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Non-invasive prenatal testing: a new era in fetal medicineBevilacqua, Elisa 21 April 2020 (has links) (PDF)
Les anomalies chromosomiques sont une cause majeure de morbidité et de mortalité périnatales. Le dépistage de ces affections a toujours été crucial pour une prise en charge optimale des femmes enceintes.Initialement, le dépistage de trisomie 21 était uniquement basé selon le risque lié à l’âge maternel. L’ajout de différents marqueurs biochimiques sériques constituant successivement les double, triple et quadruple tests a pu améliorer le taux de détection. Néanmoins, c’est en 1997 qu’apparaît un point tournant de l’histoire de dépistage des trisomies :l’introduction de la mesure de la clarté nucale à l’échographie du premier trimestre.Depuis 2011, de nouvelles recherches se sont concentrées sur le dépistage prénatal non invasif (DPNI) utilisant l'ADN fœtal libre circulant dans le sang maternel. Cette technique a suggéré directement une supériorité marquée pour la détection de la trisomie 21 comparée à toutes les autres méthodes de dépistage connues. Rapidement, ce test a été introduit en pratique clinique dans le monde entier sans une évaluation préalable et approfondie, que ce soit au niveau scientifique, technique ou éthique.Les objectifs de cette thèse étaient de fournir des réponses aux diverses questions persistantes concernant l’utilisation clinique des tests de dépistages basés sur ADN fœtal libre dans la circulation maternelle.À notre connaissance, nous sommes la première équipe à essayer d'évaluer le taux d'échec et la performance du DPNI effectué par différents laboratoires utilisant différentes méthodes analytiques. Nous avons démontré que les approches « DANSR » (approche ciblée sur les chromosomes d’intérêt) et « GW-MPS » (approche globale sur les séquences géniques reparties sur la totalité du génome par un séquençage à haut débit) offraient toutes les deux un taux échec bas avec une bonne performance dans la détection des trisomies 21, 13 et 18. Cependant, le niveau de fraction fœtale semble varier d'un laboratoire à l'autre et n’est, par conséquent, pas comparable. Nous avons également observé que le taux d'échec des laboratoires avec un test « home-brew » était 4 fois supérieur à celui des tests commerciaux développés par les laboratoires en interne. De plus, aucune pertinence clinique de la divulgation des aneuploïdies autres que les 3 trisomies communes décelées par les DPNI « GW-MPS » n’a pu être démontrée.Ensuite, nous nous sommes intéressés au groupe particulier des grossesses gémellaires. Dans ce groupe, le DPNI était faisable mais il était associé à un taux d'échec supérieur aux grossesses uniques, tout en fournissant un taux de détection moindre des trisomies communes. Un poids maternel élevé et la conception par fécondation in vitro étaient des facteurs indépendants associés à l’échec du test. De plus, il faut souligner l’importance de développer des normes de contrôle de qualité pour les analyses faites sur l’ADN fœtal libre. Nous avons aussi étudié les modifications de l’ADN fœtal libre après une mort fœtale en raison d’une aneuploïdie chez l’un des deux fœtus lors d’une grossesse gémellaire. Après le décès du fœtus atteint, la fraction fœtale de l’ADN libre circulant dans le sang maternel a montré une évolution imprévisible, pouvant augmenter, diminuer ou rester stable dans le temps. Par conséquent, ces résultats déconseillent l’utilisation du DPNI en cas de décès d’un des deux fœtus, même après un intervalle de temps de plusieurs semaines.Notre attention s’est ensuite portée vers la performance du DPNI pour le dépistage des anomalies autres que les 3 trisomies communes. Nous avons d’abord étudié la performance du DPNI pour les anomalies des chromosomes sexuels ainsi que les caractéristiques des patientes optant pour ce test. Plus de la moitié des patientes ayant eu un DPNI ont également souhaité le dépistage des anomalies des chromosomes sexuels. Les valeurs prédictives positives suivantes ont été observées :24% pour 45 X et 47 XXX et environ 71% pour 47 XXY et 47 XYY. Ainsi, la recherche d’anomalies des chromosomes sexuels peut causer la détection accidentelle d’anomalies chromosomiques sans conséquence clinique. Par conséquent, un conseil génétique est obligatoire dans toutes ces situations, de même qu’un examen invasif pour un caryotype fœtal de confirmation.Ensuite, nous avons montré que le test à ADN fœtal libre utilisant une technologie ciblée basée sur le microarray permettait d'identifier les grossesses à risque accru de délétions 22q11.2. Cependant, des données fiables sur les performances du DPNI pour le syndrome 22q11.2 sont encore manquantes, et des recherches plus poussées sont nécessaires. Depuis 2015, nous participons à une étude prospective, multicentrique et en aveugle, qui évalue cliniquement un test d’ADN fœtal libre pour la détection de délétions ou de duplications dans la région 22q11. Le recrutement s’est terminé le 1er novembre 2019.Enfin, en décrivant le profil et le choix des patientes belges soumises à un test à ADN fœtal libre, nous avons observé un changement vers une population à faible risque, ce qui peut entrainer une réduction de la valeur prédictive positive du test. Il est donc primordial que cet effet soit connu des professionnels de la santé qui conseillent, prescrivent et interprètent ces tests.En conclusion, notre recherche a démontré la complexité des tests à ADN fœtal libre circulant dans le sang maternel, non seulement du point de vue technique, mais également en termes de conseils aux patients avant et après le test, ce qui requière des connaissances et compétences spécifiques des médecins proposant ces tests.L’ère du test à ADN fœtal libre circulant dans le sang maternel n’en est qu’à ses débuts. Notre travail n’a exploré qu’une petite partie de l’énorme potentiel de ce nouvel outil de dépistage des aneuploïdies.L’intégration responsable des innovations dans la pratique clinique reste, aujourd’hui, l’un des défis majeurs. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished
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Korrelation der Plasma-Konzentration frei zirkulierender DNA mit dem Schweregrad der Nicht-alkoholischen FettlebererkrankungWeise, Lara Janika 02 March 2022 (has links)
Die Beurteilung von Fibrose und Entzündungsaktivität ist essenziell zur Feststellung eines progredienten Krankheitsverlaufes bei Patienten mit nicht-alkoholischer Fettlebererkrankung (NAFLD). Während zur Bestimmung des Fibrosegrades ultraschall-gestützte Methoden und Serum-Marker die invasive Leberbiopsie für verschiedenen Fragestellungen bereits ersetzen können, bleibt die nicht-invasive Charakterisierung der Entzündungsaktivität eine Herausforderung. Frei zirkulierende DNA (cfDNA) als neuer nicht-invasiv zu bestimmender Biomarker für zelluläre Entzündung und Zelltod wurde in Patienten mit NAFLD bisher noch nicht evaluiert.
Für die vorliegenden Dissertation wurde die Konzentration von cfDNA (90 und 222bp-Fragmente) im Plasma von 58 NAFLD-Patienten sowie 13 gesunden Kontroll-Probanden, die an zwei prospektiven Diagnostikstudien teilgenommen hatten, mittels quantitativer real-time PCR gemessen und ausgewertet. Die Ergebnisse wurden mit der Gewebesteifigkeit (Transiente Elastographie) und dem Leberfettgehalt (Controlled attenuation parameter; Magnetresonanzspektroskopie / 1H-MRS) sowie den Serumspiegeln der Aminotransferasen und des Ferritins verglichen.
Der Gehalt an 90bp-cfDNA im Plasma war bei NAFLD-Patienten signifikant höher als bei den gesunden Probanden. In der NAFLD-Kohorte korrelierte die cfDNA-Konzentration signifikant mit der Krankheitsaktivität und dem Krankheitsgrad, insbesondere bei Patienten mit erhöhter Lebersteifigkeit.
Die weitere Untersuchung von cfDNA als Biomarker für den Krankheitsverlauf von Patienten mit NAFLD ist daher empfehlenswert, insbesondere im Hinblick auf die Identifikation von Patienten mit einem erhöhten Risiko für progrediente NAFLD.:1 Vorbemerkung
2 Bibliographische Beschreibung
3 Abkürzungsverzeichnis
4 Einführung
4.1 Nicht-alkoholische Fettlebererkrankung
4.1.1 Definition und Epidemiologie
4.1.1.1 Herausforderung im Umgang mit der NAFLD
4.1.2 Pathogenese
4.1.3 Nichtinvasive Diagnostik
4.1.3.1 Bildgebende Verfahren
4.1.3.2 Serum-basierte Steatose- und Fibrose-Indices
4.1.4 Histopathologie
4.1.5 Genetische Prädisposition
4.1.6 Therapie
4.2 Frei zirkulierende DNA
4.2.1 Entdeckung und Ursprung frei zirkulierender DNA
4.2.2 Klinische Schwerpunkte in der bisherigen cfDNA-Forschung
4.2.3 Potenzielle Bedeutung der cfDNA bei NAFLD
4.3 Wissenschaftliche Zielsetzung
5 Publikation
6 Zusammenfassung und Ausblick
7 Literaturverzeichnis
8 Anhang
8.1 Erklärung über die eigenständige Abfassung der Arbeit
8.2 Erklärung über den wissenschaftlichen Beitrag der Promovendin an der ausgewählten Publikation
8.3 Teilnahmebescheinigung: Vorlesung zur „Guten wissenschaftlichen Praxis“ an der Medizinischen Fakultät der Universität Leipzig
8.4 Lebenslauf
8.5 Veröffentlichungen im Rahmen dieser Arbeit
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Detection Of Sepsis Biomarkers Using MicrofluidicsDamodara, Sreekant January 2021 (has links)
Sepsis is a “life-threatening organ dysfunction caused by a dysregulated host response to infection” that has a widespread impact on human life around the world. It affects more than 1.5 million people, killing at least 250,000 each year in the US alone and affects 90,000 people annually, with estimated mortality rates of up to 30% in Canada. Our understanding of the different biochemical pathways that in the progression of sepsis has improved patient care for sepsis patients. One part of patient care is the use of biomarkers for patient prognosis that draws on the full range of relevant and available information to model the possible outcomes for an individual. Numerous biomarkers have been studied for patient prognosis that includes Procalcitonin (PCT), C-reactive protein (CRP), TNF-α, cfDNA, protein C and PAI 1. Using a panel of multiple biomarkers provided more accuracy in patient prognosis than using individual biomarkers and one such panel that was proposed used cfDNA, protein C, platelet count, creatinine, Glasgow Coma Scale [GCS] score, and lactate. Commercial, low cost POC techniques were available for the measurement of all biomarkers besides cfDNA and protein
C. The objective of this doctoral thesis was chosen to develop low cost, microfluidic devices for the measurement of protein C and cfDNA using nonspecific fluorescence dyes that would enable the eventual integration of the systems and improve patient prognosis. The measurement of protein C in plasma required the separation of protein C from interfering proteins in plasma. This was done through the development of a two-stage separation process that included the development of tunable agarose isoelectric gates for separating proteins using their isoelectric point and the miniaturization of immobilized metal affinity chromatography and its extension to Barium for the selective binding of proteins using their chemical affinity. This was performed in a xurographically fabricated chip to reduce costs and enable the use of geometric focusing of the electric field to enable the operation of the device at a lower applied voltage. The challenges faced with cfDNA were different due to the different characteristics of the material and less interference from plasma. The requirement was to measure the total cfDNA content with minimal cost in comparison to currently available techniques. This was achieved through the development of thread microfluidic devices that showed the use of thread for automated aliquoting of samples by controlling length and twists of the thread. Preconcentration and use of external apparatus was avoided by showing that thread could be used to amplify fluorescence response to a range that was sufficient for the measurement of cfDNA in sepsis patients. A portable fluorescence imaging setup was developed for this purpose and was used in demonstration for the measurement of cfDNA in plasma with sufficient resolution. In conclusion, we developed technologies for rapid and low-cost measurement of protein C and cfDNA using xurographic and thread-based microfluidics that may serve as valuable in improving patient prognosis. / Thesis / Doctor of Philosophy (PhD) / Sepsis is a major reason for hospitalization and cause of death in hospitals worldwide. Its treatment is highly time sensitive with each hour of delay in diagnosis causing a significant increase in chances of death. Due to the wide range of symptoms that can be caused by sepsis, its diagnosis uses a scoring method that relies on the expertise of the onsite doctors and nurses increasing their workload. A more objective system for detection requires the measurement of the quantities of different biomarkers in blood. Biomarkers are proteins present in plasma that change in quantity due to the body’s reaction to sepsis. Several of these biomarkers have been identified and studied for their use in both diagnosing
the presence of sepsis and in predicting the outcome with the current treatment plan. In this PhD study, we chose two of these biomarkers – circulating free DNA (cfDNA) and protein C and developed low-cost techniques for rapidly measuring their concentration in blood plasma. To do this, we made microfluidic devices with techniques that use low-cost materials such as plastic sheets and threads.The device for the measurement of protein C required separating it from many other proteins in plasma. We showed that a device fabricated from stacked plastic sheets and integrated with agarose gels could be used for the measurement of protein C in plasma with sufficient resolution to help with treating septic patients at a cost of less $5 per device. Similarly, we showed that a device that integrated threads with plastic sheets could be used for measuring the quantity of cfDNA in plasma in a portable format within 15 minutes. Overall, we developed tools for rapid measurement of two biomarkers of sepsis using low cost device that cost under $5 to run and could led to improving the quality of care for sepsis patients.
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Circulating tumour DNA in localised urological cancersPatel, Keval Mahendra January 2017 (has links)
There is a need for informative biomarkers in localised urological cancers. At present, no method can accurately distinguish between indolent and aggressive prostate cancers, and men often require repeated biopsies. Patients with muscle invasive bladder cancer undergo neo-adjuvant chemotherapy (NAC) to improve survival. However many do not respond to NAC, delaying definitive treatment. Cell-free mutant DNA (mutDNA) analysis represents an opportunity for non-invasive monitoring of cancer through tumour genome analysis. MutDNA derived from plasma can monitor tumour burden. There is emerging evidence that mutDNA can identify mutations from multiple clones and is abundant in adjacent body fluids. This work explores the utility of plasma and urinary mutDNA in localised prostate and bladder cancers. This thesis describes the optimisation of urinary mutDNA analysis by assessing urinary DNA processing and extraction methods using healthy volunteer and bladder cancer patient urine samples. Primer panels were designed and validated to target frequently mutated regions in prostate and bladder cancers, as well as for analysis of patient-specific mutations. Sequencing-based methods and dPCR were employed to analyse clinical samples including plasma and urine, to detect and quantify mutDNA. Molecular and clinical data were integrated to explore potential areas of application of mutDNA analysis. For bladder cancer, mutDNA was analysed from liquid-biopsy samples including plasma, cell pellets from urine and urine supernatant from multiple time-points of 17 MIBC patients undergoing NAC. I showed that mutDNA was more frequently detected and was present at higher AFs in urine compared to plasma samples. Of potential clinical relevance, I showed that the presence of mutDNA after starting NAC was associated with disease recurrence. This original contribution to knowledge could offer patients an opportunity to expedite surgical resection in a timely manner, if corroborated in large-scale trials. For prostate cancer, a TP53 specific panel was applied to men with metastatic disease, to demonstrate that clones containing TP53 mutations, which are dominant in at the metastatic stage were present in historical prostatectomy samples taken when then patient was believed to have localised disease only. Furthermore, I showed that these TP53 mutations could be detected at the localised stage of disease. To investigate the ability of mutDNA detection private clonal mutations I developed a method for higher sensitivity analysis (MRD-Seq). This was applied to a clinical cohort of 2 men with multi-focal localised prostate cancer to demonstrate the though the overall levels of mutDNA is low, private clonal mutations may be detectable. Taken together, these original contributions to knowledge could allow for less invasive surveillance of men with low risk prostate cancer and warrants further investigation. In this thesis, I used a range of molecular methods were applied to small cohorts of clinical samples from patients with urological malignancies, in an exploratory analysis. The molecular data was analysed in conjunction with clinical information to draw hypotheses on the biology and natural history of these cancer, and to suggest possible utility of mutDNA analysis in their clinical management. Some of the findings suggest areas of potential utility, which merit further validation or investigation in larger cohorts or clinical studies.
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Die Rolle der zellfreien DNA in der InsulinresistenzBartels, Milena Susanne 05 March 2021 (has links)
Es wird postuliert, dass es einen positiven Zusammenhang zwischen cfDNA und Insulinresistenz gibt, bei dem über cfDNA Verbindungen zwischen Adipositas, einer Subinflammation im (Fett-) Gewebe und Insulinresistenz besteht. Durch die pathologische Dauerstimulation der Insulinrezeptoren entsteht eine Insulinresistenz, weshalb u.a. die Zellen des Fettgewebes einen relativen Mangel an dem Schlüsselhormon des Glukosestoffwechsels, dem Insulin, erleiden und in eine metabolische Mangelsituation geraten. Dieser intrinsiche Stress, ausgelöst durch Sauerstoff- und Glukosemangel, führt zu Apoptose. Dadurch wird cfDNA freigesetzt, die über eine Aktivierung von Makrophagen eine Subinflammation begünstigt. Ziel meiner Studie war es, die cfDNA-Freisetzung im Zusammenhang mit Adipositas und Insulinresistenz zu untersuchen und Korrelationen mit klinischen und metabolischen Parametern rund um die Insulinresistenz und T2D zu testen.
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Analýza volných nukleových kyselin v moči urologických pacientů. / Analysis of cell-free nucleic acids in urine of urological patients.Šantorová, Šárka January 2019 (has links)
The two studies follow free nucleic acids in urine in search for biomarkers to distinguish urinary bladder cancer patients from controls. Bladder cancer forms 4 % of newly diagnosed oncological diseases in the Czech Republic. Nowadays, there is no accredited non-invasive method for its diagnosis, which is sufficiently accurate. Urine supernatant, which is washing the bladder mucosa and which does not contain cell debris, seems to be an appropriate source of biomarkers for non-invasive diagnosis. miRNAs, as a non-invasive biomarker of urinary bladder cancer, were studied in one of the studies. miRNAs are short noncoding RNA, which block the process of translation. miRNAs occur in all body fluids and are relatively stable. A study with three phases was assessed to find a suitable miRNA marker. 109 individuals were examined in total (36 controls and 73 bladder cancer patients). The analysis of miRNAs was based on RT-PCR (Reverse Transcription Polymerase Chain Reaction). In the first phase, the urine of 59 individuals was analyzed on TaqMan array card with 381 miRNAs. In the second phase, the results of the first phase were confirmed on the same cohort by a single miRNA assay. In the third phase, a new cohort was used (23 controls and 27 bladder cancer patients), analyzed by a single miRNA assay again....
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Unleashing the potential of liquid biopsy: allele-informed evaluation of plasma samples for cancer patients managementOrlando, Francesco 23 January 2023 (has links)
Liquid biopsy and next-generation sequencing of cell-free DNA (cfDNA) in cancer patients’ plasma offer a minimally-invasive solution to detect tumor cell genomic information to aid real-time clinical decision-making. Reliability and sensitivity in the detection of genomic alterations is crucial for patient management and it is particularly relevant in the context of targeted therapies. However, biological and technical factors, such as low cfDNA tumor fraction and sequencing errors, affect the correct interpretation of genomic data limiting the utility of non-invasive cfDNA-based tumor profiling. To address these issues, we designed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation of patients’ tumor. The framework also implements ABEMUS, an ad-hoc computational procedure we specifically designed for cfDNA samples to accurately detect somatic point mutations that could emerge under treatment pressure and as drug resistance mechanism. When applied on serial plasma samples from three patients receiving PARP inhibition harboring DNA repair gene aberrations, PCF_SELECT demonstrated high sensitivity in detecting BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. As a step towards a more sensitive detection of tumor features in circulation of cancer patients, we next hypothesized that recent WGS-based approaches exploiting cfDNA fragments characteristics could be extrapolated for targeted sequencing data and that gene-region specific measures could improve detection metrics. Preliminary results suggest an increased sensitivity compared to copy-number-based methods supporting the integration at no extra cost in our targeted assay. Overall, this work is relevant to the context of precision oncology. It provides an innovative platform for the management of cancer patients, delivering detailed patient-specific molecular information which could be applied to guide treatment and improve clinical outcomes.
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Cell of Origin Identification Using Methylation Signatures from Seminal Cell-Free DNA and Heterogenous Cellular MixturesBarney, Ryan 13 November 2023 (has links) (PDF)
Infertility is an issue for approximately 12% of couples attempting to have a child. Of this group, 50% of the cases are due to male factor infertility. There are many reasons for decreased fecundity in men, but there remains 10% to 15% of infertile men that are diagnosed with the most severe form of infertility, non-obstructive azoospermia (NOA). A diagnosis of NOA implies the lack of sperm cells in the ejaculate with no physiological reason. The current diagnostic test and treatment consist of microscopic examination of seminal fluid and a biopsy to extract any viable sperm from the testis. This treatment is known to be problematic because of the destructive nature of surgery as well as expense. A non-invasive diagnostic test that could identify the presence of sperm in the testis at the beginning of fertility treatment would inform the patient and the physician about the functionality of the testis and thus lead to more informed decisions about treatment and potentially a decrease in cost. The ability to identify the tissue source of DNA present in the reproductive tract could facilitate a fertility diagnostic tool. Tissue specific epigenetic mechanisms are known to play a role in an organism's development. The identification of an epigenetic signature unique to sperm DNA would allow for the identification of sperm DNA in a heterologous mixture. Our lab has been able to identify a methylation signature that can consistently differentiate between sperm DNA and somatic DNA. We compared the sperm DNA signature with that of blood and testicular tissue and found that there was no overlap in epigenetic markers. To create an assay that could evaluate the presence of sperm DNA we used an Oxford Nanopore next-generation sequencing platform. Sequencing bisulfite converted DNA; we were able to retrieve the methylation status at locations of interest. A bioinformatic tool was created to analyze the thousands of reads obtained and analyze the individual methylation points within single molecules of DNA. To create a more accessible fertility test, we used the sperm DNA analysis tool to evaluate seminal cell-free DNA (cfDNA). The presence of sperm cfDNA in a patient's seminal fluid may indicate that there is sperm somewhere in the male reproductive tract even if the cells are not intact. A clinician could use this information to better advise the patient about treatment and potentially decrease cost of care.
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Effects of Vasoflux on DNA-Histone Complexes in Vitro and on Organ Function and Survival Outcome in a Murine Model of SepsisSharma, Neha January 2018 (has links)
Sepsis is life-threatening organ dysfunction produced by a dysregulated host response to infection in which neutrophils release neutrophil extracellular traps (NETs). NETs consist of DNA, histones, and antimicrobial peptides which kill pathogens. However, DNA and histones also exert damage by activating the intrinsic pathway of coagulation and inducing endothelial cell death, respectively. AADH, a 15kDa non-anticoagulant unfractionated heparin (UFH), prevents histone-mediated cytotoxicity in vitro and improves survival in septic mice. We explored the effectiveness of Vasoflux, a 5.5kDa low-molecular-weight-heparin as an anti-sepsis treatment as compared to enoxaparin and UFH. Vasoflux has reduced anticoagulant functions and hence reduces the risk of bleeding as compared to enoxaparin or UFH. We showed that UFH, enoxaparin, or Vasoflux at concentrations of up to 13.3uM, 40uM, or 40uM, neutralize histone-mediated cytotoxicity. These results suggest that these glycosaminoglycans (GAGs) are able to neutralize histone-mediated cytotoxicity independent of the AT-binding pentasaccharide. To quantitate the binding affinity between GAGs and histones, surface plasmon resonance was conducted. UFH is a more potent inhibitor of histone-mediated cytotoxicity compared to Vasoflux as UFH has a 10-fold greater binding affinity to histones compared to Vasoflux. To translate our in vitro findings to in vivo, Vasoflux, enoxaparin, and UFH were administered in a murine model of sepsis. Vasoflux at 8mg/kg - 50mg/kg reduced survival and exhibited damage in the lung, liver, and kidney in septic mice compared to 10 mg/kg of UFH or 8mg/kg of enoxaparin. This may be due to Vasoflux and UFH disrupting the DNA-histone complex, thereby releasing free procoagulant DNA. This is evident by our gel electrophoresis experiments, where addition of 1uM Vasoflux or 3.3uM UFH to DNA-histone complexes lead to histone dissociation from DNA. UFH bound to histones may be able to inhibit DNA-mediated thrombin generation, as it retains its anticoagulant properties, demonstrated by UFH-histone complexes attenuating DNA and TF-mediated thrombin generation. In contrast, Vasoflux may not neutralize the procoagulant DNA leading to a hypercoagulable state in the mice. Our study may have important clinical implications as there is an ongoing trial, HALO, which will administer intravenous UFH to patients suspected to have septic shock to reduce mortality. Based on our results, future clinical trials should consider the antithrombin-dependent anticoagulant activity of UFH being used as a sepsis treatment. / Thesis / Master of Science (MSc) / Sepsis is a life threatening condition caused by the body’s extreme response to microbial infection of the blood, whereby neutrophils release traps composed of cell-free DNA (cfDNA), histones, and antimicrobial proteins. In addition to fighting off infections, these traps also exert harmful effects like triggering clotting and killing host cells. Currently, no specific anti-septic drugs exist. Studies have shown that DNase1 (a recombinant protein that digests double stranded cfDNA) or a modified form of heparin (neutralizes histones) improves survival in septic mice. Our goal was to explore the protective effects of Vasoflux, (a non-anticoagulant heparin) and DNase1 in a mouse model of sepsis. We hypothesize that the combined therapy of DNase1 and Vasoflux will improve survival. We found that Vasoflux has minimal blood thinning activity and can prevent histones from killing cells. However, Vasoflux administered into septic mice worsened organ damage and decreased survival. We hypothesize that this damage may be due to Vasoflux’s ability to displace histones from histone-DNA complexes, thereby releasing free DNA, which promotes excessive blood clotting in sepsis.
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Vliv vybraných zánětlivých agens na proces osteoklastogeneze / Effect of selected inflammatory agents on the osteoclastogenesisŠkubica, Patrik January 2018 (has links)
Introduction: Bone is a highly active tissue throughout life and is a subject to constant remodelling. Main cells responsible for continuous resorption and de novo synthesis of bone matrix are osteoclast, osteoblasts and osteocytes. Osteoclasts are the only known type of cells able to resorb bone. These cells are formed by fusion of precursor cells in bone marrow or peripheral blood in a process called osteoclastogenesis. Formation of osteoclasts may be of importance concerning chronic inflammatory diseases that are linked with higher risk of developing osteoporosis during lifespan. Celiac disease is one of those diseases, which is characterized by destruction of intestinal mucosa after ingestion of gluten by susceptible individuals followed by induction of chronic inflammation. In this work, we focused on the potential role of osteoclastogenesis in the development of osteoporosis in patients with celiac disease and we studied roles of selected inflammatory agents (TNF-α, IL-6, IFN-γ a cfDNA) with supposed or hypothesised effects on osteoclastogenesis. Material & Methods: We obtained plasma and serum samples from newly diagnosed patients with celiac disease, patients on gluten free diet and healthy controls and analysed concentrations of cfDNA and inflammatory cytokines TNF-α, IL-6 and IFN-γ in...
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