Dissertation committee:
Ουρανία Τσιτσιλώνη - Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Παναγιώτα Παπαζαφείρη - Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Παρασκευή Ζησιμοπούλου - Ερευνήτρια Α’, Τμήμα Νευροβιολογίας, Ελληνικό Ινστιτούτο Παστέρ
Χαράλαμπος Αλεξόπουλος – Επίκουρος Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Πέτρος Γκιάστας – Επίκουρος Καθηγητής, Τμήμα Βιοτεχνολογίας, Γεωπονικό Πανεπιστήμιο Αθηνών
Ευδοκία Καραγκούνη – Ερευνήτρια Α’, Τμήμα Μικροβιολογίας, Ελληνικό Ινστιτούτο Παστέρ
Κωνσταντίνος Λαζαρίδης – Ερευνητής Β’, Τμήμα Ανοσολογίας, Ελληνικό Ινστιτούτο Παστέρ
Summary:
Neurological autoimmune disorders are rare disorders, that present heterogenicity to their clinical phenotype. Frequently, the diagnosis of the above disorders relies on the use of specific biomarkers. The aim of this PhD thesis was the detection of autoantibodies that could be used as biomarkers for the diagnosis of autoimmune neurological disorders. In the first part of the thesis, the specificity of autoantibodies against the nicotinic acetylcholine receptor (nAChR) derived from myasthenia gravis (MG) patients are analyzed. Although, the anti-nAChR autoantibodies are well-known biomarkers for the disease, the detection of only the pathogenic autoantibodies will contribute to the better use of these autoantibodies for the serological diagnosis of the disease. In the second part of the thesis, efforts were made to identify new autoantibodies against neuronal nAChRs in sera from patients with autoimmune encephalitis and its related syndromes (AES). After, the discovery of novel autoantibodies and the proof of their pathogenesis, these autoantibodies will be considered as novel biomarkers, which will be necessary for the serological confirmation of the disease’s diagnosis.
MG is a prototype autoimmune disorder caused by autoantibodies against proteins of the postsynaptic membrane at the neuromuscular junction. In the vast majority of MG patients (80-85%), autoantibodies against the muscle nAChR are detected and thus, these autoantibodies are used as biomarkers for the serological diagnosis of the disease. In clinical practice, heterogeneity in the presentation of the disease is observed, but this heterogeneity is not explained by the differences in antibody titers. Most likely, it is because the commercially available diagnostic methods are unable to distinguish different classes of antibodies, which have shown different pathogenicity in animal model studies. The aim of the thesis is the analysis of anti-nAChR autoantibodies based on their specificity regarding the recognition of a) intracellular or extracellular epitopes of the receptor and b) subunits of the heteropentameric receptors. To achieve this goal, different antibody detection methods were combined and used to analyze multiple blood samples from 21 MG patients (in a total of 60 serological samples). After the above analysis, it was observed that 7 MG patients had autoantibodies mainly against intracellular epitopes of the receptor. These autoantibodies are not considered pathogenic, which may explain the fact that the above patients had a mild form of MG. Regarding MG patients who had autoantibodies against extracellular epitopes of the receptor, it was found that the increase of anti-α1 autoantibodies at different periods resulted in the worsening of their symptoms. In contrast, an increase in non-anti-α1 autoantibodies was associated with no differences in patients’ clinical profiles. In addition, the presence of autoantibodies against non-α1 subunits in all blood samples was correlated with a mild form of the disease. Therefore, it seems that the analysis of anti-nAChR autoantibodies can contribute to the long-term follow-up of these patients, which offers information related to possible changes in pathogenic and non-pathogenic autoantibodies during the course of the disease.
AES is a large group of diseases caused by autoantibodies against antigens of the central nervous system. AES patients are represented with a variety of neurological and psychiatric symptoms, which can be treated with immunotherapies. In the central nervous system, neuronal-type nAChRs are expressed at high levels, mainly the α7 and α4β2 subtypes, which play an important role in interneuronal communication. The expression and/or function of neuronal nAChRs seems to be affected in neurological and psychiatric diseases, which present common symptoms with AES. Based on the above, it was hypothesized that neuronal nAChR are autoantigens in AES. Thus, the second aim of the thesis was the detection of autoantibodies against the neuronal nAChR in patients with suspected AES. To detect autoantibodies against neuronal nAChR in patient sera, cell-based specific immunofluorescence diagnostic assays were developed. Initially, the appropriate conditions for the expression of the different subtypes were determined by testing different combinations of subunits with chaperone proteins. Following the successful expression of α4β2, α4β4 and α7 nAChR subtypes, the specificity of the newly developed diagnostic assays was tested. Thus, sera from healthy donors and patients with other autoimmune neurological diseases were examined. No autoantibodies against the above receptor subtypes were detected in the tested sera, which indicates that the assays are specific and do not lead to false positive results. Subsequently, 1088 sera from patients with suspected AES were screened and 17 sera were found positive for autoantibodies against the α4 or α7 subunits of the neuronal nAChR (16 positive for anti-α4 and 1 positive for anti-α7). For the seropositive sera, the autoantibodies were further examined to determine to which immunoglobulin class they belong. It was shown that 10 patients had autoantibodies that belong to immunoglobulin classes capable of complement activation. Combining the above results with the available clinical data for seropositive patients, it appears that anti-α4 autoantibodies are potential biomarkers in AES. For the clinical exploitation of these results, additional studies are needed to examine the possible pathogenic role of these autoantibodies and prove their suitability for disease diagnosis.
In conclusion, in this PhD thesis, methodologies suitable for the detection of autoantibodies and useful for the diagnosis of neurological diseases have been developed. Specifically, in the first part, a new protocol for the analysis and categorization of anti-nAChR autoantibodies is described. This protocol can contribute to the more effective use of anti-nAChR autoantibodies as biomarkers in MG. In the second part, specific tests for the detection of autoantibodies against the neuronal nAChR subtypes α4β2, α4β4 and α7, were developed. These assays were used to test sera from patients with suspected AES for autoantibodies against α4β2, α4β4 and α7, resulting in the detection of patients with autoantibodies against the α4 subunit, which makes the anti-α4 autoantibodies potential biomarkers for AES.
