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Background: Multiple sclerosis (MS) is an autoimmune disease for which auto-antibodies fully validated as diagnostic and prognostic biomarkers are widely desired. Recently, an immunoreactivity against the inward rectifying potassium channel 4.1 (KIR4.1) has been reported in a large proportion of a group of MS patients, with amino acids 83–120 being the major epitope. Moreover, a strong correlation between anti-KIR4.183–120and anti-full-length-protein auto-antibodies titer was reported. However, this finding received limited confirmation. Objective: Validation of the diagnostic potential of anti-KIR4.183–120antibodies in 78 MS patients, 64 healthy blood donors, and 42 individuals with other neurological diseases. Methods: Analysis of anti-KIR4.183–120antibodies by enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum we produced as a new ELISA reliability control. Additionally, evaluation of reactivity against 293-T cells transiently transfected with full-length KIR4.1 by flow cytometry. Results: We found antibodies to KIR4.183–120only in 13 out of 78 (16.6%) MS patients; among these, only 2 were positive for anti-full-length KIR4.1 antibodies. Conclusion: Employing a new reliability control and a new cytofluorometric assay, we cannot support anti-KIR4.183–120auto-antibodies as a reliable biomarker in MS.

Glycosylation, oxidation and other post-translational modifications of membrane and transmembrane proteins can alter lipid density, packing and interactions, and are considered an important factor that affects fluidity variation in membranes. Red blood cells (RBC) membrane physical state, showing pronounced alterations in Type 1 diabetes mellitus (T1DM), could be the ideal candidate for monitoring the disease progression and the effects of therapies. On these grounds, the measurement of RBC membrane fluidity alterations can furnish a more sensitive index in T1DM diagnosis and disease progression than Glycosylated hemoglobin (HbA1c), which reflects only the information related to glycosylation processes. Here, through a functional two-photon microscopy approach we retrieved fluidity maps at submicrometric scale in RBC of T1DM patients with and without complications, detecting an altered membrane equilibrium. We found that a phase separation between fluid and rigid domains occurs, triggered by systemic effects on membranes fluidity of glycation and oxidation. The phase separation patterns are different among healthy, T1DM and T1DM with complications patients. Blood cholesterol and LDL content are positively correlated with the extent of the phase separation patterns. To quantify this extent a machine learning approach is employed to develop a Decision-Support-System (DSS) able to recognize different fluidity patterns in RBC. Preliminary analysis shows significant differences(p<0.001) among healthy, T1DM and T1DM with complications patients. The development of an assay based on Phase separation of the plasma membrane of the Red Blood cells is a potential tool for diagnosis and progression monitoring of type 1 diabetes mellitus, and could allow customization and the selection of medical treatments in T1DM in clinical settings, and enable the early detection of complications.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease for which auto-antibodies fully validated as diagnostic and prognostic biomarkers are widely desired. Recently, an immunoreactivity against the inward rectifying potassium channel 4.1 (KIR4.1) has been reported in a large proportion of a group of MS patients, with amino acids 83-120 being the major epitope. Moreover, a strong correlation between anti-KIR4.183-120 and anti-full-length-protein auto-antibodies titer was reported. However, this finding received limited confirmation. OBJECTIVE: Validation of the diagnostic potential of anti-KIR4.183-120 antibodies in 78 MS patients, 64 healthy blood donors, and 42 individuals with other neurological diseases. METHODS: Analysis of anti-KIR4.183-120 antibodies by enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum we produced as a new ELISA reliability control. Additionally, evaluation of reactivity against 293-T cells transiently transfected with full-length KIR4.1 by flow cytometry. RESULTS: We found antibodies to KIR4.183-120 only in 13 out of 78 (16.6%) MS patients; among these, only 2 were positive for anti-full-length KIR4.1 antibodies. CONCLUSION: Employing a new reliability control and a new cytofluorometric assay, we cannot support anti-KIR4.183-120 auto-antibodies as a reliable biomarker in MS.

Adult neurogenesis plays increasingly recognized roles in brain homeostasis and repair and is profoundly affected by energy balance and nutrients. We found that the expression of Hes-1 (hairy and enhancer of split 1) is modulated in neural stem and progenitor cells (NSCs) by extracellular glucose through the coordinated action of CREB (cyclic AMP responsive element binding protein) and Sirt-1 (Sirtuin 1), two cellular nutrient sensors. Excess glucose reduced CREB-activated Hes-1 expression and results in impaired cell proliferation. CREB-deficient NSCs expanded poorly in vitro and did not respond to glucose availability. Elevated glucose also promoted Sirt-1-dependent repression of the Hes-1 promoter. Conversely, in low glucose, CREB replaced Sirt-1 on the chromatin associated with the Hes-1 promoter enhancing Hes-1 expression and cell proliferation. Thus, the glucose-regulated antagonism between CREB and Sirt-1 for Hes-1 transcription participates in the metabolic regulation of neurogenesis.

p66(shc), a member of the ShcA protein family, is essential for cellular response to oxidative stress, and elicits the formation of mitochondrial Reactive Oxygen Species (ROS), thus promoting vasomotor dysfunction and inflammation. Accordingly, mice lacking the p66 isoform display increased resistance to oxidative tissue damage and to cardiovascular disorders. Oxidative stress also contributes to noise-induced hearing loss (NIHL); we found that p66(shc) expression and serine phosphorylation were induced following noise exposure in the rat cochlea, together with markers of oxidative stress, inflammation and ischemia as indicated by the levels of the hypoxic inducible factor (HIF) and the vascular endothelial growth factor (VEGF) in the highly vascularised cochlear lateral region and spiral ganglion. Importantly, p66(shc) knock-out (p66 KO) 126 SvEv adult mice were less vulnerable to acoustic trauma with respect to wild type controls, as shown by preserved auditory function and by remarkably lower levels of oxidative stress and ischemia markers. Of note, decline of auditory function observed in 12 month old WT controls was markedly attenuated in p66KO mice consistent with delayed inner ear senescence. Collectively, we have identified a pivotal role for p66(shc) -induced vascular dysfunction in a common pathogenic cascade shared by noise-induced and age-related hearing loss.

Short-term persistence of transplanted cells during early post-implant period limits clinical efficacy of cell therapy. Poor cell survival is mainly due to the harsh hypoxic microenvironment transplanted cells face at the site of implantation and to anoikis, driven by cell adhesion loss. We evaluated the hypothesis that viral-mediated expression of a gene conferring hypoxia resistance to cells before transplant could enhance survival of grafted cells in early stages after implant. We used adipose tissue as cell source because it consistently provides high yields of adipose-tissue-derived stromal and vascular cells (ASCs), suitable for regenerative purposes. Luciferase positive cells were transduced with lentiviral vectors expressing either green fluorescent protein as control or human manganese superoxide dismutase (SOD2). Cells were then exposed in vitro to hypoxic conditions, mimicking cell transplantation into an ischemic site. Cells overexpressing SOD2 displayed survival rates significantly greater compared to mock transduced cells. Similar results were also obtained in vivo after implantation into syngeneic mice and assessment of cell engraftment by in vivo bioluminescent imaging. Taken together, these findings suggest that ex vivo gene transfer of SOD2 into ASCs before implantation confers a cytoprotective effect leading to improved survival and engraftment rates, therefore enhancing cell therapy regenerative potential.

Although numerous techniques have been developed to monitor autophagy and to probe its cellular functions, these methods cannot evaluate in sufficient detail the autophagy process, and suffer limitations from complex experimental setups and/or systematic errors. Here we developed a method to image, contextually, the number and pH of autophagic intermediates by using the probe mRFP-GFP-LC3B as a ratiometric pH sensor. This information is expressed functionally by AIPD, the pH distribution of the number of autophagic intermediates per cell. AIPD analysis reveals how intermediates are characterized by a continuous pH distribution, in the range 4.5-6.5, and therefore can be described by a more complex set of states rather than the usual biphasic one (autophagosomes and autolysosomes). AIPD shape and amplitude are sensitive to alterations in the autophagy pathway induced by drugs or environmental states, and allow a quantitative estimation of autophagic flux by retrieving the concentrations of autophagic intermediates.

Myasthenia gravis (MG) is an autoimmune disease in which 90% of patients have autoantibodies against the muscle nicotinic acetylcholine receptor (AChR), while autoantibodies to muscle-specific tyrosine kinase (MuSK) have been detected in half (5%) of the remaining 10%. Recently, the low-density lipoprotein receptor-related protein 4 (LRP4), identified as the agrin receptor, has been recognized as a third autoimmune target in a significant portion of the double sero-negative (dSN) myasthenic individuals, with variable frequency depending on different methods and origin countries of the tested population. There is also convincing experimental evidence that anti-LRP4 autoantibodies may cause MG.