Thesis (Ph.D.)--Indiana University, 2009. / Title from screen (viewed on February 2, 2010). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Mervin C. Yoder, Jr., David A. Ingram, Jr., Lawrence A. Quilliam, Mark D. Pescovitz. Includes vitae. Includes bibliographical references (leaves 171-194).

No description available.

L’hypoxo-ischémie (HI) cérébrale néonatale représente une des principales causes de mortalité et de morbidité chez les nouveau-nés. Sa physiopathologie implique différents processus délétères menant vers la perte neuronale et responsables de séquelles neuro-cognitives. L'hypothermie thérapeutique est le seul traitement actuel mais est insuffisant. Cette étude a caractérisé et comparé l’effet de deux types de cellules issues du sang de cordon humain, les cellules mononuclées (HUCBCs) et les progéniteurs endothéliaux tardifs (ECFCs) sur l’amélioration des scores neuro-comportementaux mais aussi à l’échelle moléculaire et fonctionnelle dans le modèle d’hypoxo-ischémie néonatale à court (7 jours après l’épisode ischémique) et long terme (12 semaines après l’épisode ischémique).L’injection intrapéritonéale d'ECFCs ou de HUCBCs, 2 jours après HI, améliore les capacités de motricité et de mémorisation précoce et tardive des animaux à l’âge adulte, et diminue les comportements anxieux. Ces résultats sont associés à une augmentation de la densité capillaire en temps précoce et tardif. L’imagerie de perfusion cérébrale SPECT/CT a objectivé une restauration complète de la perfusion cérébrale de l’hémisphère lésé à l’âge adulte par les deux types cellulaires. Ces observations tardives sont associées à un effet protecteur précoce de ces cellules sur l’augmentation de la survie neuronale et la diminution de l’astrogliose réactionnelle ou encore sur la composante inflammatoire par diminution de l’activation microgliale pro-inflammatoire au niveau striatal. Les résultats de cette étude ouvrent ainsi de nouvelles perspectives pour l’usage des ECFCs dans le traitement de l’HI néonatale. / Neonatal hypoxic-ischemic encephalopathy (NHIE) is a dramatic perinatal complication, associated with poor neurological prognosis despite neuroprotection by therapeutic hypothermia, in the absence of an available curative therapy. We evaluated and compared ready-to-use human umbilical cord blood cells (HUCBCs) and bankable but allogeneic endothelial progenitors (ECFCs) as cell therapy candidate for NHIE. We compared benefits of HUCBC and ECFC transplantation 48 hours after injury in male rat NHIE model, based on the Rice-Vannucci approach. Based on behavioral tests, immune-histological assessment and metabolic imaging of brain perfusion using SPECT, HUCBC or ECFC administration provided equally early and sustained functional benefits, up to 8 weeks after injury. These results were associated with total normalization of injured hemisphere cerebral blood flow assessed by SPECT/CT imaging. In conclusion, even if ECFCs represent an efficient candidate, HUCBCs’ autologous criteria and easier availability make them the ideal candidate for hypoxic-ischemic cell therapy.

Biologische Systeme sind in der Lage sich an eine Niedrig-Dosis-Bestrahlung anzupassen und eine geringere Sensitivität gegenüber einer nachfolgenden Hoch-Dosis-Bestrahlung zu entwickeln. Dieses Phänomen wird als adaptive Antwort bezeichnet und wurde nach der Bestrahlung mit externen Strahlungsquellen wiederholt in vivo und in vitro untersucht. Im Gegensatz dazu gibt es für die Bestrahlung mit offenen Radionukliden keine systematischen und vergleichenden Untersuchungen. Im Mittelpunkt dieser Arbeit standen der Nachweis sowie die Analyse der adaptiven Antwort an PC Cl3-Zellen nach Bestrahlung mit den offenen Radionukliden Re-188 und Tc-99m. Die Zellschädigung wurde mit dem alkalischen Komet-Assay, zur Bestimmung des initialen DNA-Schadens und dem Koloniebildungstest, zur Ermittlung des klonogenen Überlebens, untersucht. Zur Aufklärung von möglichen Regulationsmechanismen der adaptiven Antwort wurde die Induktion und Reparatur von DSB mit dem gamma-H2AX-Immunfluoreszenz-Assay und die intrazelluläre Radionuklidaufnahme betrachtet. In dieser Arbeit erfolgte erstmals eine systematische Untersuchung der adaptiven Antwort nach Bestrahlung mit offenen Radionukliden in vitro. Insgesamt zeigen diese Ergebnisse, dass nach Bestrahlung mit offenen Radionukliden eine adaptive Antwort ausgebildet wird. Diese ist von der Strahlenqualität während Vor- und Folgebestrahlung sowie der Art der DNA-Schädigung und den initiierten Reparaturprozessen abhängig. Weiter Einflussfaktoren sind die Erholungszeit, die Vorbestrahlung (Dosis, Strahlenqualität) und die Art des Schadensnachweises. Neben den bekannten Regulationsmechanismen wurde erstmals die Reduktion der intrazellulären Radionuklidaufnahme als weitere mögliche adaptive Antwort beschrieben.

Indiana University-Purdue University Indianapolis (IUPUI) / A hierarchy of endothelial colony forming cells (ECFCs) with different levels of proliferative potential has been identified in human circulating blood and blood vessels. High proliferative potential ECFCs (HPP-ECFCs) display properties (robust proliferative potential in vitro and vessel-forming ability in vivo) consistent with stem/progenitor cells for the endothelial lineage. Corneal endothelial cells (CECs) are different from circulating and resident vascular endothelial cells (ECs). Whereas systemic vascular endothelium slowly proliferates throughout life, CECs fail to proliferate in situ and merely expand in size to accommodate areas of CEC loss due to injury or senescence. However, we have identified an entire hierarchy of ECFC resident in bovine CECs. Thus, this study provides a new conceptual framework for defining corneal endothelial progenitor cell potential. The identification of persistent corneal HPP-ECFCs in adult subjects might contribute to regenerative medicine in corneal transplantation. While human cord blood derived ECFCs are able to form vessels in vivo, it is unknown whether they are committed to an arterial or venous fate. We have demonstrated that human cord blood derived ECFCs heterogeneously express gene transcripts normally restricted to arterial or venous endothelium. They can be induced to display an arterial gene expression pattern after vascular endothelial growth factor 165 (VEGF165) or Notch ligand Dll1 (Delta1ext-IgG) stimulation in vitro. However, the in vitro Dll1 primed ECFCs fail to display significant skewing toward arterial EC phenotype and function in vivo upon implantation, suggesting that in vitro priming is not sufficient for in vivo specification. Future studies will determine whether ECFCs are amenable to specification in vivo by altering the properties of the implantation microenvironment. There is emerging evidence suggesting that the concentration of circulating ECFCs is closely related to the adverse progression of cardiovascular disorders. In a pig model of acute myocardial ischemia (AMI), we have demonstrated that AMI rapidly mobilizes ECFCs into the circulation, with a significant shift toward HPP-ECFCs. The exact role of the mobilized HPP-ECFCs in homing and participation in repair of the ischemic tissue remains unknown. In summary, these studies contribute to an improved understanding of ECFCs and suggest several possible therapeutic applications of ECFCs.

The history of surgery is nearly as old as the human race. Control of wound infection has always been an essential part of any surgical procedure, and is still an important challenge in hospital operating rooms today. For patients undergoing surgery there is always a risk that they will develop some kind of postoperative complication. It is widely accepted that airborne bacteria reaching a surgical site are mainly staphylococci released from the skin flora of the surgical staff in the operating room and that even a small fraction of those particles can initiate a severe infection at the surgical site.  Wound infections not only impose a tremendous burden on healthcare resources but also pose a major threat to the patient. Hospital-acquired infection ranks amongst the leading causes of death within the surgical patient population. A broad knowledge and understanding of sources and transport mechanisms of infectious particles may provide valuable possibilities to control and minimize postoperative infections. This thesis contributes to finding solutions, through analysis of such mechanisms for a range of ventilation designs together with investigation of other factors that can influence spread of infection in hospitals, particularly in operating rooms. The aim of this work is to apply the techniques of computational fluid dynamics in order to provide better understanding of air distribution strategies that may contribute to infection control in operating room and ward environments of hospitals, so that levels of bacteria-carrying particles in the air can be reduced while thermal comfort and air quality are improved.  A range of airflow ventilation principles including fully mixed, laminar and hybrid strategies were studied. Airflow, particle and tracer gas simulations were performed to examine contaminant removal and air change effectiveness. A number of further influential parameters on the performance of airflow ventilation systems in operating rooms were examined and relevant measures for improvement were identified. It was found that airflow patterns within operating room environments ranged from laminar to transitional to turbulent flows. Regardless of ventilation system used, a combination of all airflow regimes under transient conditions could exist within the operating room area. This showed that applying a general model to map airflow field and contaminant distribution may result in substantial error and should be avoided. It was also shown that the amount of bacteria generated in an operating room could be minimized by reducing the number of personnel present. Infection-prone surgeries should be performed with as few personnel as possible. The initial source strength (amount of colony forming units that a person emits per unit time) of staff members can also be substantially reduced, by using clothing systems with high protective capacity. Results indicated that horizontal laminar airflow could be a good alternative to the frequently used vertical system. The horizontal airflow system is less sensitive to thermal plumes, easy to install and maintain, relatively cost-efficient and does not require modification of existing lighting systems. Above all, horizontal laminar airflow ventilation does not hinder surgeons who need to bend over the surgical site to get a good view of the operative field. The addition of a mobile ultra-clean exponential laminar airflow screen was also investigated as a complement to the main ventilation system in the operating room. It was concluded that this system could reduce the count of airborne particles carrying microorganisms if proper work practices were maintained by the surgical staff. A close collaboration and mutual understanding between ventilation experts and surgical staff would be a key factor in reducing infection rates. In addition, effective and frequent evaluation of bacteria levels for both new and existing ventilation systems would also be important. / Tidigt i mänsklighetens utveckling har kirurgin funnits med i bilden. Hantering av infektioner har genom tiderna varit en oundviklig del av alla kirurgiska ingrepp, och finns kvar ännu idag som en viktig utmaning i operationssalar på sjukhus. För patienter som genomgår kirurgi finns alltid en risk att de efter ingreppet utvecklar någon behandlingsrelaterad komplikation. Allmänt accepterat är att de luftburna bakterier som når operationsområdet huvudsakligen består av stafylokocker frigjorda från hudfloran av operationspersonalen i operationssalen, och att endast en liten del av dessa partiklar behövs för att initiera en allvarlig infektion i det behandlade området. Sårinfektioner innebär inte bara en enorm börda för hälso- och sjukvårdsresurser, utan utgör också en betydande risk för patienten. På sjukhus förvärvad infektion finns bland de främsta dödsorsakerna i kirurgiska patientgrupper.. En bred kunskap och förståelse av spridningsmekanismer och källor till infektionsspridande partiklar kan ge värdefulla möjligheter att kontrollera och minimera postoperativa infektioner. Denna avhandling bidrar till lösningar genom analys av en rad olika ventilationssystem tillsammans med undersökning av andra faktörer som kan påverka infektionsspridningen på sjukhus, främst i operationssalar. Syftet med arbetet är att med hjälp av CFD-teknik (Computational Fluid Dynamics) få bättre förståelse för olika luftspridningsmekanismers betydelse vid ventilation av operationssalar och vårdinrättningar på sjukhus, så att halten av bacteriebärande partiklar i luften kan minskas samtidigt som termisk komfort och luftkvalité förbättras.  Flera luftflödesprinciper för ventilation inklusive omblandade strömning, riktad (laminär) strömning och hybridstrategier har studerats. Simuleringar av luft-, partikel- och spårgasflöden gjordes för alla fallstudier för att undersöka partikelevakuering och luftomsättning i rummet. Flera viktiga parametrar som påverkar detta undersöktes och relevanta förbättringar  föreslås i samarbete med industrin. Av resultaten framgår att mängden genererade bakterier i en operationssal  kan begränsas genom att minska antalet personer i operationsteamet. Infektionsbenägna operationer skall utföras med så lite personal som möjligt. Den initiala källstyrkan (mängden kolonibildande enheter som en person avger per tidsenhet) från operationsteamet kan avsevärt minskas om högskyddande kläder används. Av resultaten framgår också att ett horisontellt (laminärt) luftflöde kan vara ett bra alternativ till det ofta använda vertikala luftflödet. Ett horisontellt luftflöde är mindre känsligt för termisk påverkan från omgivningen, enkelt att installera och underhålla, relativt kostnadseffektivt och kräver vanligen ingen förändring av befintlig belysningsarmatur. Framför allt begränsar inte denna ventilationsprincip kirurgernas rörelsemönster. De kan luta kroppen över operationsområdet utan att hindra luftflödet. En flyttbar flexibel skärm för horisontell spridning av ultraren ventilationsluft i tillägg till ordinarie ventilation undersöktes också. Man fann att denna typ av tilläggsventilation kan minska antalet luftburna partiklar som bär mikroorganismer om operationspersonalen följer en strikt arbetsordning. Bra samarbete och förståelse mellan ventilationsexperter och operationsteamet på sjukhuset är nyckeln till att få ner infektionsfrekvensen. Det är också viktigt med effektiva och frekventa utvarderingar av bakteriehalten i luften, för såväl nya  som befintliga ventilationssystem. / <p>QC 20160129</p>

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Endothelial Colony Forming Cells (ECFCs) are highly proliferative endothelial progenitor cells with clonal proliferative potential and in vivo vessel forming ability. While endothelial cells have been derived from human induced pluripotent stem cells (hiPS) or human embryonic stem cells (hES), they are not highly proliferative and require ectopic expression of a TGFβ inhibitor to restrict plasticity. Neuropilin-1 (NRP-1) has been reported to identify the emergence of endothelial precursor cells from human and mouse ES cells undergoing endothelial differentiation. However, the protocol used in that study was not well defined, used uncharacterized neuronal induction reagents in the culture medium, and failed to fully characterize the endothelial cells derived. We hypothesize that NRP-1 expression is critical for the emergence of stable endothelial cells with ECFC properties from hES cells. We developed a novel serum and feeder free defined endothelial differentiation protocol to induce stable endothelial cells possessing cells with cord blood ECFC-like properties from hES cells. We have shown that Day 12 hES cell-derived endothelial cells express the endothelial markers CD31+ NRP-1+, exhibit high proliferative potential at a single cell level, and display robust in vivo vessel forming ability similar to that of cord blood-derived ECFCs. The efficient production of the ECFCs from hES cells is 6 logs higher with this protocol than any previously published method. These results demonstrate progress towards differentiating ECFC from hES and may provide patients with stable autologous cells capable of repairing injured, dysfunctional, or senescent vasculature if these findings can be repeated with hiPS.

Endothelzell-basierte Therapien vermitteln regenerative Effekte hinsichtlich der Revaskularisierung von ischämischen Geweben. Doch ist die Verfügbarkeit von autologen Endothelzellen aufgrund einer krankheitsbedingt reduzierten Frequenz im peripheren Blut oder einer verminderten Integrität der endogenen Endothelzell-Populationen eingeschränkt. Hingegen ist es möglich, allogene endotheliale Vorläuferzellen aus der Nabelschnur in zelltherapeutisch relevanten Mengen zu isolieren. In der vorliegenden Arbeit wurden zunächst die Eigenschaften allogener humaner Nabelschnur (NS)-abgeleiteter sog. Endothelial Colony-Forming Cells (ECFCs) mit denen von venösen NS-abgeleiteten Endothelzellen verglichen. Aufgrund der nachgewiesenen Immunogenität von allogenen ECFCs wurde eine weiterführende Strategie zur Reduktion dieser immunogenen Eigenschaften durch die Koapplikation mit Mesenchymalen Vorläuferzellen (MSCs) verfolgt. Humane ECFCs wurden mit MSCs desselben Spenders kombiniert und in funktionellen in vitro- und in vivo-Assays untersucht. Dadurch konnte nachgewiesen werden, dass IFNγ-stimulierte ECFC/MSC-Kokulturen eine reduzierte Expression von HLA-Molekülen zeigen. Entsprechend induzierten spezifische CD8+ T-Zellen eine reduzierte Lyse der kokultivierten ECFCs und MSCs. Die Kokultur von ECFCs und MSCs mit allogenen Immunzellen führte zu einer nahezu vollständigen Inhibition der T-Zell-Proliferation. Um die reduzierte Immunogenität von ECFC und MSC in vivo zu verifizieren, wurden die Zellen in immundefiziente Mäuse injiziert, welche nachfolgend mit humanen PBMCs rekonstituiert wurden. So konnte nachgewiesen werden, dass die Koapplikation von ECFCs und MSCs nicht nur die Entstehung von stabilen Gefäßnetzwerken begünstigt, sondern zudem in den Transplantaten zu einer verringerten Immunzell-Infiltration führte. Die Koapplikation von ECFCs mit MSCs könnte daher eine klinische Nutzung dieser allogenen Quelle für die therapeutische Unterstützung der Vaskularisierung ermöglichen. / Endothelial cell-based therapies promote tissue regeneration and vascularization after ischemic damage. The availability of autologous endothelial progenitor cells is restricted in diseased patients, however therapeutically relevant numbers of allogeneic Endothelial Progenitor Cells can be isolated from an umbilical cord (UC). In the present study, the immunogenic properties of these Endothelial Colony Forming Cells (ECFCs) were first compared to human umbilical vein endothelial cells (HUVECs). Both cytokine-treated endothelial cells induced CD4+ and CD8+ T cell proliferation after coculture with allogeneic immune cells. So far, the potential interactions between ECFCs and Mesenchymal Stem/Progenitor Cells (MSCs) concerning their immunological features is poorly understood, but we hypothesize that MSCs might improve the immune compatibility and vessel building characteristics of ECFCs. Therefore, human UC-derived ECFC and MSC cocultures from the same donor were analyzed using various functional in vitro and in vivo assays. Stimulation of these cocultures with IFNγ caused strongly reduced expression levels of HLA-molecules compared to ECFC monocultures. The decreased molecular density on the cocultured ECFCs resulted in reduced cytotoxic CD8+ T cell-mediated lysis. Further, during IFNγ stimulation, the combination of ECFCs with MSCs prevented initiation of allogeneic T cell proliferation. To verify this concept in vivo, ECFCs and MSCs were co-transplanted in a humanized allograft mouse model in immunodeficient mice in order to effectively induce stable microvessels. These experiments demonstrate that when MSCs are co-applied with ECFCs, they not only support the formation of stable blood vessels, but also lead to fewer HLA-DR+ human vascular structures and fewer infiltrating human leukocytes. The data presented indicate that crosstalk between UC-derived ECFCs and MSCs might lower the risk of allogeneic ECFC rejection.