Peripheral vascular diseases such as peripheral artery disease (PAD) and critical limb ischemia (CLI) are growing at an ever-increasing rate in the Western world due to an aging population and the incidence of type II diabetes. A growing economic burden continues because these diseases are common indicators of future heart attack or stroke. Common therapies are generally limited to pharmacologic agents or endovascular therapies which have had mixed results still ending in necrosis or limb loss. Therapeutic angiogenic strategies have become welcome options for patients suffering from PAD due to the restoration of blood flow in the extremities. Capillary sprouting and a return to normoxic tissue states are also demonstrated by the use of angiogenic cytokines in conjunction with bone marrow cell populations. To this point, it has been determined that spatial and temporal controlled release of growth factors from vehicles provides a greater therapeutic and angiogenic effect than growth factors delivered intramuscularly, intravenously, or intraarterialy due to rapid metabolization of the cytokine, and non-targeted release. Furthermore, bone marrow cells have been implicated to enhance angiogenesis in numerous ischemic diseases due to their ability to secrete angiogenic cytokines and their numerous cell fractions present which are implicated to promote mature vessel formation. Use of angiogenic peptides, in conjunction with bone marrow cells, has been hypothesized in EPC mobilization from the periphery and marrow tissues to facilitate neovessel formation. For this purpose, controlled release of angiogenic peptides basic fibroblast growth factor (FGF-2) and granulocyte-colony stimulating factor (G-CSF) was performed using tunable ionic gelatin hydrogels or fibrin scaffolds with ionic albumin microspheres. The proliferation of endothelial cell culture was determined to have an enhanced effect based on altering concentrations of growth factors and method of release: co-delivery versus sequential. Scaffolds with these angiogenic peptides were implanted in young balb/c mice that underwent unilateral hindlimb ischemia by ligation and excision of the femoral artery. Endpoints for hindlimb reperfusion and angiogenesis were determined by Laser Doppler Perfusion Imaging and immunohistochemical staining for capillaries (CD-31) and smooth muscle cells (alpha-SMA). In addition to controlled release of angiogenic peptides, further studies combined the use of a fibrin co-delivery scaffold with FGF-2 and G-CSF with bone marrow stem cell transplantation to enhance vessel formation following CLI. Endpoints also included lipophilic vascular painting to evaluate the extent of angiogenesis and arteriogenesis in an ischemic hindlimb. Tissue engineering strategies utilizing bone marrow cells and angiogenic peptides demonstrate improved hindlimb blood flow compared to BM cells or cytokines alone, as well as enhanced angiogenesis based on immunohistochemical staining and vessel densities.

Background: In childhood acute lymphoblastic leukemia (ALL), the cure rate has now reached 80% in the western world. Even so, 15¬–20% will die from the disease or treatment-related causes, among them children who did not present any known unfavorable features at diagnosis. Treatment of childhood ALL is risk-adapted, meaning that certain factors that are related to the child or the leukemic blasts stratifies to more or less intensive treatment. In this thesis, characteristics of the bone marrow (BM) stroma, reflecting the interaction between the leukemic cells and their microenvironment, were evaluated. The aims were to investigate these factors in relation to other known data in order to further understand the biology of leukemia, and to suggest additional risk factors that would further improve decision making for the treatment of individual children diagnosed with ALL. Methods: We retrospectively investigated microvessel density (MVD), blast-congested vessel fraction (BCVF), and degree of fibrosis – reticulin fiber density (RFD) – in sections from diagnostic BM biopsies from children diagnosed in Umeå, Uppsala, and Stockholm. RFD was also studied in BM sections from treatment day 29. Results: RFD had prognostic impact in patients with high-hyperdiploid (HeH) leukemia. Moreover, rapid reduction of RFD during induction treatment was associated with a favorable prognosis compared to slow reduction, in B-cell precursor (BCP) ALL patients. There was also a correlation between RFD at diagnosis and minimal residual disease (MRD) measured by flow cytometry on treatment day 29 in BCP patients. BCP patients with high RFD and high MVD had an unfavorable outcome compared to all other BCP patients. In addition, MVD and RFD were both associated with immunophenotype, and MVD with cytogenetic aberrations. There was a correlation between MVD and WBC count in BCP high-risk patients. There was also a strong correlation between BCVF and WBC count in all BCP patients, but not between BCVF and MVD or RFD. There was a negative correlation between MVD and in vitro cellular resistance to several drugs in BCP patients. A drug-resistance score combining the drugs most strongly correlated to MVD – cytarabine, doxorubicin, and dexametasone (ADD score) – identified the prognostic potential of ADD score in HeH patients with no unfavorable features. Conclusions: Taken together, these studies indicate that stroma factors in leukemia are related to both phenotypic and genotypic features of acute leukemia. Stroma factors also seem to influence the response to induction treatment, in vitro drug resistance, and outcome in certain subgroups of childhood ALL patients. The results emphasize the importance of BM stroma in leukemia and the need for greater use of BM biopsy at diagnosis.

Mesenchymale Stromazellen (MSCs) werden beim Pferd bereits mit vielversprechenden Ergebnissen zur Behandlung von muskuloskelettalen Erkrankungen, insbesondere von Sehnenerkrankungen, eingesetzt. In bisherigen klinischen Studien lag das Hauptaugenmerk auf der Behandlung von Erkrankungen der Oberflächlichen Beugesehne bei Rennpferden, die jedoch in Deutschland nur einen verhältnismäßig kleinen Anteil des Patientenaufkommens darstellen. Die zu erwartenden Ergebnisse nach MSC-Behandlung von Fesselträgererkrankungen sind dagegen noch nicht bekannt. Darüber hinaus sind die grundlegenden Kenntnisse zur Biologie equiner MSCs noch unzureichend, was Verständnis und Optimierung des bestehenden Therapiekonzeptes erschwert. Häufig wird die Verwendung alternativer Gewebequellen für MSCs diskutiert, wobei jedoch nur wenige vergleichende Daten zu den jeweiligen zellulären Eigenschaften vorliegen. Ziel dieser Arbeit war es daher, zum einen mehr Kenntnisse über die zu erwartenden klinischen Ergebnisse nach MSC-Behandlung von Sehnenerkrankungen zu erlangen, einschließlich Erkrankungen des Fesselträgers, zum anderen den Wissensstand hinsichtlich der in-vitro-Charakterisierung equiner MSCs zu erweitern, wobei ein Vergleich klinisch relevanter Charakteristika zwischen MSCs aus verschiedenen Gewebequellen angestrebt wurde. In die klinische Studie wurden 98 Pferde, die aufgrund von Sehnen- und Banderkrankungen mit MSCs behandelt worden waren, einbezogen. Von 58 dieser Tiere konnten Langzeitergebnisse nach einem Beobachtungszeitraum von mindestens einem Jahr erhoben werden. Diese wurden hinsichtlich des Behandlungserfolges sowie möglicher Einflussfaktoren ausgewertet, wobei die Behandlung als erfolgreich bewertet wurde, wenn die Patienten nach dem Beobachtungszeitraum voll trainiert oder im Sport eingesetzt werden konnten und dabei kein Rezidiv aufgetreten war. Die Behandlung mit MSCs wurde bei 84,5 % der Pferde als erfolgreich eingestuft, wobei Erkrankungen der Oberflächlichen Beugesehne mit 84,2 % und Erkrankungen des Fesselträgers mit 83,3 % gleichermaßen gute Ergebnisse zeigten. Tendenziell beeinflussten Nutzungsdisziplin, Erkrankungsstadium und Patientenalter das klinische Ergebnis ebenso wie bei konventioneller Behandlung. Insgesamt war nach MSC-Behandlung das Auftreten von Rezidiven deutlich seltener zu beobachten als in der Literatur für die konventionelle Behandlung beschrieben wird. Für die in-vitro-Studie zur vergleichenden Charakterisierung equiner MSCs aus verschiedenen Quellen wurden Knochenmark, Fett- und Sehnengewebe sowie Nabelschnurblut und -gewebe gewonnen. Aus diesen Proben wurden jeweils die plastikadhärenten MSCs isoliert und hinsichtlich Zellausbeute, Proliferations- und Migrationseigenschaften, tripotentem Differenzierungspotential sowie der Expression der Sehnenmarker Kollagen 1A2 und Skleraxis vergleichend untersucht. Die Ausbeute an MSCs war bei allen soliden Geweben (Fett-, Sehnen-, und Nabelschnurgewebe) hochsignifikant höher (p < 0,001). Ebenso proliferierten MSCs aus Fett- und Sehnengewebe signifi-kant schneller als MSCs aus Knochenmark oder Nabelschnurblut (p < 0,01). Von letzteren wurden darüber hinaus etwa drei viertel aller Zellkulturen vor der achten Passage seneszent. Das höchste Migrationspotential zeigten wiederum MSCs aus Sehnen- und Fettgewebe, wobei hier MSCs aus Nabelschnurgewebe das ungünstigste Ergebnis erzielten (p < 0,01). Die adipogene Differenzierung gelang bei MSCs aus allen Quellen vergleichbar gut. Bei der osteogenen Differenzierung erreichten MSCs aus Knochenmark das beste Ergebnis, während MSCs aus Nabelschnurblut und –gewebe nur schwach osteogen differenzierten (Tag 21: p < 0,01; Tag 35: p < 0,05). Im Gegensatz dazu erreichten MSCs aus Nabelschnurblut bei der chondrogenen Differenzierung die meisten Scorepunkte, MSCs aus Knochenmark dagegen die wenigsten (p < 0,05). Kollagen 1A2 wurde von MSCs aus Fettgewebe am höchsten exprimiert, Skleraxis von MSCs aus Nabelschnurblut. MSCs aus Sehnengewebe exprimierten beide Sehnenmarker auf fast ebenso hohem Level. MSCs aus Knochenmark dagegen zeigten hier jeweils die niedrigste Expression (p < 0,05 für Kollagen 1A2). Basierend auf den Ergebnissen der klinischen Studie ist die MSC-Therapie nach wie vor als vielversprechende Behandlungsoption für Sehnenerkrankungen anzusehen und ist auch für die Behandlung von Fesselträgererkrankungen geeignet. Zukünftige, kontrollierte klinische Studien müssen jedoch die Wirksamkeit der MSC-Therapie noch weitergehend bestätigen. Die in-vitro-Studie zeigte signifikante Unterschiede zwischen equinen MSCs aus verschiedenen Quellen auf, die bei der Auswahl einer Gewebequelle für die MSC-Isolierung für klinische Anwendungen berücksichtigt werden sollten. MSCs aus Fettgewebe erscheinen aufgrund ihrer sehr guten Proliferations- und zuverlässigen Differenzierungseigenschaften als eine gute Alternative zu MSCs aus Knochenmark für autologe Therapien. MSCs aus Sehnengewebe sind den hier vorliegenden Ergebnissen zufolge besonders gut für die Behandlung von Sehnenerkrankungen geeignet; vor einer routinemäßigen Anwendung dieser MSCs sollten jedoch ihre Eigenschaften weiterführend untersucht werden. / In horses, mesenchymal stromal cells (MSCs) are used for the treatment of musculoskeletal diseases, especially tendon injuries, with promising results. Previous clinical studies mainly focused on the treatment of superficial digital flexor tendon injuries in racehorses, which, however, represent only a relatively small percentage of the overall equine case load in Germany. Average outcome to be expected following MSC treatment of suspensory ligament injuries was not yet determined. Moreover, basic knowledge on equine MSC biology is still deficient, hampering the understanding and thus the optimisation of the existing treatment regime. The use of alternative MSC sources is frequently discussed, yet to date, only few data comparing the cellular properties of equine MSCs from different sources have been published. The aim of this study was, on the one hand, to gain more knowledge concerning the expected outcome after MSC treatment of tendon injuries, including injuries to the suspensory ligament. On the other hand, it was aimed at expanding the knowledge on equine MSC characterisation in vitro, thereby focusing on the comparison of clinically relevant properties of MSCs derived from different sources. In the clinical study, 98 horses were included, all of which had received MSC treatment for tendon or ligament injuries. In 58 of these horses, long term results after a follow-up period of at least one year could be collected. These data were analysed with respect to treatment outcome and potential influencing factors. Treatment was considered successful when horses were back to full training or competition after the follow-up period, without having suffered a re-injury. The overall success rate was 84.5 %. Success rates in horses suffering from superficial digital flexor tendon injuries and in horses suffering from suspensory ligament injuries were comparably good (84.2 % and 83.3 %, respectively). Similar to conventional therapies, the sports discipline in which the horses performed, age and disease stage tended to influence the outcome. Overall, re-injury rates after MSC treatment were considerably lower than those described in the literature following conventional treatment. For the comparative characterisation of MSCs from different sources in vitro, samples of bone marrow, adipose and tendon tissue, as well as umbilical cord blood and –tissue were collected. Plastic-adherent MSCs were isolated out of these samples and comparatively characterised focusing on cell yields, proliferation and migration properties, trilineage differentiation potential and the expression of the tendon markers collagen 1A2 and scleraxis. MSC yields were significantly higher in all solid tissues (adipose, tendon and umbilical cord tissue) (p < 0.001). Further, MSCs from adipose and tendon tissue proliferated significantly faster than MSCs from bone marrow or umbilical cord blood (p < 0.01). Moreover, approximately three quarters of the samples derived from the latter sources underwent senescence before reaching passage eight. The highest migration potential was found in MSCs derived from tendon and adipose tissue again, while MSCs from umbilical cord tissue showed the least (p < 0.01). The adipogenic differentiation potential was comparably good in MSCs from all different sources. The osteogenic differentiation was most distinct in MSCs from bone marrow, while MSCs from umbilical cord blood and tissue showed only weak evidence of differentiation (day 21: p < 0.01; day 35: p < 0.05). In contrast, following chondrogenic differentiation, MSCs from umbilical cord blood scored highest and MSCs from bone marrow scored lowest (p < 0.05). Collagen 1A2 was most highly expressed in MSCs from adipose tissue, highest scleraxis expression levels were found in MSCs from umbilical cord blood. MSCs from tendon tissue, however, expressed both markers at almost evenly high levels. Contrastingly, lowest expression levels of both markers were found in MSCs derived from bone marrow (p < 0.05 for collagen 1A2). Based on the results of the clinical study, MSC therapy can still be considered a very promising treatment option for tendon diseases and is also a suitable treatment for suspensory ligament injuries. In the future, controlled clinical studies will have to further confirm the efficacy of this treatment regime. The in-vitro-study showed significant differences between equine MSCs derived from different sources, which should be considered when choosing a MSC source for clinical applications. For autologous therapies, MSCs derived from adipose tissue appear to be a good alternative to MSCs derived from bone marrow, due to their remarkable proliferation and reliable differentiation capacities. Furthermore, according to this study, MSCs derived from tendon tissue are especially suitable for treating tendon injuries. Prior to routine clinical applicability of these MSCs, however, their properties should be further investigated.

Cartilage regeneration is one of the most widely studied areas in tissue-engineering. Despite significant progress, most efforts to date have only focused on generating homogenous tissues whose bulk properties are similar to articular cartilage. However, anatomically and functionally, articular cartilage consists of four spatially distinct regions: the superficial, transitional, deep, and calcified zones. Each zone is characterized by unique extra-cellular matrix (ECM) compositions, mechanical properties, and cellular organization. The ECM is primarily composed of type II collagen and glycosaminoglycans (GAGs), whose relative concentrations vary between zones and therefore lead to distinctive mechanical properties. One of the major unsolved challenges in engineering cartilage has been the inability to regenerate tissue that mimics the zonal architecture of articular cartilage. Recent studies have attempted to imitate this spatial organization using zone-specific chondrocytes isolated from donor animal cartilage. Directed differentiation of a single stem population into zonally organized native-like articular cartilage has not yet been reported. This dissertation reports that hydrogels, incorporating both synthetic and natural polymers as well as cell-induced degradability, are suitable for generating zone-specific chondrogenic phenotypes from a single MSC population. Specifically, cues provided from the unique combinations of chondroitin sulfate (CS), hyaluronic acid (HA), and MMP-sensitive peptide (MMP-pep) within a PEG-based hydrogel, direct the chondrogenic differentiation of MSCs. The findings of this dissertation demonstrate the capability of creating native-like and mechanically relevant articular cartilage consisting of zone specific layers. This ability provides a new direction in cartilage tissue engineering and could be invaluable for cartilage repair if incorporated with current minimally invasive surgical techniques. / text

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Main aim: To supply recommendations to implement a Hazard Analysis of Critical Control Points (HACCP) system in a hospital food service unit serving low bacterial diets in order to prevent or decrease the infection rates in Hematopoietic Stem Cell Transplant (HSCT) patients. Objectives: Firstly, to investigate the current food safety and hygiene status in a hospital food service unit, serving low bacterial diets, by means of a questionnaire and bacterial swabs taken from the food service unit. Secondly, to utilize the gathered information in a structured action plan to implement HACCP standards successfully in the appointed food service unit. The implementation of HACCP will not be done by the author. Design: The primary research was done in a food service unit of a 350 bed private hospital. One unsuspected audit with a pre-designed audit form was done. The audit consisted out of ten categories. A percentage was allocated to each category. Four swabs, as well as four food samples, were taken during the audit. The swabs and samples were tested to assess the microbiological safety of the foods prepared in the appointed hospital food service unit. The results of the audit, swabs and food samples were used to evaluate the current Food and Safety System of the hospital food service unit according to internationally approved HACCP standards. Setting: The study was conducted in the metropolitan area of Gauteng, South Africa. Results: None of the ten areas audited was of an acceptable standard and an average of 37% was scored. Category 5, the service and distribution area, scored the highest (69%) and category 10, the quality procedures and records division, scored the lowest (6%). According to United States Food and Drug Administration Baseline Report five forbidden policies could lead to increased risk of food borne illnesses. All five forbidden policies were detected in the food service unit during the audit. The microbiological tests showed relatively high microbial counts. Conclusion: The results of the study confirmed that instead of focusing mainly on the selection of food items allowed, and the cooking methods used in HSCT diets, the type of food service, together with the food and safety protocol that the food service follows, could play an important role in providing food that is safe for HSCT patient use. / Thesis (M.Sc. (Dietetics))--North-West University, Potchefstroom Campus, 2007.

Lic. -avh. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (Ph.D. in Toxicology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 224-253). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;