Improvement of synaptic plasticity and cognitive function in RASopathies-a monocentre, randomized, double-blind, parallel-group, placebo-controlled, cross-over clinical trial (SynCoRAS).

BACKGROUND: Cognitive impairment is a common medical issue in rat sarcoma (RAS) pathway disorders, so-called RASopathies, like Neurofibromatosis type 1 (NF1) or Noonan syndrome (NS). It is presumed to be caused by impaired synaptic plasticity. In animal studies, pathway-specific pharmacological interventions with lovastatin (LOV) and lamotrigine (LTG) have been shown to improve synaptic plasticity as well as cognitive function. The aim of this clinical trial is to translate the findings of animal studies to humans and to probe the effect of lovastatin (NS) and lamotrigine (NS and NF1) on synaptic plasticity and cognitive function/alertness in RASopathies. METHODS: Within this phase IIa, monocentre, randomized, double-blind, parallel-group, placebo-controlled, cross-over clinical trial (syn. SynCoRAS), three approaches (approaches I-III) will be carried out. In patients with NS, the effect of LTG (approach I) and of LOV (approach II) is investigated on synaptic plasticity and alertness. LTG is tested in patients with NF1 (approach III). Trial participants receive a single dose of 300 mg LTG or placebo (I and III) and 200 mg LOV or placebo (II) daily for 4 days with a cross-over after at least 7 days. Synaptic plasticity is investigated using a repetitive high-frequency transcranial magnetic stimulation (TMS) protocol called quadri-pulse theta burst stimulation (qTBS). Attention is examined by using the test of attentional performance (TAP). Twenty-eight patients are randomized in groups NS and NF1 with n = 24 intended to reach the primary endpoint (change in synaptic plasticity). Secondary endpoints are attention (TAP) and differences in short interval cortical inhibition (SICI) between placebo and trial medication (LTG and LOV). DISCUSSION: The study is targeting impairments in synaptic plasticity and cognitive impairment, one of the main health problems of patients with RASopathies. Recent first results with LOV in patients with NF1 have shown an improvement in synaptic plasticity and cognition. Within this clinical trial, it is investigated if these findings can be transferred to patients with NS. LTG is most likely a more effective and promising substance improving synaptic plasticity and, consecutively, cognitive function. It is expected that both substances are improving synaptic plasticity as well as alertness. Changes in alertness may be a precondition for improvement of cognition. TRIAL REGISTRATION: The clinical trial is registered in ClinicalTrials.gov (NCT03504501; https://www. CLINICALTRIALS: gov ; date of registration: 04/11/2018) and in EudraCT (number 2016-005022-10).

Effects of BisGMA on glutathione metabolism and apoptosis in human gingival fibroblasts in vitro.

Aim of this study was to investigate the effects of the resin monomer BisGMA on the glutathione concentration (monobromobimane assay) and apoptosis (Annexin V/PI-assay) of cultured primary human gingival fibroblasts. Cells were treated for up to 24h with 0.001-0.25 mM BisGMA to determine growth curves using the DNA stain H33342. Subsequent Annexin V/PI-assays revealed that fibroblasts exposed to concentrations of 0.005-0.01 mM (non-cytotoxic) and 0.05 mM (ED(10)-concentration) showed no increase of the share of apoptotic cells compared to non-treated controls (5-8%), while 0.1 mM BisGMA (approximately ED(50)-concentration) caused a significant increase of the percentage of apoptotic cells (50%). Simultaneously to the induction of apoptosis, 0.1 and 0.25 mM of BisGMA caused a significant depletion of the intracellular GSH content after 18 h of incubation. Our results indicate that BisGMA at concentrations >0.1 mM causes an extreme depletion of the intracellular GSH pool as well as apoptosis.

UBD, a downstream element of FOXP3, allows the identification of LGALS3, a new marker of human regulatory T cells.

Here, we report the identification of the ubiquitin-like gene UBD as a downstream element of FOXP3 in human activated regulatory CD4(+)CD25(hi) T cells (T(reg)). Retroviral transduction of UBD in human allo-reactive effector CD4(+) T helper (T(h)) cells upregulates CD25 and mediates downregulation of IL4 and IL5 expression similar to overexpression of FOXP3. Moreover, UBD impairs T(h) cell proliferation without upregulation of FOXP3 and impairs calcium mobilization. In the presence of ionomycin, overexpression of UBD in T(h) cells leads to the induction of IL1R2 that resemble FOXP3-transduced T(h) cells and naturally derived T(reg) cells. A comparison of the transcriptome of FOXP3- and UBD-transduced T(h) cells with T(reg) cells allowed the identification of the gene LGALS3. However, high levels of LGALS3 protein expression were observed only in human CD4(+)CD25(hi) derived T(reg) cells and FOXP3-transduced T(h) cells, whereas little was induced in UBD-transduced T(h) cells. Thus, UBD contributes to the anergic phenotype of human regulatory T cells and acts downstream in FOXP3 induced regulatory signaling pathways, including regulation of LGALS3 expression. High levels of LGALS3 expression represent a FOXP3-signature of human antigen-stimulated CD4(+)CD25(hi) derived regulatory T cells.

Conformational restraints and flexibility of 14-meric peptides in complex with HLA-B*3501.

Human HLA-B*3501 binds an antigenic peptide of 14-aa length derived from an alternative reading frame of M-CSF with high affinity. Due to its extraordinary length, the exact HLA binding mode was unpredictable. The crystal structure of HLA-B*3501 at 1.5 A shows that the N and C termini of the peptide are embedded in the A and F pockets, respectively, similar to a peptide of normal length. The central part of the 14-meric peptide bulges flexibly out of the groove. Two variants of the alternative reading frame of M-CSF peptide substituted at P2 or P2 and P9 with Ala display weak or no T cell activation. Their structure differs mainly in flexibility and conformation from the agonistic peptide. Moreover, the variants induce subtle changes of MHC alpha-helical regions implicated as critical for TCR contact. The TCR specifically recognizing this peptide/MHC complex exhibits CDR3 length within the normal range, suggesting major conformational adaptations of this receptor upon peptide/MHC binding. Thus, the potential antigenic repertoire recognizable by CTLs is larger than currently thought.