Μελέτη της μεθυλίωσης του DNA σε ασθενείς με αλληλοεπικαλυπτόμενα μυελικά νεοπλάσματα

Σκοπός: Τα μυελοδυσπλαστικά/μυελουπερπλαστικά νεοπλάσματα (ΜΔΣ/ΜΥΝ) είναι μυελικές νεοπλασίες στις οποίες παρατηρούνται χαρακτηριστικά τόσο των μυελοδυσπλαστικών (ΜΔΣ), όσο και των μυελοϋπερπλαστικών νεοπλασμάτων (ΜΥΝ). Σε μεγάλο ποσοστό των ασθενών συχνά ανιχνεύονται σημειακές ή άλλες μεταλλάξεις μικρής κλίμακας σε γονίδια που συμμετέχουν στην κυτταρική σηματοδότηση, στη ρύθμιση του επιγενετικού μηχανισμού του κυττάρου (TET2 και IDH1/2), στην ωρίμανση του RNA (SRSF2, SF3B1, U2AF1). Μάλιστα, οι τελευταίες δεν είναι αποκλειστικές, αλλά μπορεί να συνοδεύονται από μεταλλάξεις DNMT3, JAK2, ASXL1 και TET2. Σκοπός της παρούσας Διπλωματικής Εργασίας ήταν η κατά προσέγγιση εκτίμηση της μεθυλίωσης του γονιδιώματος μέσω ανάλυσης των ρετρομεταθετών στοιχείων LINE-1, τα οποία απαντώνται διάσπαρτα στο γονιδίωμα, με την τεχνική post Real-time PCR HRMΑ σε δείγματα από ασθενείς με ΜΔΣ/ΜΥΝ. Υλικά και Μέθοδοι: Σε αυτή την εργασία χαρακτηρίστηκαν 60 ασθενείς με βάσει τα κριτήρια του WHO 2016 για τα ΜΔΣ/ΜΥΝ και σύμφωνα με το μοριακό τους προφίλ, επιλέχθηκαν 15 ασθενείς κατά τη διάγνωση. Για τον προσδιορισμό της κατάστασης μεθυλίωσης των LINE-1, για τις αναλύσεις του πρότυπου της καμπύλης τήξης, χρησιμοποιήθηκαν ¬6 δείγματα DNΑ ατόμων χωρίς αιματολογικές διαταραχές. Για το σκοπό αυτό χρησιμοποιήθηκε η μέθοδος MS-HRMA και η μέθοδος MSP για την επαλήθευση των δεδομένων. Αποτελέσματα: Τα μεταθετά στοιχεία LINE-1, των ασθενών με ΜΔΣ/ΜΥΝ εμφανίζουν στατιστικώς σημαντικά (p-value=0.0071), αυξημένα επίπεδα μεθυλίωσης (88.47%), συγκριτικά με τα αντίστοιχα επίπεδα στα άτομα του φυσιολογικού πληθυσμού (85.81%). Επιπλέον, τα μεταθετά στοιχεία LINE-1, των ασθενών με ΜΔΣ/ΜΥΝ, που έχουν μετάλλαξη στο γονίδιο DNMT3A, εμφανίζουν στατιστικώς σημαντικά (p-value=0.0322), μειωμένα επίπεδα μεθυλίωσης (86.77%), συγκριτικά με τα αντίστοιχα επίπεδα στους ασθενείς με μετάλλαξη σε άλλο γονίδιο (88.73%). Τα αποτελέσματα επαληθεύτηκαν με τη μέθοδο MSP. Συμπεράσματα: Στην παρούσα εργασία, παρατηρήθηκε στατιστικώς σημαντική διαφορά των επιπέδων μεθυλίωσης (p-value=0.0036) των ασθενών (έχουν παραληφθεί αυτοί με μετάλλαξη στο γονίδιο DNMT3A), σε σχέση με τα άτομα του φυσιολογικού πληθυσμού. Τα αποτελέσματα της παρούσας εργασίας υποδηλώνουν ότι είναι απαραίτητο να διεξαχθούν περαιτέρω μελέτες, προκειμένου να εξαχθούν ασφαλή συμπεράσματα.Objective: Μyelodysplastic/myeloproliferative neoplasms are myeloid disorders characterized by both dysplastic and proliferative features. Molecular lesions in genes involved in cell signaling, epigenetic regulation (TET2 and IDH1 / 2), and RNA splicing (SRSF2, SF3B1, U2AF1) are often detected in a large proportion of patients. The latter are not exclusive, and are usually detected with mutation in other genes, i.e. DNMT3, JAK2, ASXL1 and TET2. The purpose of this thesis was to estimate the gene methylation by analyzing the LINE-1 retrotransposon elements that are dispersed in the genome utilizing α post-Real-Time PCR HRMA technique in samples of patients with MDS/MPN. Methods: In this study, 60 patients were included based on WHO 2016 criteria for MDS/MPN and according to their molecular profile, 15 patients were selected for methylation analysis. To determine the methylation status of the LINE-1, a standard curve was prepared using 6 DNA samples from healthy donors. For this purpose, the MS-HRMA method was used and the MSP method was used to verify the data. Results: The LINE-1 retrotransposon elements of MDS/MPN patients showed statistically significant (p-value = 0.0071), increased methylation levels (88.47%) in samples from patients with MDS/MPN, compared to the methylation levels iDNA samples from healthy donors (85.81%). Additionally, in MDS/MPN patients with a mutation in the DNMT3A gene, the LINE-1 transposons methylation status significantly reduced (86.77%;p-value = 0.0322), in comparison to methylation levels in patients with mutations in other genes (88.73%). The results were verified by the MSP method. Conclusions: In this study, we showed a statistically significant difference in the methylation levels of LINE-1 retrotransposon elements (p-value = 0.0036) in patients with MDS/MPN compared to the normal population. Further analysis should be performed in larger cohorts of patients to confirm these results

Genome organization of epidemic Acinetobacter baumannii strains

<p>Abstract</p> <p>Background</p> <p><it>Acinetobacter baumannii </it>is an opportunistic pathogen responsible for hospital-acquired infections. <it>A. baumannii </it>epidemics described world-wide were caused by few genotypic clusters of strains. The occurrence of epidemics caused by multi-drug resistant strains assigned to novel genotypes have been reported over the last few years.</p> <p>Results</p> <p>In the present study, we compared whole genome sequences of three <it>A. baumannii </it>strains assigned to genotypes ST2, ST25 and ST78, representative of the most frequent genotypes responsible for epidemics in several Mediterranean hospitals, and four complete genome sequences of <it>A. baumannii </it>strains assigned to genotypes ST1, ST2 and ST77. Comparative genome analysis showed extensive synteny and identified 3068 coding regions which are conserved, at the same chromosomal position, in all <it>A. baumannii </it>genomes. Genome alignments also identified 63 DNA regions, ranging in size from 4 o 126 kb, all defined as genomic islands, which were present in some genomes, but were either missing or replaced by non-homologous DNA sequences in others. Some islands are involved in resistance to drugs and metals, others carry genes encoding surface proteins or enzymes involved in specific metabolic pathways, and others correspond to prophage-like elements. Accessory DNA regions encode 12 to 19% of the potential gene products of the analyzed strains. The analysis of a collection of epidemic <it>A. baumannii </it>strains showed that some islands were restricted to specific genotypes.</p> <p>Conclusion</p> <p>The definition of the genome components of <it>A. baumannii </it>provides a scaffold to rapidly evaluate the genomic organization of novel clinical <it>A. baumannii </it>isolates. Changes in island profiling will be useful in genomic epidemiology of <it>A. baumannii </it>population.</p

Virulence-related traits of epidemic Acinetobacter baumannii strains belonging to the international clonal lineages I-III and to the emerging genotypes ST25 and ST78

BACKGROUND: Acinetobacter baumannii is responsible for large epidemics in hospitals, where it can persist for long time on abiotic surfaces. This study investigated some virulence-related traits of epidemic A. baumannii strains assigned to distinct MLST genotypes, including those corresponding to the international clones I-III as well as emerging genotypes responsible for recent epidemics. METHODS: Genotyping of bacteria was performed by PFGE analysis and MLST according to the Pasteur’s scheme. Biofilm formation on polystyrene plates was assessed by crystal violet staining; resistance to desiccation was evaluated on glass cover-slips when kept at room-temperature and 31% relative humidity; adherence to and invasion of A549 human alveolar epithelial cells were determined by the analysis of viable bacteria associated with or internalized by A549 human alveolar epithelial cells; Galleria mellonella killing assays were used to analyze the virulence of A. baumannii in vivo. RESULTS: The ability to form biofilm was significantly higher for A. baumannnii strains assigned to ST2 (international clone II), ST25 and ST78 compared to other STs. All A. baumannii strains survived on dry surfaces for over 16 days, and strains assigned to ST1 (international clone I) and ST78 survived for up to 89 and 96 days, respectively. Adherence to A549 pneumocytes was higher for strains assigned to ST2, ST25 and ST78 than other genotypes; a positive correlation exists between adherence and biofilm formation. Strains assigned to ST78 also showed significantly higher ability to invade A549 cells. No significant differences in the killing of G. mellonella worms were found among strains. CONCLUSIONS: Elevated resistance to desiccation, high biofilm-forming capacity on abiotic surfaces and adherence to A549 cells might have favoured the spread and persistence in the hospital environment of A. baumannii strains assigned to the international clones I and II and to the emerging genotypes ST25 and ST78

Molecular epidemiological investigation of multidrug-resistant Acinetobacter baumannii strains in four Mediterranean countries with a multilocus sequence typing scheme

Thirty-five multidrug-resistant Acinetobacter baumannii strains, representative of 28 outbreaks involving 484 patients from 20 hospitals in Greece, Italy, Lebanon and Turkey from 1999 to 2009, were analysed by multilocus sequence typing. Sequence type (ST)2, ST1, ST25, ST78 and ST20 caused 12, four, three, three and two outbreaks involving 227, 93, 62, 62 and 31 patients, respectively. The genes bla oxa-58, bla oxa-23 and bla oxa-72 were found in 27, two and one carbapenem-resistant strain, respectively. In conclusion, A. baumannii outbreaks were caused by the spread of a few strains

Use of larvae of the wax moth Galleria mellonella as an in vivo model to study the virulence of Helicobacter pylori

BACKGROUND: Helicobacter pylori is the first bacterium formally recognized as a carcinogen and is one of the most successful human pathogens, as over half of the world’s population is colonized by the bacterium. H. pylori-induced gastroduodenal disease depends on the inflammatory response of the host and on the production of specific bacterial virulence factors. The study of Helicobacter pylori pathogenic action would greatly benefit by easy-to-use models of infection. RESULTS: In the present study, we examined the effectiveness of the larvae of the wax moth Galleria mellonella as a new model for H. pylori infection. G. mellonella larvae were inoculated with bacterial suspensions or broth culture filtrates from either different wild-type H. pylori strains or their mutants defective in specific virulence determinants, such as VacA, CagA, CagE, the whole pathogenicity island (PAI) cag, urease, and gamma-glutamyl transpeptidase (GGT). We also tested purified VacA cytotoxin. Survival curves were plotted using the Kaplan-Meier method and LD(50) lethal doses were calculated. Viable bacteria in the hemocoel were counted at different time points post-infection, while apoptosis in larval hemocytes was evaluated by annexin V staining. We found that wild-type and mutant H. pylori strains were able to survive and replicate in G. mellonella larvae which underwent death rapidly after infection. H. pylori mutant strains defective in either VacA, or CagA, or CagE, or cag PAI, or urease, but not GGT-defective mutants, were less virulent than the respective parental strain. Broth culture filtrates from wild-type strains G27 and 60190 and their mutants replicated the effects observed using their respective bacterial suspension. Also, purified VacA cytotoxin was able to kill the larvae. The killing of larvae always correlated with the induction of apoptosis in hemocytes. CONCLUSIONS: G. mellonella larvae are susceptible to H. pylori infection and may represent an easy to use in vivo model to identify virulence factors and pathogenic mechanisms of H. pylori. The experimental model described can be useful to screen a large number of clinical H. pylori strain and to correlate virulence of H. pylori strains with patients’ disease status

Carbapenem resistance in Acinetobacter baumannii: the molecular epidemic features of an emerging problem in health care facilities

Acinetobacter baumannii is an opportunistic gram-negative pathogen with increasing relevance in a variety of nosocomial infections especially among intensive-care-unit (ICU) patients. Carbapenems have been widely used to treat serious multidrug-resistant A. baumannii infections; however, incidences of carbapenem-resistant A. baumannii are rising in several parts of the world and large and sustained outbreaks caused by such bacteria have been described. Carbapenem-resistant A. baumannii epidemics are sustained by clusters of highly similar strains that successfully spread among different cities and countries; their resistance phenotype is mainly due to the acquisition of carbapenem-hydrolyzing class D β-lactamase (CHDL) genes flanked by insertion sequence (IS) elements. Multi-facility outbreaks can be also sustained by inter-hospital transfer of colonized patients. Here, we review the global epidemiology of carbapenem-resistant A. baumannii, with the emphasis on the molecular epidemiology and genetic characterization of carbapenem resistance in epidemic strains

Specific myeloid signatures in peripheral blood differentiate active and rare clinical phenotypes of multiple sclerosis

Current understanding of Multiple Sclerosis (MS) pathophysiology implicates perturbations in adaptive cellular immune responses, predominantly T cells, in Relapsing-Remitting forms (RRMS). Nevertheless, from a clinical perspective MS is a heterogeneous disease reflecting the heterogeneity of involved biological systems. This complexity requires advanced analysis tools at the single-cell level to discover biomarkers for better patient-group stratification. We designed a novel 44-parameter mass cytometry panel to interrogate predominantly the role of effector and regulatory subpopulations of peripheral blood myeloid subsets along with B and T-cells (excluding granulocytes) in MS, assessing three different patient cohorts: RRMS, PPMS (Primary Progressive) and Tumefactive MS patients (TMS) (n=10, 8, 14 respectively). We further subgrouped our cohort into inactive or active disease stages to capture the early underlying events in disease pathophysiology. Peripheral blood analysis showed that TMS cases belonged to the spectrum of RRMS, whereas PPMS cases displayed different features. In particular, TMS patients during a relapse stage were characterized by a specific subset of CD11c+CD14+ CD33+, CD192+, CD172+-myeloid cells with an alternative phenotype of monocyte-derived macrophages (high arginase-1, CD38, HLA-DR-low and endogenous TNF-a production). Moreover, TMS patients in relapse displayed a selective CD4 T-cell lymphopenia of cells with a Th2-like polarised phenotype. PPMS patients did not display substantial differences from healthy controls, apart from a trend toward higher expansion of NK cell subsets. Importantly, we found that myeloid cell populations are reshaped under effective disease-modifying therapy predominantly with glatiramer acetate and to a lesser extent with anti-CD20, suggesting that the identified cell signature represents a specific therapeutic target in TMS. The expanded myeloid signature in TMS patients was also confirmed by flow cytometry. Serum neurofilament light-chain levels confirmed the correlation of this myeloid cell signature with indices of axonal injury. More in-depth analysis of myeloid subsets revealed an increase of a subset of highly cytolytic and terminally differentiated NK cells in PPMS patients with leptomeningeal enhancement (active-PPMS), compared to those without (inactive-PPMS). We have identified previously uncharacterized subsets of circulating myeloid cells and shown them to correlate with distinct disease forms of MS as well as with specific disease states (relapse/remission)