Remitting–relapsing multiple sclerosis patient refractory to conventional treatments and bone marrow transplantation who responded to natalizumab

Bone marrow transplantation (BMT) was introduced as a treatment option 15 years ago for severe, drug-resistant multiple sclerosis (MS). Up until now, BMT has been undertaken in relatively few patients worldwide, with moderate success, and recent studies suggest that patients with early, highly aggressive MS benefit most from this treatment. In this work, we determined peripheral blood lymphocyte populations in a patient (patient A) with remitting–relapsing multiple sclerosis (RR-MS), refractory to conventional treatments, and who underwent BMT, relapsed, and has been treated with natalizumab for the last 22 months. Eleven other RR-MS patients in the acute phase of the disease, untreated or treated with interferon-beta, and 20 healthy subjects served as controls. Natalizumab treatment in patient A resulted in lymphocytosis and increased levels of CD20+/CD20+CD5+ B cells and T regulatory cells (Tregs). The patient maintained relatively low levels of T cells, T helper cells, memory T helper cells, and naive cytotoxic T cells, and very low levels of naive T helper cells and natural killer cells throughout. The Tregs of patient A post-treatment with natalizumab responded well in culture to a peptide mapping to a myelin basic protein antigenic epitope (mean 42% increase) compared with Tregs of healthy controls (mean 15% increase) whereas Tregs of the RR-MS controls or patient A prenatalizumab treatment either did not respond or responded adversely to the peptide (mean 3% and 21% decreases, respectively). Since the beginning of natalizumab treatment, patient A has had no relapses, and his Expanded Disability Status Score has improved. From the parameters studied, Treg responsiveness to autoantigens seems to be an important differentiating factor in RR-MS progression

Cytokines as Biomarkers of Treatment Response to IFN in Relapsing-Remitting Multiple Sclerosis

Background. MS patients show a remarkable heterogeneity in their response to disease modifying treatments. Given the need for early treatment initiation and the diversity of available options, a predictive marker that indicates good or poor response to treatment is highly desirable. Objective. To find a biomarker for treatment response to IFN among pro-and anti-inflammatory cytokines. Materials and Methods. IFN-, TNF-, IL-2, IL-4, IL-6, IL-10, IL-17A, and TGF-1 levels were measured in serum and CSF of 43 patients with RR-MS who were followed up for a mean period of 5.3 years. Thirty-five patients received IFN treatment and were divided into good responders (GR, n = 19) and poor responders (PR, n = 16). The remaining 8 patients showed a very favorable outcome and remained untreated (noRx). Results. GR had significantly higher serum baseline levels of IL-17A than PR and significantly higher serum levels of IL-17A, IFN-, TNF-, and IL-2 than noRx. PR had significantly higher IFN-serum levels than noRx. No significant differences were observed in serum levels of IL-6, IL-4, IL-10, and TGF-1 or the levels of all cytokines measured in CSF between the 3 groups of patients. Conclusions. Baseline serum levels of IL-17A can be used as a biomarker of IFN treatment response

The role of helper T-cells and their cytokines in the pathogenesis course and prognosis of multiple sclerosis

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS) in the pathogenesis of which the immune system plays a crucial role. Although it is still considered to be an autoimmune disorder, the responsible autoantigen(s) remains elusive and the indications for autoimmunity are indirect. Therefore, it is more appropriate to consider it an immune-mediated disease. A central role in the immunological reaction in MS is attributed to helper T- cells (CD4+, mainly Th1 and Th17), which initiate and maintain the immune cascade of events that result in the destruction of myelin in the CNS. A possible mechanism for this could be an imbalance between CNS-specific CD4+ autoreactive T-cells and peripheral suppressive mechanisms, mediated mainly by regulatory T-cells (Tregs). Cytokines are secreted molecules that mediate the functions of cells that produce them, mainly cells of the immune system. The aim of this thesis was to investigate the role of helper T- cells and their cytokines in the pathogenesis, course and prognosis of MS. Because of the complexity of the disease pathogenesis and the number of immune cells involved, we decided to divide our research into three separate studies. The first study (Study A) focused on the regulatory T- cells (CD4+, nTregs) in an attempt to assess their frequencies under different disease states and therapies and their functionality. The second study (Study B) investigated the cytokine profile, both pro- and anti-inflammatory, of newly diagnosed MS patients compared to that of patients with other inflammatory and non- inflammatory neurological diseases and symptomatic controls, in order to test whether there is a particular immunological profile in MS patients, distinguishing them from other patients. The third study (Study C) was a prospective, longterm observational study of MS patients, whose cytokine profile had been assessed at disease diagnosis. The aim of the study was to investigate whether cytokines can be used as biological markers of disease progression and treatment response to IFNβ.In RRMS nTregs are downregulated but functional during the acute phase, and are restored to normal levels in patients in remission or treated with interferon.The results of our study indicate that MS patients have levels of pro- and anti-inflammatory cytokines comparable to SC and patients with NIND. However, they have a Th2 deviation compared to all other subgroups, especially to IND patients, which show the highest Th1 and Th17 deviation. This finding correlates with the increased IgG index in MS patients. Baseline serum IL-17A levels distinguish GR from PR patients, serum IFN-γ levels distinguish PR from noRx patients, and noRx patients (with minimal disability and a benign course) also exhibit minimal inflammation. The cytokine profiles of GR patients vs PR/noRx patients, discriminate the latter groups as those who should obviate treatment with IFNβ.Η Πολλαπλή Σκλήρυνση (ΠΣ) είναι μια χρόνια, φλεγμονώδης, απομυελινωτική νόσος του κεντρικού νευρικού συστήματος (ΚΝΣ), στην παθογένεση της οποίας το ανοσοποιητικό σύστημα διαδραματίζει κεντρικό ρόλο. Αν και συνεχίζει μέχρι και σήμερα να θεωρείται αυτοάνοση νόσος, το(α) υπεύθυνο(α) αντιγόνο(α) δεν έχει ταυτοποιηθεί και οι ενδείξεις περί αυτοανοσίας είναι κυρίως έμμεσες. Έτσι, είναι ίσως ορθότερο να θεωρείται μια ανοσομεσολαβούμενη νόσος. Κεντρικός ρόλος στην ανοσολογική αντίδραση στην ΠΣ έχει αποδοθεί στα βοηθητικά Τ- κύτταρα (CD4+), τα οποία θεωρείται ότι είναι υπεύθυνα για την έναρξη και συντήρηση του ανοσολογικού καταρράκτη, που οδηγεί τελικά στην προσβολή του ελύτρου της μυελίνης εντός του ΚΝΣ. Από την άλλη, η διάσπαση της ανοσολογικής ανοχής, που μεσολαβείται από τα ρυθμιστικά Τ- κύτταρα, θεωρείται ότι συμβάλλει στην έναρξη της αυτοάνοσης αντίδρασης. Οι κυτταροκίνες είναι διαλυτά μόρια που μεσολαβούν τις δράσεις των κυττάρων που τα παράγουν, κυρίως των κυττάρων του ανοσοποιητικού συστήματος.Σκοπός της παρούσας διατριβής ήταν να μελετηθεί ο ρόλος των βοηθητικών Τ- κυττάρων και των κυτταροκινών τους στην παθογένεση, πορεία και πρόγνωση της ΠΣ. Εξαιτίας της πολυπλοκότητας του παθογενετικού μηχανισμού και του πλήθους των κυττάρων που ενέχονται σε αυτόν προτιμήσαμε να χωρίσουμε την ερευνητική προσπάθεια σε τρεις επιμέρους μελέτες. Η πρώτη μελέτη (Μελέτη Α) αφορούσε στα ρυθμιστικά Τ- κύτταρα (CD4+, nTregs), σε μια προσπάθεια να διαπιστωθεί αν τα ποσοστά τους επηρεάζονται από το στάδιο της νόσου και τις θεραπείες και αν διατηρούν τη λειτουργικότητά τους. Η δεύτερη μελέτη (Μελέτη Β) εξέτασε το κυτταροκινικό προφίλ, φλεγμονώδες και αντιφλεγμονώδες, νεοδιαγνωσμένων ασθενών με ΠΣ συγκριτικά με μάρτυρες με άλλες, φλεγμονώδεις και μη φλεγμονώδεις νευρολογικές παθήσεις και με συμπτωματικούς μάρτυρες, προκειμένου να διερευνηθεί ο τύπος της ανοσολογικής αντίδρασης και αν υπάρχει ένα διακριτό κυτταροκινικό, και επομένως ανοσολογικό, προφίλ στους ασθενείς με ΠΣ. Η τρίτη μελέτη (Μελέτη Γ) ήταν μια προοπτική, μακροπρόθεσμη μελέτη παρακολούθησης ασθενών με ΠΣ, των οποίων είχε μελετηθεί το κυτταροκινικό προφίλ κατά τη διάγνωση της νόσου. Στόχος ήταν να διαπιστωθεί εάν ένα συγκριμένο κυτταροκινικό προφίλ συνδέεται με καλή ή κακή πρόγνωση καθώς και να αναζητηθεί ένας πιθανός βιολογικός δείκτης ανταπόκρισης στις τροποποιητικές της νόσου θεραπείες, εν προκειμένω στην IFNβ.Στην RRMS τα nTregs είναι μειωμένα αλλά λειτουργικά κατά τη διάρκεια της φάσης υποτροπής, και επανέρχονται σε φυσιολογικά επίπεδα σε ασθενείς σε ύφεση ή υπό θεραπεία με ιντερφερόνη. Oι ασθενείς με ΠΣ έχουν επίπεδα φλεγμονωδών και αντιφλεγμονωδών κυτταροκινών παρόμοια με αυτά των SC και NIND ασθενών. Ωστόσο, εμφανίζουν συνολικά μια ανοσολογική στροφή προς Th2 φαινότυπο συγκριτικά με τις άλλες υποομάδες, ειδικά με τους IND ασθενείς, οι οποίοι εμφανίζουν τη μεγαλύτερη στροφή προς Th1 και Th17 φαινότυπο. Το εύρημα αυτό συσχετίζεται με τον αυξημένο δείκτη IgG στους ασθενείς με ΠΣ. Οι ασθενείς με καλή ανταπόκριση (GR) στη θεραπεία με IFNβ ξεχώρισαν από τους ασθενείς με φτωχή ανταπόκριση (PR) από τα σημαντικά υψηλότερα επίπεδα ορού της IL-17A στους πρώτους πριν την έναρξη της θεραπείας

Cytokines as Biomarkers of Treatment Response to IFNβ in Relapsing-Remitting Multiple Sclerosis

Background. MS patients show a remarkable heterogeneity in their response to disease modifying treatments. Given the need for early treatment initiation and the diversity of available options, a predictive marker that indicates good or poor response to treatment is highly desirable. Objective. To find a biomarker for treatment response to IFNβ among pro- and anti-inflammatory cytokines. Materials and Methods. IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10, IL-17A, and TGF-β1 levels were measured in serum and CSF of 43 patients with RR-MS who were followed up for a mean period of 5.3 years. Thirty-five patients received IFNβ treatment and were divided into good responders (GR, n = 19) and poor responders (PR, n = 16). The remaining 8 patients showed a very favorable outcome and remained untreated (noRx). Results. GR had significantly higher serum baseline levels of IL-17A than PR and significantly higher serum levels of IL-17A, IFN-γ, TNF-α, and IL-2 than noRx. PR had significantly higher IFN-γ serum levels than noRx. No significant differences were observed in serum levels of IL-6, IL-4, IL-10, and TGF-β1 or the levels of all cytokines measured in CSF between the 3 groups of patients. Conclusions. Baseline serum levels of IL-17A can be used as a biomarker of IFNβ treatment response

Correction: Immune Parameters That Distinguish Multiple Sclerosis Patients from Patients with Other Neurological Disorders at Presentation.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system. Effector T helper cells, mainly Th1 and Th17, cytotoxic T-cells, B-cells, macrophages, microglia, and the cytokines they secrete, are implicated in the initiation and maintenance of a deregulated immune response to myelin antigens and the ensuing immune-mediated demyelination. In this study, we investigated whether signature cytokines exist in MS patients at presentation to gain an insight into the underlying immunopathogenic processes at the early stage of the disease.We collected serum and cerebrospinal fluid (CSF) samples from 123 patients at presentation, eventually diagnosed with MS or non-inflammatory (NIND) or inflammatory neurological diseases (IND) or symptomatic controls (SC). The levels of cytokines IFN-γ, TNF-α, TGF-β1, IL-2, IL-4, IL-6, IL-10 and IL-17 were measured, and cytokine ratios, such as Th1/Th2, Th1/Th17, and Type-1/Type-2, were calculated. All parameters were tested for their correlations with the intrathecal IgG synthesis.Cytokine levels in CSF were lower than in serum in all the patients, with the exception of IL-6. Serum or CSF cytokine levels of MS patients did not differ significantly from NIND or SC, with the exception of serum IFN-γ and TNF-α that were significantly higher in NIND. IND patients presented with the highest levels of all cytokines in serum and CSF, with the exception of serum IL-10 and CSF IL-17. MS patients had a significantly lower serum Th1/Th2 ratio compared to the NIND and IND groups, and significantly lower serum Type-1/Type-2, IFN-γ/IL-10 and CSF Th1/Th17 ratios compared to IND patients. MS patients had a significantly higher CSF IL-17/IL-10 ratio compared to IND patients. The IgG index was higher in MS patients compared to the control groups; the differences reached statistical significance between the MS and the NIND and SC groups. Reiber-Felgenhauer analysis of the QIgG and QAlb indices revealed higher intrathecal IgG synthesis in MS patients, and higher blood-CSF barrier dysfunction in IND patients. The IgG index correlated with CSF IL-4 in MS patients only.We found no signature cytokines or profiles thereof in MS patients at presentation. Only IND patients presented with a clear Th1 cytokine polarization in serum and CSF. The parameters that distinguished MS patients from patients with other neurological disorders were IgG intrathecal synthesis, the IgG index and its correlation with CSF IL-4 levels

ERb-Dependent Direct Suppression of Human and Murine Th17 Cells and Treatment of Established Central Nervous System Autoimmunity by a Neurosteroid

Multiple sclerosis (MS), an autoimmune disease of the CNS, is mediated by autoreactive Th cells. A previous study showed that the neurosteroid dehydroepiandrosterone (DHEA), when administered preclinically, could suppress progression of relapsing-remitting experimental autoimmune encephalomyelitis (EAE). However, the effects of DHEA on human or murine pathogenic immune cells, such as Th17, were unknown. In addition, effects of this neurosteroid on symptomatic disease, as well as the receptors involved, had not been investigated. In this study, we show that DHEA suppressed peripheral responses from patients with MS and reversed established paralysis and CNS inflammation in four different EAE models, including the 2D2 TCR-transgenic mouse model. DHEA directly inhibited human and murine Th17 cells, inducing IL-10–producing regulatory T cells. Administration of DHEA in symptomatic mice induced regulatory CD4+ T cells that were suppressive in an IL-10–dependent manner. Expression of the estrogen receptor b by CD4+ T cells was necessary for DHEA-mediated EAE amelioration, as well as for direct downregulation of Th17 responses. TGF-b1 as well as aryl hydrocarbon receptor activation was necessary for the expansion of IL-10–producing T cells by DHEA. Thus, our studies demonstrate that compounds that inhibit pathogenic Th17 responses and expand functional regulatory cells could serve as therapeutic agents for autoimmune diseases, such as MS