The predictive role of proinflammatory and neuroendocrine factors in the acute phases of bipolar disorder: study protocol of a longitudinal controlled trial.

INTRODUCTION AND AIMS: Bipolar disorder (BD) is a severe and recurring mental illness associated with a significant personal and social burden. It has been recently hypothesized that increased levels of pro-inflammatory cytokines and cortisol, which is also associated with a reduced expression of the brain-derived neurotrophic factor (BDNF), may influence affective recurrences in BD. Our study aims to: 1) assess changes in the levels of peripheral cytokines, BDNF and salivary cortisol during acute and euthymic phases of bipolar disorder, compared to that of a sample oh healthy controls; 2) evaluate whether these changes represent a biosignature for the different phases of the illness. MATERIALS AND METHODS: Patients aged 18-65 years old, with a diagnosis of BD I or II types, will be enrolled during an acute episode, according to DSM-5 criteria, together with age- and gender-matched healthy controls. Blood and salivary samples will be collected at baseline and after 3 and 6 months. Validated assessment instruments will be administered to all participants for the evaluation of symptom severity, global functioning, suicidal risk, stress levels and physical comorbidities. EXPECTED RESULTS: We expect changes in inflammatory and neuroendocrine indices to be predictive of the onset of an acute phase of bipolar disorder and that overall levels of cytokines, cortisol and BDNF are overall significantly different between BD patients and healthy controls. CONCLUSIONS: The longitudinal design of the study will allow to assess whether the presence of acute affective symptoms in BD patients correlates with significantly higher levels of cytokines and salivary cortisol and with reduced BDNF levels compared to euthymic phases. Moreover, the comparison with healthy control subjects will allow to understand if inflammatory mediators as well as the hypothalamic-pituitary-adrenal (HPA) axis are chronically elevated in BD patients and are independent from mood swings.

Blood immune cells as potential biomarkers predicting relapse-free survival of stage III/IV resected melanoma patients treated with peptide-based vaccination and interferon-alpha.

Introduction: Despite the recent approval of several therapies in the adjuvant setting of melanoma, tumor relapse still occurs in a significant number of completely resected stage III-IV patients. In this context, the use of cancer vaccines is still relevant and may increase the response to immune checkpoint inhibitors. We previously demonstrated safety, immunogenicity and preliminary evidence of clinical efficacy in stage III/IV resected melanoma patients subjected to a combination therapy based on peptide vaccination together with intermittent low-dose interferon-α2b, with or without dacarbazine preconditioning (https://www.clinicaltrialsregister.eu/ctr-search/search, identifier: 2008-008211-26). In this setting, we then focused on pre-treatment patient immune status to highlight possible factors associated with clinical outcome. Methods: Multiparametric flow cytometry was used to identify baseline immune profiles in patients' peripheral blood mononuclear cells and correlation with the patient clinical outcome. Receiver operating characteristic curve, Kaplan-Meier survival and principal component analyses were used to evaluate the predictive power of the identified markers. Results: We identified 12 different circulating T and NK cell subsets with significant (p ≤ 0.05) differential baseline levels in patients who later relapsed with respect to patients who remained free of disease. All 12 parameters showed a good prognostic accuracy (AUC>0.7, p ≤ 0.05) and 11 of them significantly predicted the relapse-free survival. Remarkably, 3 classifiers also predicted the overall survival. Focusing on immune cell subsets that can be analyzed through simple surface staining, three subsets were identified, namely regulatory T cells, CD56dimCD16- NK cells and central memory γδ T cells. Each subset showed an AUC>0.8 and principal component analysis significantly grouped relapsing and non-relapsing patients (p=0.034). These three subsets were used to calculate a combination score that was able to perfectly distinguish relapsing and non-relapsing patients (AUC=1; p=0). Noticeably, patients with a combined score ≥2 demonstrated a strong advantage in both relapse-free (p=0.002) and overall (p=0.011) survival as compared to patients with a score <2. Discussion: Predictive markers may be used to guide patient selection for personalized therapies and/or improve follow-up strategies. This study provides preliminary evidence on the identification of peripheral blood immune biomarkers potentially capable of predicting the clinical response to combined vaccine-based adjuvant therapies in melanoma.

DNA repair protein DNA-PK protects PC12 cells from oxidative stress-induced apoptosis involving AKT phosphorylation.

BACKGROUND: Emerging evidence suggest that DNA-PK complex plays a role in the cellular response to oxidative stress, in addition to its function of double strand break (DSB) repair. In this study we evaluated whether DNA-PK participates in oxidative stress response and whether this role is independent of its function in DNA repair. METHODS AND RESULTS: We used a model of H2O2-induced DNA damage in PC12 cells (rat pheochromocytoma), a well-known neuronal tumor cell line. We found that H2O2 treatment of PC12 cells induces an increase in DNA-PK protein complex levels, along with an elevation of DNA damage, measured both by the formation of γΗ2ΑX foci, detected by immunofluorescence, and γH2AX levels detected by western blot analysis. After 24 h of cell recovery, γΗ2ΑX foci are repaired both in the absence and presence of DNA-PK kinase inhibitor NU7026, while an increase of apoptotic cells is observed when DNA-PK activity is inhibited, as revealed by counting pycnotic nuclei and confirmed by FACS analysis. Our results suggest a role of DNA-PK as an anti-apoptotic factor in proliferating PC12 cells under oxidative stress conditions. The anti-apoptotic role of DNA-PK is associated with AKT phosphorylation in Ser473. On the contrary, in differentiated PC12 cells, were the main pathway to repair DSBs is DNA-PK-mediated, the inhibition of DNA-PK activity causes an accumulation of DNA damage. CONCLUSIONS: Taken together, our results show that DNA-PK can protect cells from oxidative stress induced-apoptosis independently from its function of DSB repair enzyme.

The Fatty Acid and Protein Profiles of Circulating CD81-Positive Small Extracellular Vesicles Are Associated with Disease Stage in Melanoma Patients.

The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is fundamental to increasing survival and therapeutic adjustment. In stages II-IV especially, additional indications for adjuvant therapy purposes after resection and for treatment of metastatic patients are urgently needed. We investigated whether the fatty acid (FA) and protein compositions of small extracellular vesicles (sEV) derived from the plasma of stage 0-I, II and III-IV melanoma patients (n = 38) could reflect disease stage. The subpopulation of sEV expressing CD81 EV marker (CD81sEV) was captured by an ad hoc immune affinity technique from plasma depleted of large EV. Biological macromolecules were investigated by gas chromatography and mass spectrometry in CD81sEV. A higher content of FA was detectable in patients with respect to healthy donors (HD). Moreover, a higher C18:0/C18:1 ratio, as a marker of cell membrane fluidity, distinguished early (stage 0-I) from late (III-IV) stages' CD81sEV. Proteomics detected increases in CD14, PON1, PON3 and APOA5 exclusively in stage II CD81sEV, and RAP1B was decreased in stage III-IV CD81sEV, in comparison to HD. Our results suggest that stage dependent alterations in CD81sEV' FA and protein composition may occur early after disease onset, strengthening the potential of circulating sEV as a source of discriminatory information for early diagnosis, prediction of metastatic behavior and following up of melanoma patients.

Corrigendum to "Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring".

[This corrects the article DOI: 10.1155/2020/1938704.].

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring.

BACKGROUND: Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. METHODS: The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. RESULTS: Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. CONCLUSION: Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

Type E Botulinum Neurotoxin-Producing Clostridium butyricum Strains Are Aerotolerant during Vegetative Growth.

Clostridium butyricum, the type species of the genus Clostridium, is considered an obligate anaerobe, yet it has been shown to grow in the presence of oxygen. C. butyricum strains atypically producing the botulinum neurotoxin type E are the leading cause of type E human botulism in Italy. Here, we show that type E botulinum neurotoxin-producing C. butyricum strains growing exponentially were able to keep growing and producing toxin in vitro upon exposure to air, although less efficiently than under ideal oxygen-depleted conditions. Bacterial growth in air was maintained when the initial cell density was higher than 103 cells/ml. No spores were detected in the cultures aerated for 5 h. To understand the biological mechanisms allowing the adaptation of vegetative cells of C. butyricum type E to oxygen, we compared the proteome and metabolome profiles of the clostridial cultures grown for 5 h under either aerated or anaerobic conditions. The results indicated that bacterial cells responded to oxygen stress by slowing growth and modulating the expression of proteins involved in carbohydrate uptake and metabolism, redox homeostasis, DNA damage response, and bacterial motility. Moreover, the ratio of acetate to butyrate was significantly higher under aeration. This study demonstrates for the first time that a botulinum neurotoxin-producing Clostridium can withstand oxygen during vegetative growth. IMPORTANCE Botulinum neurotoxins, the causative agents of the potentially fatal disease of botulism, are produced by certain Clostridium strains during vegetative growth, usually in anaerobic environments. Our findings indicate that, contrary to current understanding, the growth of neurotoxigenic C. butyricum strains and botulinum neurotoxin type E production can continue upon transfer from anaerobic to aerated conditions and that adaptation of strains to oxygenated environments requires global changes in proteomic and metabolic profiles. We hypothesize that aerotolerance might constitute an unappreciated factor conferring physiological advantages on some botulinum toxin-producing clostridial strains, allowing them to adapt to otherwise restrictive environments.

Corticomuscular coherence is tuned to the spontaneous rhythmicity of speech at 2-3 Hz.

Human speech features rhythmicity that frames distinctive, fine-grained speech patterns. Speech can thus be counted among rhythmic motor behaviors that generally manifest characteristic spontaneous rates. However, the critical neural evidence for tuning of articulatory control to a spontaneous rate of speech has not been uncovered. The present study examined the spontaneous rhythmicity in speech production and its relationship to cortex-muscle neurocommunication, which is essential for speech control. Our MEG results show that, during articulation, coherent oscillatory coupling between the mouth sensorimotor cortex and the mouth muscles is strongest at the frequency of spontaneous rhythmicity of speech at 2-3 Hz, which is also the typical rate of word production. Corticomuscular coherence, a measure of efficient cortex-muscle neurocommunication, thus reveals behaviorally relevant oscillatory tuning for spoken language.

Some considerations about the biological appearance of pacing stimuli in visuomotor finger-tapping tasks.

Sensorimotor synchronization is a crucial function for human daily activities, which relies on the ability of predicting external events. Synchronization performance, as assessed in finger-tapping (FT) tasks, is characterized by an anticipation tendency, as the tap generally precedes the pacing event. This synchronization error (SE) depends on many factors, in particular on the features of the pacing stimulus. Interest is growing in the facilitation effect that action observation has on motor execution. So far, neuroimaging and neurophysiology studies of motor priming via action observation have mainly employed tasks requiring single action instances. The impact of action observation on motor synchronization to periodic stimuli has not yet been tested; to this aim, a synchronization FT task may be an eligible probing task. The purpose of this study was to characterize a biological pacer at the behavioral level and provide information for those interested in studying the brain processes of continuous observation/execution coupling in timed actions using FT tasks. We evaluated the influence of the biological appearance of a pacer (a tapping finger) on SE, when compared to an abstract, kinematically equivalent pacer (a tilting hinged bar) and a more standard stimulus (a pulsating dot). We showed that the continuous visual display of a biological pacer yields comparable results to the abstract pacer, and a more robust performance and larger anticipations than a traditional pulsating stimulus.

The functional role of the ventral premotor cortex in a visually paced finger tapping task: a TMS study.

The accurate control of timed actions is a fundamental aspect of our daily activities. Repetitive movements can be either self-paced or synchronized with an external stimulus. Finger tapping (FT) is a suitable task to study the mechanisms of motor timing in both conditions. The neuronal network supporting motor timing in FT tasks comprises the lateral cerebellum, the lateral and mesial premotor areas as well as parietal sites. It has been suggested that lateral premotor cortices (PMC) are involved in time representation and sensorimotor transformations needed for synchronization. Most studies have focused on the dorsal aspect of PMC (dPMC) whereas the ventral PMC (vPMC) function has been poorly investigated. Here we used an online transcranial magnetic stimulation (TMS) protocol to probe the role of vPMC in an FT task, as compared to a functionally relevant site (dPMC) and an unrelated one. According to the synchronization-continuation paradigm, subjects had to synchronize their tapping to a periodic continuous visual stimulus, and then continue without the external pacer. Two different visual pacers were used: a tapping finger and a hinged tilting bar. We show that TMS reduced the synchronization error when delivered to the vPMC. This effect was larger when the more abstract hinged tilting bar was used as a pacer instead of the finger. No effects were observed in the continuation phase. We hereby offer the first online-TMS evidence of the involvement of vPMC in visually cued FT tasks.

Checking an integrated model of web accessibility and usability evaluation for disabled people.

A combined objective-oriented and subjective-oriented method for evaluating accessibility and usability of web pages for students with disability was tested. The objective-oriented approach is devoted to verifying the conformity of interfaces to standard rules stated by national and international organizations responsible for web technology standardization, such as W3C. Conversely, the subjective-oriented approach allows assessing how the final users interact with the artificial system, accessing levels of user satisfaction based on personal factors and environmental barriers. Five kinds of measurements were applied as objective-oriented and subjective-oriented tests. Objective-oriented evaluations were performed on the Help Desk web page for students with disability, included in the website of a large Italian state university. Subjective-oriented tests were administered to 19 students labeled as disabled on the basis of their own declaration at the University enrolment: 13 students were tested by means of the SUMI test and six students by means of the 'Cooperative evaluation'. Objective-oriented and subjective-oriented methods highlighted different and sometimes conflicting results. Both methods have pointed out much more consistency regarding levels of accessibility than of usability. Since usability is largely affected by individual differences in user's own (dis)abilities, subjective-oriented measures underscored the fact that blind students encountered much more web surfing difficulties.

Distribution of GD3 in DPPC monolayers: a thermodynamic and atomic force microscopy combined study.

Gangliosides are the main component of lipid rafts. These microdomains, floating in the outer leaflet of cellular membrane, play a key role in fundamental cellular functions. Little is still known about ganglioside and phospholipid interaction. We studied mixtures of dipalmitoylphosphatidylcholine and GD3 (molar fraction of 0.2, 0.4, 0.6, 0.8) using complementary techniques: 1), thermodynamic properties of the Langmuir-Blodgett films were assessed at the air-water interface (surface tension, surface potential); and 2), three-dimensional morphology of deposited films on mica substrates were imaged by atomic force microscopy. Mixture thermodynamics were consistent with data in the literature. In particular, excess free energy was negative at each molar fraction, thus ruling out GD3 segregation. Atomic force microscopy showed that the height of liquid-condensed domains in deposited films varied with GD3 molar fraction, as compatible with a lipid aggregation model proposed by Maggio. No distinct GD3-rich domain was observed inside the films, suggesting that GD3 molecules gradually mix with dipalmitoylphosphatidylcholine molecules, confirming DeltaG data. Morphological analysis revealed that the shape of liquid-condensed domains is strongly influenced by the amount of GD3, and an interesting stripe-formation phenomenon was observed. These data were combined with the thermodynamic results and interpreted in the light of McConnell's model.

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer.

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.