To investigate in vitro the pathogenic mechanism of anti-PS/PT antibodies to better define their role in the diagnosis of APS syndrome

1. ABSTRACT Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by vascular thrombosis (venous or arterial) and/or adverse obstetric outcomes accompanied by persistent and elevated levels of antiphospholipid (aPL) antibodies. According to the 2006 revised international classification criteria, the presence of one among anti-beta2 glycoprotein I (a\u3b22GPI) IgG or IgM, anti-cardiolipin (aCL) IgG or IgM and the lupus anticoagulant (LA) is indicated for a definite diagnosis of APS. However, not infrequently, none of the \u201ccriteria\u201d antibodies can be demonstrated. Only recently the so-called \u201cseronegative APS\u201d was definitely recognized as a distinctive setting, or better re-defined by the demonstration of new classes of aPL antibodies, such as the autoantibodies directed against prothrombin (aPT and aPS/PT). In the next future, these autoantibodies, particularly aPS/PT, could become additional serological classification criteria for APS especially to recognized patients negative for classical aPL. The combination of a\u3b22GPI, aPS/PT and LA demonstrates the best diagnostic accuracy for APS and aPS/PT were recently recommended as a surrogate of LA when specific inhibitors and/or analytical variables may affect its interpretation. Despite these recommendations, very few clinical laboratories include aPS/PT in routine analyzes so far. Moreover, no definite recommendations are available to guide the therapeutic approach in patients positive only for aPS/PT antibodies. To clarify their role in APS diagnosis and treatment, a better comprehension of its pathogenic mechanisms is needed. Thus, the principal aim of this thesis is to investigate the pathogenic mechanism underlying the thrombotic manifestations associated to the presence of aPS/PT. To address this issue, the biological effects sustained in vitro by aPS/PT were compared to those sustained by a\u3b22GpI, the most studied and recognized player in APS, by developing an experimental model able to investigate the thrombotic effect of these autoantibodies on monocytes and endothelial cells. Beside this principal study, to improve the risk management of APS patients, the plasmatic activity of the PAF-AH (Platelet Activating Factor Acetylhydrolase) was investigated as a new potential prognostic biomarker. PAF-AH is a specific marker of vascular inflammation dependent to common lipid metabolism markers (i.e. LDL) which is involved in the atherosclerotic plaque instability. Obtained data on the TF mRNA expression and Nitric Oxide production (colorimetric assay), confirmed that aPS/PT and a\u3b22GpI exert similar pro-thrombotic effects on monocytes and endothelial cells. On the contrary, the different effect of a\u3b22GpI and aPS/PT on mRNA expression of IL1\u3b2 and NLRP3, and the different impact on PAF-AH activity (colorimetric assay), may suggest that these classes of antibodies probably activate different metabolic pathways. Moreover, plasmatic PAF-AH activity in patients with positive aPL antibodies appeared to be independent to common lipid metabolism markers (i.e. LDL). Based on these results, PAF-AH plasmatic activity may represent a new prognostic biomarker also in the context of aPL antibodies, to identify patients at major risk and favouring more tailored therapeutic interventions. Further prospective studies on selected patients are ongoin

Pseudofinite and pseudocompact metric structures

We initiate the study of pseudofiniteness in continuous logic. We introduce a related concept, namely that of pseudocompactness, and investigate the relationship between the two concepts. We establish some basic properties of pseudofiniteness and pseudocompactness and provide many examples. We also investigate the injective-surjective phenomenon for definable endofunctions in pseudofinite structures.Comment: Second version. Some typos fixed. Easier proofs in Section

The influence of a stressful microenvironment on tumor exosomes: a focus on the DNA cargo

Exosomes secreted by tumor cells, through the transport of bioactive molecules, reprogram the surroundings, building a microenvironment to support the development of the tumor. The discovery that exosomes carry genomic DNA reflecting that of the tumor cell of origin has encouraged studies to use them as non-invasive biomarkers. The exosome-mediated transfer of oncogenes suggested a new mechanism of malignant transformation that could play a role in the formation of metastases. Several studies have examined the role of tumor exosomes on the modulation of the tumor microenvironment, but relatively few have been directed to assess how stressful stimuli can influence their production and cargo. Understanding the changes in exosome loads and the production pattern of the stressed tumor cell may uncover actionable mechanisms responsible for tumor progression

Incorporating Reflective Writing into the Clerkship

During the last decade, medical schools have turned to writing exercises as a means for encouraging students to reflect on their learning experiences during clinical clerkships. The reasons for the increased popularity of reflective writing are broad. Approaches to encouraging reflective writing are quite varied. Recently, three internal medicine clerkships (University of Chicago Pritzker School of Medicine, University of Florida College of Medicine, and University of Massachusetts Medical School) independently implemented reflective writing activities in the clerkship curriculum

Parkinson’s Disease and Forced Exercise: A Preliminary Study

Objective. The concept of forced exercise has drawn attention for the treatment of Parkinson’s disease symptoms with anecdotal reports of success. This study sought to ascertain any significant effect of forced exercise using a motorized stationary bicycle when compared to controls on Parkinson’s disease symptoms in a blinded, randomized, and controlled setting. Setting. Parkinson’s disease outpatient clinic, Veterans Administration Medical Center. Method. We assessed 23 patients (13 experimental and 10 controls) on a number of standard Parkinson’s measures at baseline, after participation in eight weeks of twice weekly forced exercise or eight weeks of conventional clinic care, and then after a three-month period had elapsed. Dependent measures were UPDRS-III, Berg Balance Scale, finger taping test, and the PDQ-39. Results. Results did not demonstrate any main effect differences between the exercise and control groups on any measure at any point in time. A within subjects effect was demonstrated for the forced exercise group on overall UPDRS-III scores at the three-month end point. No other within group effects were noted. Results suggest that early enthusiasm for forced exercise may need tempering. Limitations of the study are discussed as well as numerous logistical challenges to this type of study

Validation of a One-Step Reverse Transcription-Droplet Digital PCR (RT-ddPCR) Approach to Detect and Quantify SARS-CoV-2 RNA in Nasopharyngeal Swabs

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. Quantitative reverse transcription-PCR (RT-qPCR) is, to this day, the preferred methodology for viral RNA detection, even if not without problems. To overcome some of the limitations still existing for the detection and quantification of nucleic acids in various applications, the use of one-step reverse transcription-droplet digital PCR (RT-ddPCR) has been established. The purpose of this study was, then, to evaluate the efficacy of ddPCR for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs, optimizing the detection of low-viral load-burdened samples. Methods. The RT-ddPCR workflow was validated for sensitivity, specificity, linearity, reproducibility, and precision using samples from 90 COVID-19-infected patients referred to the Department of Laboratory Medicine of the University Hospital of Udine (Italy). Results. The present study shows that RT-ddPCR allows the detection of as low as 10.3 copies of a SARS-COV-2 E-gene per sample with a higher level of accuracy and precision, especially at low concentration. Conclusion. During the postpeak phase of the SARS-CoV-2 pandemic, it is essential to rely on a highly robust molecular biology method to identify infected subjects, whether they have symptoms or not, in order to prepare appropriate containment measures

Differential Eye Movements in Mild Traumatic Brain Injury vs. Normal Controls

Objective measures to diagnose and to monitor improvement of symptoms following mild traumatic brain injury (mTBI) are lacking. Computerized eye tracking has been advocated as a rapid, user friendly and field ready technique to meet this need. Eye tracking data collected via a head mounted, video-based binocular eye tracker was used to examine saccades, fixations and smooth pursuit movement in 60 military Service Members with post concussive syndrome (PCS) and 26 asymptomatic control subjects in an effort to determine if eye movement differences could be found and quantified. The diagnosis of mTBI was confirmed by the study physiatrist’s history, physical examination, and a review of any medical records. Results demonstrated that subjects with symptomatic mTBI had statistically larger position errors, smaller saccadic amplitudes, smaller predicted peak velocities, smaller peak accelerations, and longer durations. Subjects with symptomatic mTBI were also less likely to follow a target movement (less primary saccades). In general, symptomatic mTBI tracked the stepwise moving targets less accurately, revealing possible brain dysfunction. A reliable, standardized protocol that appears to differentiate mTBI from normals was developed for use in future research. This investigation represents a step toward objective identification of those with PCS. Future studies focused on increasing the specificity of eye movement differences in those with PCS are needed

Effects of hyperbaric oxygen on eye tracking abnormalities in males after mild traumatic brain injury

The effects of hyperbaric oxygen (HBO2) on eye movement abnormalities in 60 military servicemembers with at least one mild traumatic brain injury (mTBI) from combat were examined in a single-center, randomized, double-blind, sham-controlled, prospective study at the Naval Medicine Operational Training Center. During the 10 wk of the study, each subject was delivered a series of 40, once a day, hyperbaric chamber compressions at a pressure of 2.0 atmospheres absolute (ATA). At each session, subjects breathed one of three preassigned oxygen fractions (10.5%, 75%, or 100%) for 1 h, resulting in an oxygen exposure equivalent to breathing either surface air, 100% oxygen at 1.5 ATA, or 100% oxygen at 2.0 ATA, respectively. Using a standardized, validated, computerized eye tracking protocol, fixation, saccades, and smooth pursuit eye movements were measured just prior to intervention and immediately postintervention. Between- and within-groups testing of pre- and postintervention means revealed no significant differences on eye movement abnormalities and no significant main effect for HBO2 at either 1.5 ATA or 2.0 ATA equivalent compared with the sham-control. This study demonstrated that neither 1.5 nor 2.0 ATA equivalent HBO2 had an effect on postconcussive eye movement abnormalities after mTBI when compared with a sham-control