Bettina Balint

Glatiramer acetate improves regulatory T-cell function by expansion of naive CD4(+)CD25(+)FOXP3(+)CD31(+) T-cells in patients with multiple sclerosis.

Naturally occurring regulatory T-cells (Treg) exhibit impaired function in patients with relapsing-remitting multiple sclerosis (RRMS) resulting from an age-inappropriate disproportion between prevalences of newly generated CD31-coexpressing naive Treg and long-lived memory Treg in the periphery. Recent evidence suggests that the immunomodulatory action of glatiramer acetate (GA) includes effects on Treg function and frequencies. We prospectively assessed suppressive activities and frequencies of Treg and Treg subsets in 15 patients with RRMS undergoing long-term therapy with GA. Treatment for up to six months reconstituted naive Treg and increased total Treg numbers with concomitant reversion of the Treg defect.

Interferon Beta–Induced Restoration of Regulatory T-Cell Function in Multiple Sclerosis Is Prompted by an Increase in Newly Generated Naive Regulatory T Cells

BACKGROUND Naturally occurring regulatory T (T(reg)) cells are functionally impaired in patients with relapsing-remitting multiple sclerosis. We recently showed that prevalences of newly generated CD31-coexpressing naive T(reg) cells (recent thymic emigrant-T(reg) cells) are critical for suppressive function of circulating T(reg) cells, and a shift in the homeostatic composition of T(reg)-cell subsets related to a reduced de novo generation of recent thymic emigrant-T(reg) cells may contribute to the multiple sclerosis (MS)-related T(reg)-cell dysfunction. Interferon beta, an immunomodulatory agent with established efficacy in MS, lowers relapse rates and slows disease progression. Emerging evidence suggests that T(reg)-cell suppressive capacity may increase in patients with MS undergoing treatment with interferon beta, although the mechanisms mediating this effect are uncertain. OBJECTIVE To evaluate the effect of interferon beta treatment on the suppressive activity and the homeostasis of circulating T(reg) cells in patients with MS. PARTICIPANTS Twenty patients with relapsing-remitting MS and 18 healthy control subjects. INTERVENTIONS Administration of interferon beta. MAIN OUTCOME MEASURES Effect of interferon beta on T(reg)-cell homeostasis and suppressive capacity. RESULTS Suppressive capacities of T(reg) cells were consistently upregulated at 3 and 6 months after treatment with interferon beta. The restoration of T(reg)-cell function was paralleled by increased naive recent thymic emigrant-T(reg) cells and a coincidental reduction in memory T(reg) cells. CONCLUSION The increase in T(reg)-cell inhibitory capacity mediated by interferon beta treatment can be explained by its effect on the homeostatic balance within the T(reg) cell compartment.

Isolated and combined dystonia syndromes – an update on new genes and their phenotypes

Recent consensus on the definition, phenomenology and classification of dystonia centres around phenomenology and guides our diagnostic approach for the heterogeneous group of dystonias. Current terminology classifies conditions where dystonia is the sole motor feature (apart from tremor) as ‘isolated dystonia’, while ‘combined dystonia’ refers to dystonias with other accompanying movement disorders. This review highlights recent advances in the genetics of some isolated and combined dystonic syndromes. Some genes, such as ANO3, GNAL and CIZ1, have been discovered for isolated dystonia, but they are probably not a common cause of classic cervical dystonia. Conversely, the phenotype associated with TUBB4A mutations expanded from that of isolated dystonia to a syndrome of hypomyelination with atrophy of the basal ganglia and cerebellum (H‐ABC syndrome). Similarly, ATP1A3 mutations cause a wide phenotypic spectrum ranging from rapid‐onset dystonia‐parkinsonism to alternating hemiplegia of childhood. Other entities entailing dystonia‐parkinsonism include dopamine transporter deficiency syndrome (SLC63 mutations); dopa‐responsive dystonias; young‐onset parkinsonism (PARKIN, PINK1 and DJ‐1 mutations); PRKRA mutations; and X‐linked TAF1 mutations, which rarely can also manifest in women. Clinical and genetic heterogeneity also characterizes myoclonus‐dystonia, which includes not only the classical phenotype associated with epsilon‐sarcoglycan mutations but rarely also presentation of ANO3 gene mutations, TITF1 gene mutations typically underlying benign hereditary chorea, and some dopamine synthesis pathway conditions due to GCH1 and TH mutations. Thus, new genes are being recognized for isolated dystonia, and the phenotype of known genes is broadening and now involves different combined dystonia syndromes.

B cells undergo unique compartmentalized redistribution in multiple sclerosis

Increasing evidence fosters the role of B cells (BC) in multiple sclerosis (MS). The compartmentalized distribution of BC in blood and cerebrospinal fluid (CSF) is incompletely understood. In this study, we analyzed BC-patterns and BC-immunoreactivity at these sites during active and during stable disease and the impact of disease modifying drugs (DMD) on peripheral BC-homeostasis. For this purpose we assessed BC-subsets in blood and CSF from patients with clinically isolated syndrome (CIS), relapsing remitting MS (RRMS), rheumatoid arthritis (RA), and healthy controls (HC) by flow cytometric detection of whole (W-BC), naïve, transitional (TN-BC), class-switched memory (CSM-BC), unswitched memory (USM-BC), double-negative memory (DNM-BC) BC-phenotypes, plasma blasts (PB), and plasma cells (PC). FACS-data were correlated with BC-specific chemotactic activities in CSF, intrathecal CXCL13-levels, and immunoreactivity of peripheral W-BC. Our study revealed that frequencies of systemic CSM-BC/USM-BC became contracted in active CIS/MS while proportions of naive BC, TN-BC and DNM-BC were reciprocally expanded. Moreover, the shifted BC-composition promoted reduced immunoreactivity of W-BC and resolved during remission. Cross-over changes in CSF included privileged accumulation of CSM-BC linked to intrathecal CXCL13-concentrations and expansion of PB/PC. Treatment with interferon-beta and natalizumab evoked distinct though differing redistribution of circulating BC-subsets. We conclude that symptomatic CIS and MS are accompanied by distinctive changes in peripheral and CSF BC-homeostasis. The privileged reciprocal distribution between naïve versus CSM-phenotypes in both compartments together with the marked chemotactic driving force towards BC prompted by CSF supernatants renders it likely that CSF BC are mainly recruited from peripheral blood during active CIS/MS, whereas constantly low percentages of circulating PB/PC and their failure to respond to migratory stimuli favors intrathecal generation of antibody secreting cells. Notably, BC-redistribution closely resembles alterations detectable in systemic autoimmunity associated with active RA and impacts BC-function Together with unique effects of DMDs on BC-homeostasis these findings underline the important role of BC in MS.

ADCY5 mutations are another cause of benign hereditary chorea

Objective: To determine the contribution of ADCY5 mutations in cases with genetically undefined benign hereditary chorea (BHC). Methods: We studied 18 unrelated cases with BHC (7 familial, 11 sporadic) who were negative for NKX2-1 mutations. The diagnosis of BHC was based on the presence of a childhood-onset movement disorder, predominantly characterized by chorea and no other major neurologic features. ADCY5 analysis was performed by whole-exome sequencing or Sanger sequencing. ADCY5 and NKX2-1 expression during brain development and in the adult human brain was assessed using microarray analysis of postmortem brain tissue. Results: The c.1252C>T; p.R418W mutation was identified in 2 cases (1 familial, 1 sporadic). The familial case inherited the mutation from the affected father, who had a much milder presentation, likely due to low-grade somatic mosaicism. The mutation was de novo in the sporadic case. The clinical presentation of these cases featured nonparoxysmal generalized chorea, as well as dystonia in the most severely affected, but no facial myokymia. We observed significant progression of symptoms in ADCY5 mutation carriers, in contrast to BHC secondary to NKX2-1 mutations. The difference in the clinical course is mirrored by the brain expression data, showing increasing ADCY5 expression in the striatum during brain development, whereas NKX2-1 shows an opposite trend. Conclusions: Our study identifies mutations in ADCY5, the gene previously linked to familial dyskinesia with facial myokymia, as a cause of familial and sporadic BHC. ADCY5 genetic analysis should be performed in cases with a benign choreiform movement disorder even in the absence of facial myokymia.

De Novo Mutations in PDE10A Cause Childhood-Onset Chorea with Bilateral Striatal Lesions

Chorea is a hyperkinetic movement disorder resulting from dysfunction of striatal medium spiny neurons (MSNs), which form the main output projections from the basal ganglia. Here, we used whole-exome sequencing to unravel the underlying genetic cause in three unrelated individuals with a very similar and unique clinical presentation of childhood-onset chorea and characteristic brain MRI showing symmetrical bilateral striatal lesions. All individuals were identified to carry a de novo heterozygous mutation in PDE10A (c.898T>C [p.Phe300Leu] in two individuals and c.1000T>C [p.Phe334Leu] in one individual), encoding a phosphodiesterase highly and selectively present in MSNs. PDE10A contributes to the regulation of the intracellular levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Both substitutions affect highly conserved amino acids located in the regulatory GAF-B domain, which, by binding to cAMP, stimulates the activity of the PDE10A catalytic domain. In silico modeling showed that the altered residues are located deep in the binding pocket, where they are likely to alter cAMP binding properties. In vitro functional studies showed that neither substitution affects the basal PDE10A activity, but they severely disrupt the stimulatory effect mediated by cAMP binding to the GAF-B domain. The identification of PDE10A mutations as a cause of chorea further motivates the study of cAMP signaling in MSNs and highlights the crucial role of striatal cAMP signaling in the regulation of basal ganglia circuitry. Pharmacological modulation of this pathway could offer promising etiologically targeted treatments for chorea and other hyperkinetic movement disorders.

Fine-Tuning of Regulatory T Cell Function: The Role of Calcium Signals and Naive Regulatory T Cells for Regulatory T Cell Deficiency in Multiple Sclerosis

The suppressor function of regulatory T cells (Tregs) is impaired in multiple sclerosis (MS), but the mechanisms underlying this deficiency are not fully understood. As Tregs counteract the sustained elevation of intracellular calcium, which is indispensable for full activation of conventional T cells (Tcons), we hypothesized that interference with this pathway might prompt MS-related Treg dysfunction. Using single-cell live imaging, we observed that Tregs rapidly reduce Ca2+ influx and downstream signals in Tcons upon cell contact, yet differ in their potency to efficiently suppress several target cells at the same time. Strikingly, individual Tregs harboring a CD4+CD25+FOXP3+CD45RA+ naive phenotype suppressed significantly more adjacent Tcons than did CD4+CD25+FOXP3+CD45RA− memory Tregs. Some constituents even completely failed to dampen Tcon Ca2+ influx and were contained exclusively in the memory subset. In accordance with their more powerful suppressive performance, the Ca2+ signature was considerably enhanced in naive Tregs in response to TCR triggering, compared with the memory counterparts. MS Tregs displayed a significantly diminished suppression of mean Ca2+ influx in the sum of individual Tcons recorded. This reduced inhibitory activity was closely linked to decreased numbers of individual Tcons becoming suppressed by adjacent Tregs and, in turn, correlated with a marked reduction of naive subtypes and concomitant expansion of nonsuppressive memory phenotypes. We conclude that the superior achievement of naive Tregs is pivotal in maintaining Treg efficiency. As a consequence, MS Tregs become defective because they lack naive subtypes and are disproportionately enriched in memory cells that have lost their inherent downregulatory activity.

Anti-DPPX encephalitis Pathogenic effects of antibodies on gut and brain neurons

Objective: To characterize pathogenic effects of antibodies to dipeptidyl-peptidase-like protein 6 (DPPX), a subunit of Kv4.2 potassium channels, on gut and brain neurons. Methods: We identified a new patient with anti-DPPX encephalitis and analyzed the effects of the patients serum and purified immunoglobulin G (IgG), and of serum of a previous patient with anti-DPPX encephalitis, on the activity of enteric neurons by voltage-sensitive dye imaging in guinea pig myenteric and human submucous plexus preparations. We studied the subcellular localization of DPPX by immunocytochemistry in cultured murine hippocampal neurons using sera of 4 patients with anti-DPPX encephalitis. We investigated the influence of anti-DPPX-containing serum and purified IgG on neuronal surface expression of DPPX and Kv4.2 by immunoblots of purified murine hippocampal neuron membranes. Results: The new patient with anti-DPPX encephalitis presented with a 2-month episode of diarrhea, which was followed by tremor, disorientation, and mild memory impairment. Anti-DPPX-IgG-containing sera and purified IgG increased the excitability and action potential frequency of guinea pig and human enteric nervous system neurons. Patient sera revealed a somatodendritic and perisynaptic neuronal surface staining that colocalized with the signal of commercial anti-DPPX and Kv4.2 antibodies. Incubation of hippocampal neurons with patient serum and purified IgG resulted in a decreased expression of DPPX and Kv4.2 in neuronal membranes. Conclusions: Hyperexcitability of enteric nervous system neurons and downregulation of DPPX and Kv4.2 from hippocampal neuron membranes mirror the clinical phenotype of patients with anti-DPPX encephalitis and support a pathogenic role of anti-DPPX antibodies in anti-DPPX encephalitis.

The interleukin-7 receptor α chain contributes to altered homeostasis of regulatory T cells in multiple sclerosis.

Treg homeostasis is disturbed in multiple sclerosis (MS). Frequencies of recent thymic emigrant (RTE)‐Treg are reduced and the disparity between RTE‐Treg and long‐lived memory Treg coincides with the MS‐associated Treg defect, as shown previously. Recent studies demonstrate that IL‐7 and thymic stromal lymphopoietin (TSLP) are critical for Treg maturation. Therefore, altered signaling through their receptors (IL‐7R, TSLP receptor (TSLPR)), sharing the IL‐7Rα‐chain (IL‐7Rα), might contribute to impaired Treg development. Using blood samples from 56 patients with MS and 33 healthy controls, we assessed IL‐7Rα‐expression on conventional T cells; frequencies, phenotypes and suppressive activities of Treg, plasma levels of IL‐7 and soluble IL‐7Rα; and screened for MS‐associated IL‐7RA gene polymorphism rs6897932. Moreover, we determined Treg expressing two different TCR Vα‐chains designating thymus‐originated cells. As TSLP/TSLPR signaling in thymic myeloid dendritic cells (MDCs) promotes Treg differentiation, we measured TSLPR expression on peripheral MDCs to indirectly test whether altered TSLPR expression might add to compromised Treg neogenesis. We found reduced IL‐7Rα expression on conventional T cells and upregulated IL‐7 plasma levels together with reduction of RTE‐Treg frequencies and Treg function in MS, without clear genetic influence. Decreased IL‐7Rα expression in MS correlated with declined dual‐receptor‐Treg and reduced MDC TSLPR expression, indicating contracted thymic Treg output. We suggest that altered IL‐7R/TSLPR signaling contributes to impaired Treg neogenesis in MS, which is compensated by expanded memory‐Treg and finally results in dysfunctional Treg.

T-cell homeostasis in pediatric multiple sclerosis Old cells in young patients

Objective: To assess pediatric patients with multiple sclerosis (MS) for early signs of homeostatic and functional abnormalities in conventional (Tcon) and regulatory T cells (Treg). Methods: We studied the composition of the peripheral T-cell compartment and Treg function in a cross-sectional study with 30 pediatric MS (pMS) patients by multicolor flow cytometry and proliferation assays. Data were compared to those obtained from adult patients (n = 26) and age-matched control donors (n = 67). Results: Proportions of naive T cells were 10%–20% higher in children than in adults, reflecting the age-related decline. pMS patients, however, had clearly lower numbers of naive T cells, among them recent thymic emigrants (RTE), whereas percentages of memory T cells were increased. In the Treg compartment, reduced RTE numbers coincided with markedly dampened suppressive capacities of total Treg. These homeostatic changes in circulating T cells precisely paralleled the pattern seen in adult MS. As in adults, treatment with immunomodulatory drugs attenuated these alterations. Conclusion: The homeostatic changes detected in the T-cell compartment in pMS are similar to those in adult-onset disease. With ratios between naive and memory T-cell subsets matching those of 20- to 30-years-older controls, signs of early thymic involution are already found in pMS, suggesting that an intrinsic compromise in thymic-dependent T-cell neogenesis might contribute to MS pathogenesis.