, però encara quedava més d’un 50% d’aquests malalts, amb afecció principalment al sistema límbic i una resposta favorable al tractament amb immunosupressors, en els quals no se’ls hi havia detectat cap Ac onconeuronal associat. Amb el nou grup d’Acs dirigits contra Ags de membrana, aquell grup de malalts amb encefalitis als quals no se’ls hi havia detectat la presencia d’Acs onconeuronals associats començava a definir-se. En total, des del 2007 s’havien identificat 7 autoantígens de membrana: el R-NMDA, RAMPA, R-GABAB, LGI1, Caspr2, R-Glicina i mGluR5. Totes aquestes dianes antigèniques estan implicades en la transmissió sinàptica, la plasticitat o l’excitabilitat nerviosa, i tot i que les síndromes associades a aquests autoantígens són síndromes severes, que afecten a la memòria, comportament, cognició, i molts cops van acompanyats d’alteracions psiquiàtriques, el tractament amb immunoteràpia acostuma a ser efectiu. Els objectius d’aquesta tesi van ser principalment tres. En referència al primer objectiu, vam contribuir a caracteritzar el perfil clínic i immunològic de les encefalitis associades a anticossos contra antígens de membrana, concretament de les encefalitis associades als Acs contra el receptor AMPA i el receptor GABAB. En referència al segon objectiu, vam identificar un nou anticòs contra la proteïna de membrana DPPX, associat a pacients amb encefalitis. I en referència al tercer objectiu vam caracteritzar el perfil clínic de pacients amb esclerosi múltiple i anticossos contra la proteïna contactina-2. A partir dels estudis realitzats en aquesta tesi vam concloure que: El receptor del GABAB és l’autoantígen més comú en el grup de pacients caracteritzats per encefalitis límbica associada a càncer de pulmó de cèl•lula petita, prèviament considerats “seronegatius”. En malalts amb anticossos conta el GAD, la freqüència de tenir també Acs contra el receptor GABAB és baixa, i només s’observa en un context paraneoplàsic. La detecció d’Acs contra el receptor AMPA s’hauria de considerar en pacients, particularment dones, majors de 50 anys que presentin encefalitis límbica i canvis ràpids i progressius de comportaments anormals que recorden una psicosi aguda. També vam identificar un nou autoantígen de membrana associat a l’encefalitis caracteritzada per hiperexcitabilitat del sistema nerviós central, precedida de diarrea desconeguda. I en el cas dels pacients amb esclerosi múltiple, la freqüència dels anticossos contra la contactina-2 és baixa, els Acs persisteixen en el temps, i no s’associen a un perfil clinico-radiològic característic.The clinical profile of a particular group of patients with paraneoplastic limbic encephalitis and antibodies (Abs) against different onconeural proteins were initially described. Almost fifty percent of these patients had small cell lung cancer and Abs against Hu antigen (Ag), but there remained the other fifty percent of the patients, with limbic system dysfunction, good response to the treatment, and no Abs associated. With the identification of the new group of Abs against cell surface Ags, the group of patients with encephalitis without onconeural Abs, starts to define. Since 2007, seven synaptic autoantigens have been identified: NMDA-R, AMPA-R, GABAB-R, LGI1, Caspr2, Glycine-R, mGluR5. All of these targets are related with synaptic transmission, plasticity, or nervous excitability. Although these syndromes are severe and affect the memory, the behavior, the cognition, and some times also have psychic alterations, they have good response to the treatment. We had three objectives in this thesis. The first objective was to characterize the clinical and immunological profile of patients with encephalitis and Abs against cell surface Ag. The second objective was to identify a new Ab against cell surface Ags. And the third objective was to characterize the clinical profile of patients with multiple sclerosis and antibodies against contactin-2. Based on this thesis we concluded that GABAB-R is the most common autoantigen in patients characterized by limbic encephalitis and small cell lung cancer, previously considered “seronegative”. And in patients with GAD-Abs, the frequency of also having Abs against GABAB-R is low, and only observed in a paraneoplastic context. The detection of AMPAR-Abs should be considered in patients, particularly women older than 50 years old, who present, not only limbic encephalitis, but also with rapidly progressive abnormal behavior resembling acute psychosis. We also identified a new cell surface Ag, in patients with limbic encephalitis characterized by central nervous system hiperexcitability, preceded by diarrhea of unknown etiology. In the case of patients with multiple sclerosis the frequency of Abs against contatin2 is low, and, their presence is not associated with a particular clinical-radiological profile, although it persist long time

Global proteomic and methylome analysis in human induced pluripotent stem cells reveals overexpression of a human TLR3 affecting proper innate immune response signaling

When considering the clinical applications of autologous cell replacement therapy of human iPSC‐derived cells there is a clear need to better understand what the immune response will be before we embark on extensive clinical trials to treat or model human disease. We performed a detailed assessment comparing human fibroblast cell lines (termed F1) reprogrammed into human iPSC and subsequently differentiated back to fibroblast cells (termed F2) or other human iPSC‐derived cells including neural stem cells made from either retroviral, episomal or synthetic mRNA cell reprogramming methods. Global proteomic analysis reveals the main differences in signal transduction and immune cell protein expression between F1 and F2 cells, implicating wild type toll like receptor protein 3 (TLR3). Furthermore, global methylome analysis identified an isoform of the human TLR3 gene that is not epigenetically reset correctly upon differentiation to F2 cells resulting in a hypomethylated transcription start site in the TLR3 isoform promoter and overexpression in most human iPSC‐derived cells not seen in normal human tissue. The human TLR3 isoform in human iPSC‐NSC functions to suppress NF‐KB p65 signaling pathway in response to virus (Poly IC), suggesting suppressed immunity of iPSC‐derived cells to viral infection. The sustained wild type TLR3 and TLR3 isoform over expression is central to understanding the altered immunogenicity of human iPSC‐derived cells calling for screening of human iPSC‐derived cells for TLR3 expression levels before applications

Global proteomic and methylome analysis in human induced pluripotent stem cells reveals overexpression of a human TLR3 affecting proper innate immune response signaling

Altres ajuts: the E-Rare (ERA-Net for research programs on rare diseases) and EuroRETT (a European network on Rett syndrome, funded by the European Commission under its 6th Framework Program since 2006)When considering the clinical applications of autologous cell replacement therapy of human induced pluripotent stem cells (iPSC)-derived cells, there is a clear need to better understand what the immune response will be before we embark on extensive clinical trials to treat or model human disease. We performed a detailed assessment comparing human fibroblast cell lines (termed F1) reprogrammed into human iPSC and subsequently differentiated back to fibroblast cells (termed F2) or other human iPSC-derived cells including neural stem cells (NSC) made from either retroviral, episomal, or synthetic mRNA cell reprogramming methods. Global proteomic analysis reveals the main differences in signal transduction and immune cell protein expression between F1 and F2 cells, implicating wild type (WT) toll like receptor protein 3 (TLR3). Furthermore, global methylome analysis identified an isoform of the human TLR3 gene that is not epigenetically reset correctly upon differentiation to F2 cells resulting in a hypomethylated transcription start site in the TLR3 isoform promoter and overexpression in most human iPSC-derived cells not seen in normal human tissue. The human TLR3 isoform in human iPSC-NSC functions to suppress NF-KB p65 signaling pathway in response to virus (Poly IC), suggesting suppressed immunity of iPSC-derived cells to viral infection. The sustained WT TLR3 and TLR3 isoform overexpression is central to understanding the altered immunogenicity of human iPSC-derived cells calling for screening of human iPSC-derived cells for TLR3 expression levels before applications. Stem Cells 2019;37:476-488