Carla Mucignat

Increased spontaneous activity of a network of hippocampal neurons in culture caused by suppression of inhibitory potentials mediated by anti-gad antibodies

Introduction: Anti-glutamic acid decarboxylase autoantibodies (GAD-Ab) are commonly considered the marker of autoimmune diabetes; they were first described in patients affected by stiff-person syndrome and recently, in ataxic or epileptic patients. The pathogenetic role of GAD-Ab remains unclear but inhibition of GABA synthesis or interference with GABA exocytosis are hypothesized. The aim of the study was to assess whether GAD-Ab interfere with neuronal transmission. Patients and methods: Serum from a GAD-Ab positive epileptic patient (by IHC and RIA), serum from a GAD-positive (only by RIA) diabetic case, sera from two epileptic GAD-Ab negative patients and a normal control were selected. Post-synaptic inhibitory potentials (IPSPs) were registered on hippocampal neurons in culture before and after the application of diluted sera in a patch clamp study. Results: A significant increase in the frequency of IPSPs was observed after application of GAD-positive epileptic serum, while no effect was noted using sera from negative controls. Conclusion: The inhibition in neuronal transmission only after application of GAD-positive epileptic serum, suggests an interference with GABA function and consequently with neuronal inhibition supporting a pathogenetic role of GAD-Ab in the development of epilepsy.

Peculiar labeling of cultured hippocampal neurons by different sera harboring anti-glutamic acid decarboxylase autoantibodies (GAD-Ab)

Immunological derangement is assumed to be present in a subgroup of patients affected by drug-resistant epilepsy with serum harboring anti-glutamic acid decarboxylase autoantibodies (GAD-Ab). To further investigate the specific reactivity of GAD-Ab with target cells, we tested sera from drug-resistant epileptics harboring GAD-Ab on cultured fetal rat hippocampal neurons. As a control, we tested sera from GAD-Ab-negative epileptics and GAD-Ab-positive patients affected by Stiff Person Syndrome (SPS), ataxia or diabetes. A specific pattern of reactivity, varying according to disease, was detected on application of sera from GAD-Ab-positive patients with epilepsy, SPS and ataxia, but no specific labeling was found on application of sera from patients with GAD-Ab-negative epilepsy or from GAD-Ab-positive diabetic controls.

The aurora kinase inhibitor VX-680 shows anti-cancer effects in primary metastatic cells and the SW13 cell line

New therapeutic targets are needed to fight cancer. Aurora kinases (AK) were recently identified as vital key regulators of cell mitosis and have consequently been investigated as therapeutic targets in preclinical and clinical studies. Aurora kinase inhibitors (AKI) have been studied in many cancer types, but their potential capacity to limit or delay metastases has rarely been considered, and never in adrenal tissue. Given the lack of an effective pharmacological therapy for adrenal metastasis and adrenocortical carcinoma, we assessed AKI (VX-680, SNS314, ZM447439) in 2 cell lines (H295R and SW13 cells), 3 cell cultures of primary adrenocortical metastases (from lung cancer), and 4 primary adrenocortical tumor cell cultures. We also tested reversan, which is a P-gp inhibitor (a fundamental efflux pump that can extrude drugs), and we measured AK expression levels in 66 adrenocortical tumor tissue samples. Biomolecular and cellular tests were performed (such as MTT, thymidine assay, Wright’s staining, cell cycle and apoptosis analysis, Western blot, qRT-PCR, and mutation analysis). Our results are the first to document AK overexpression in adrenocortical carcinoma as well as in H295R and SW13 cell lines, thus proving the efficacy of AKI against adrenal metastases and in the SW13 cancer cell model. We also demonstrated that reversan and AKI Vx-680 are useless in the H295R cell model, and therefore should not be considered as potential treatments for ACC. Serine/threonine AK inhibition, essentially with VX-680, could be a promising, specific therapeutic tool for eradicating metastases in adrenocortical tissue.

Mitogen-Activated Protein Kinase Pathway: Genetic Analysis of 95 Adrenocortical Tumors

Mitogen-activated protein kinase (MAPK) pathway is often deregulated in adrenocortical tumors (ACT) but with no concrete data confirming alteration rate. The objective of this study was to evaluate genetic alterations in key components of MAPK pathway. We found one BRAF mutation (p.V600E) and four HRAS silent mutations. No alteration was found in NRAS, KRAS, EGFR genes. The patient carrying BRAF mutation was further characterized by investigating his biomolecular and clinico-pathological findings. Therefore, even if MAPK signaling is activated in ACT, our results suggest that genetic alterations do not seem to represent a frequent mechanism of ACT tumorigenesis.

Major Urinary Proteins on Nanodiamond-Based Resonators Toward Artificial Olfaction

A new bio-sensing platform based on major urinary proteins (MUPs) from the mouse as chemical recognition elements has been developed. The transducers were surface acoustic devices coated with diamond nanoparticles as an intermediate layer enabling covalent attachment of the proteins. The resulting sensors detected 2,4-Dinitrotoluene, 4-Nitrotoluene, and 2-Isobutyl-3-methoxypyrazine at ppb levels. The best sensor showed a sensitivity of 24000 Hz · ppm-1 to 2, 4-DNT when grafted with the protein MUP20. Trends in the sensitivity of the various VOC sensors were compared with the association constant values Ka of the proteins to target ligands measured by competitive assay in liquid phase. The system is able to detect analytes both in liquid as well as vapor phase and indicate that MUPs are robust bio-recognition elements that can be utilized in artificial olfaction applications.

Drug-induced Parkinson’s disease modulates protein kinase A and Olfactory Marker Protein in the mouse olfactory bulb

BackgroundOlfaction is often affected in parkinsonian patients, but dopaminergic cells in the olfactory bulb are not affected by some Parkinson-inducing drugs. We investigated whether the drug MPTP produces the olfactory deficits typical of Parkinson and affects the olfactory bulb in mice.FindingsLesioned and control mice were tested for olfactory search, for motor and exploratory behavior. Brains and olfactory mucosa were investigated via immunohistochemistry for thyrosine hydroxylase, Olfactory Marker Protein and cyclic AMP-dependent protein kinase as an intracellular pathway involved in dopaminergic neurotransmission. MPTP induced motor impairment, but no deficit in olfactory search. Thyrosine hydroxylase did not differ in olfactory bulb, while a strong decrease was detected in substantia nigra and tegmentum of MPTP mice. Olfactory Marker Protein decreased in the olfactory bulb of MPTP mice, while a cyclic AMP-dependent protein kinase increased in the inner granular layer of MPTP mice.ConclusionsMPTP mice do not present behavioural deficits in olfactory search, yet immunoreactivity reveals modifications in the olfactory bulb, and suggests changes in intracellular signal processing, possibly linked to neuron survival after MPTP.