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Identification of Epo-independent red cell progenitors : the E-cad+ progenitorsLemke, Britt January 2004 (has links)
Erythrozyten zählen zu den am häufigsten vorkommenden terminal differenzierten Zelltypen des menschlichen Körpers. Durchschnittlich werden täglich ca. 2 x 1011 von ihnen im Körper eines erwachsenen Menschen produziert. Die reifen Erythrozyten entstehen aus multipotenten hämatopoetischen Stammzellen, die über Stadien von erythroiden Vorläuferzellen, erst den sogenannten burst forming units-erythroid (BFU-E) und später den colony forming units-erythroid (CFU-E), zu kernlosen hämoglobinisierten Zellen differenzieren. <br />
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Für die Untersuchung der molekularen Mechanismen der humanen Erythropoese ist die effektive in vitro Amplifizierung einer weitgehend homogenen Population der Vorläuferzellen der einzelnen Entwicklungsstadien notwendig. Den Wachstumsfaktoren stem cell factor (SCF) und Erythropoietin (Epo) fällt dabei eine entscheidende Rolle zu. Unter ihrem synergistischen Einfluß lassen sich Epo-abhängige Zellpopulationen, die sich aus BFU-E und CFU-E Typ Zellen zusammensetzen, ausreichend amplifizieren (Panzenböck et al., 1998). Freyssinier et al., 1999 beschrieb erstmals die Isolierung einer Epo-unabhängigen Population von Vorläuferzellen (CD36+ Vorläuferzellen), die ebenfalls erythroide Eigenschaften aufweisen.<br />
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Ziel dieser Arbeit war die Isolierung und Charakterisierung von Epo-unabhängigen Vorläuferzellen, die eine frühe erythroide und möglichst homogene Vorläuferzellpopulation darstellen und möglicherweise ein höheres Proliferationspotential aufweisen.<br />
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Für die Identifizierung der Epo-unabhängigen Vorläuferzellen, wurden CD34+ Zellen aus Nabelschnurblut aufgereinigt und unter serumfreien Kulturbedingungen und unter Zusatz der Wachstumsfaktoren SCF, Interleukin 3 (IL-3) und eines Fusionsproteins aus IL-6 und löslichem IL-6 Rezeptor (hyper-IL-6) über einen Zeitraum von 8 Tagen kultiviert. Anschließend wurde eine Population von E-cadherin positiven (E-cad+) Zellen über immunomagnetische Selektion isoliert. Diese neu gewonnenen Epo-unabhängigen E-cad+ Vorläuferzellen wurden hinsichtlich ihres proliferativen Potentials und ihrer Differenzierungseigenschaften mit SCF/Epo-Vorläuferzellen und CD36+ Vorläuferzellen verglichen. Von allen drei Zelltypen wurden des weiteren detailierte molekulargenetische Analysen mittels DNA microarray Technologie durchgeführt und die resultierenden Genexpressionsmuster miteinander verglichen.<br />
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Die Ergebnisse zeigen, dass die E-cad+ Vorläuferzellen eine frühe, weitgehend homogene Epo-unabhängige Population vom BFU-E Typ darstellen und durch entsprechende Änderungen der Kulturbedingungen zu einer in vitro Differenzierung angeregt werden können. Die E-cad+ Vorläuferzellen sind hinsichtlich ihres proliferativen Potentials, ihrer Reaktion auf verschiedene Wachstumsfaktoren, der Expression spezifischer Oberflächenmoleküle und ihrer Genexpressionsmuster mit SCF/Epo-Vorläuferzellen und CD36+ Vorläuferzellen vergleichbar.<br />
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Aufgrund der Identifizierung unterschiedlich exprimierter Gene zwischen den Epo-unabhängigen E-cad+ und den Epo-abhängigen SCF/Epo Vorläuferzellen konnten Kanditatengene wie Galectin-3, Cyclin D1, der Anti-Müllerian Hormonrezeptor, Prostata-Differenzierungsfaktor und insulin-like growth factor binding protein 4 identifiziert werden, die als potentielle Regulatoren der Erythropoese in Betracht kommen könnten. Es konnte weiterhin gezeigt werden, dass CD36+ Vorläuferzellen, die aus der selben Zellpopulation wie die E-cad+ Vorläuferzellen immunomagnetisch selektioniert wurden, eine heterogene Population darstellen, die sowohl E-cadherin positive als auch negative Zellen enthält. Die Analyse der Genexpressionsmuster zeigte, dass in den CD36+ Vorläuferzellen zwar auch die Expression erythroid-spezifischen Gene nachgewiesen werden kann, hier aber im Gegensatz zu den E-cad+ Vorläuferzellen auch für Megakaryozyten spezifische Gene stark exprimiert sind.<br />
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Die Ergebnisse dieser Arbeit tragen zu einem neuen Modell der in vivo Abläufe der Entwicklung roter Blutzellen bei und werden der weiteren Untersuchung der molekularen Mechanismen der Erythropoese dienen. / Red cell development in adult humans results in the mean daily production of 2x1011 erythrocytes. Mature hemoglobinized and enucleated erythrocytes develop from multipotent hematopoietic stem/progenitor cells through more committed progenitor cell types such as BFU-E and CFU-E. The studies on the molecular mechanisms of erythropoiesis in the human system require a sufficient number of purified erythroid progenitors of the different stages of erythropoiesis. Primary human erythroid progenitors are difficult to obtain as a homogenous population in sufficiently high cell numbers. Various culture conditions for the in vitro cell culture of primary human erythroid progenitors have been previously described. Mainly, the culture resulted in the generation of rather mature stages of Epo-dependent erythroid progenitors. In this study our efforts were directed towards the isolation and characterization of more early red cell progenitors that are Epo-independent.<br />
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To identify such progenitors, CD34+ cells were purified from cord blood and cultured under serum free conditions in the presence of the growth factors SCF, IL-3 and hyper-IL-6, referred to as SI2 culture conditions. By immunomagnetic bead selection of E-cadherin (E-cad) positive cells, E-cad+ progenitors were isolated. These Epo-independent E-cad+ progenitors have been amplified under SI2 conditions to large cell numbers. The E-cad+ progenitors were characterized for surface antigen expression by flow cytometry, response to growth factors in proliferation assay and for their differentiation potential into mature red cells. Additionally, the properties of E-cad+ progenitors were compared to those of two other erythroid progenitors: Epo-dependent progenitors described by Panzenböck et al. (referred to as SCF/Epo progenitor), and CD36+ progenitors described by Freyssinier et al. (Panzenböck et al., 1998; Freyssinier et al., 1999). Finally, the gene expression profile of E-cad+ progenitors was compared to the profiles of SCF/Epo progenitors and CD36+ progenitors using the DNA microarray technique.<br />
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Based on our studies we propose that Epo-independent E-cad+ progenitors are early stage, BFU-E like progenitors. They respond to Epo, despite the fact that they were generated in the absence of Epo, and can completely undergo erythroid differentiation. Furthermore, we demonstrate that the growth properties, the growth factor response and the surface marker expression of E-cad+ progenitors are similar to those of the SCF/Epo progenitors and the CD36+ progenitors. By the comparison of gene profiles, we were also able to demonstrate that the Epo-dependent and Epo-independent red cell progenitors are very similar. Analyzing the molecular differences between E-cad+ and SCF/Epo progenitors revealed several candidate genes such as galectin-3, cyclin D1, AMHR, PDF and IGFBP4, which are potential regulators involved in red cell development. We also demonstrate that the CD36+ progenitors, isolated by immunomagentic bead selection, are a heterogeneous progenitor population containing an E-cad+ and an E-cad- subpopulation. Based on their gene expression profile, CD36+ progenitors seem to exhibit both erythroid and megakaryocytic features.<br />
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These studies led to a more updated model of erythroid cell development that should pave the way for further studies on molecular mechanisms of erythropoiesis.
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Development Of Wiskott-Aldrich Syndrome Knock Out Protocol For Drug Substance Assay DevelopmentHanna, Julia C 01 June 2023 (has links) (PDF)
Wiskott-Aldrich Syndrome (WAS) is a rare X-linked primary immunodeficiency affecting approximately 1 in 100,000 live XY births in North America and is caused by a mutation to the WAS gene which is expressed across hematopoietic lineages. The WAS protein (WASp) plays a role in regulating actin polymerization. On a cellular level, there are a variety of effects of a lack of WASp or expression of a dysfunctional WASp protein for patients including issues with migration, adhesion, chemotactic response, phagocytosis, activation, and proliferation across different cell types in addition to reduced platelet size and output. This can lead to several systematic effects for the patients however because mutations to the WAS gene are not limited to one location or type there is a great amount of variability between patient symptoms making it challenging to diagnose. Major symptoms include frequent and recurrent infections, uncontrolled bleeding episodes, issues associated with autoimmunity, and malignancy, the most common form being lymphoma. Without treatment, the life expectancy of an individual diagnosed with WAS is 14 years of age, and the only curative treatment strategy available is hematopoietic stem and progenitor cell transfer (HSPCT). If not performed with an HLA-identical donor, which is available to less than 10% of patients, and within the first two years of life, the risk of graft versus host disease (GvHD) increases drastically for the patient. A gene therapy using autologous and genetically corrected CD34+ cells would be advantageous to the patients due to a reduction in preparative conditioning, immunosuppressive aftercare, and the risk of GvHD. CSL Behring is currently in the development of a lentiviral gene therapy to fulfill this gap in care, however, to develop the assays required to assess and characterize the drug substance usually an uncorrected patient sample is compared with a gene-edited sample. The limitation here is that due to the risk of infection and bleeding patient sample is very limited and therefore the development of a mock patient sample is necessary for early development. The goal of the project is to develop a WAS-KO protocol utilizing CRISPR/Cas9 and its characterization.
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Toxicological and antifertility investigations of oleanolic acid in male vervet monkeys (chlorocebus aethiops)Mdhluli, Mongezi January 2003 (has links)
Philosophiae Doctor - PhD / Introduction: Plant extracts and herbal preparations are often marketed as natural and safe alternatives to conventional medicines for the prevention and treatment of a variety of ailments, without proof of efficacy and safety. Cardiovascular, hematopoetic, hepatic and renal impairment resulting from the use of conventional drugs is widely acknowledged. However, there is less awareness of the potential toxicity of herbal preparations and other botanicals, many of which are widely perceived by the public as being effective and harmless, and are commonly used for self medication without supervision. In addition, potential interactions between herbal medicines and conventional drugs may compromise with patient management. In the safety evaluation of most substances, non human primates are preferred to rodent species for preclinical animal safety studies, because of their biological similarity to humans. They are regarded to be the best metabolic models for humans in a broad range of investigations. Additionally, a disadvantage of using small animal species in toxicological testing is that they require higher doses of drugs and more frequent administrations than in larger species. In light of these considerations, vervet monkeys are used here to investigate toxicity of a plant-derived triterpene, oleanolic acid. The focus is to determine effects of different concentrations of this triterpene on the cardiovascular, hematopoetic, hepatic and renal systems. Materials and methods: 12 male vervet monkeys used in this study were equally divided into four groups, i.e. three treatment groups (4, 10 and 25 mg/kg bodyweight), and one control group. Each individual in a treatment group received a specified concentration of oleanolic acid in food for 16 weeks. Monkeys in the control group received the vehicle (food) alone. Bodyweight, body temperature, respiratory rate, heart rate, systolic pressure, diastolic pressure, and mean arterial pressure were recorded from ketamine-anaethetized monkeys at baseline and every second week until week 16. In addition, blood samples were collected at baseline and every fourth week for clinical biochemistry indicators (serum electrolytes, enzymes, proteins, lipids, nitrogenous compounds, bilirubins and glucose) and hematological tests (red cell count and its indices, hemoglobin, haematocrit, white blood cell and differential count and platelet count). Results: No animal showed deviation from their normal behavioral patterns, food and water intake, was in poor health or died during and after completion of the study. The average bodyweights were not statistically significantly different between controls and the treated groups. The biphasic changes in the average body temperature of treated monkeys were similar to those seen in the control group during the first eight weeks of the study. No statistically significant differences were found in body temperature determinations between controls and the treated groups. Fluctuations observed in the respiratory rates of the treated monkeys were not statistically significantly different from that of the control group. Although not statistically significantly different from the controls, the systolic, diastolic and mean arterial pressures in the group treated with 25 mg/kg oleanolic acid were lower at week 16 compared to baseline, while those of the groups treated with 4 and 10 mg/kg oleanolic acid were relatively unchanged. Except for a reduction in systolic pressure of the control group, other blood pressure parameters were stable. Heart rates in the treated groups were not statistically significantly different from those in the controls. In all groups, except the control, high density lipoprotein concentrations were higher at week 16 compared to baseline. Fluctuations in low-density lipoprotein and total cholesterol concentrations were similar between controls and the treated groups. The triglycerides were lower at week 16 compared to baseline for all four groups. Upward trends from baseline to the end of the study were observed in creatine kinase concentrations of the controls and the groups that received 4 and 25 mg/kg. Concentrations of this enzyme were unchanged in the group that received 10 mg/kg oleanolic acid between baseline and the end of the study. No statistically significant differences were found with cholesterol, triglyceride
and creatine kinase concentrations between treated groups and the controls. Serum concentrations of aspartate aminotransferase were unchanged in the controls and the groups treated with 4 and 10 mg/kg oleanolic acid, but changes in this parameter over time were
statistically significantly different (P = 0.0452) from the controls in the group that received 25 mg/kg oleanolic acid. Despite wide fluctuations in the alanine aminotransferase concentrations in the groups that received 4 and 25 mg/kg oleanolic acid, no statistically significant differences were found with any of the treated groups compared to the controls. No statistically significantly different changes were seen in alkaline phosphatase activities between controls and the treated groups. Reductions in gamma-glutamyl transferase activities in the groups that received 4 and 25 mg/kg oleanolic acid were not statistically significantly different from concentrations of this enzyme in the controls. In addition, no statistically significant differences were evident between controls and the group that received 10 mg/kg oleanolic acid. There were no statistically significantly different changes in the total and conjugated bilirubin and glucose concentrations between controls and the treated groups. Fluctuations over time in the serum albumin and globulin concentrations were similar between treated groups and the controls, whereas total protein concentrations were relatively constant. Consequently, no statistically significant differences were
found between controls and the treated groups. Wide fluctuations were observed in the creatinine concentrations of the groups that received 4 mg/kg oleanolic acid, while no such changes were encountered in the controls and the group that received 10 and 25 mg/kg oleanolic acid. Serum urea concentrations increased in all groups over time, except for the group that received 10 mg/kg oleanolic acid. Both urea and creatinine concentrations in the treated groups were not statistically significantly different from concentrations in the controls.
Serum concentrations of sodium, chloride, potassium, calcium and magnesium and phosphate in the treated groups were not statistically significantly different from these electrolyte concentrations in the controls. Decline in red cell and hemoglobin concentrations of the controls and the group that received 25 mg/kg oleanolic acid were not statistically significantly different between these groups. In addition,
no statistical significant differences were found in red cell and hemoglobin concentrations between controls and the groups that received 4 and 10 mg/kg oleanolic acid. Controls and the treated groups showed upward trends in haematocrit concentrations. Mean corpuscular volumes were statistically significantly increased; P = 0.0027 (4 mg/kg), P = 0.0010 (10 mg/kg), and P = 0.0022 (25 mg/kg), while mean corpuscular hemoglobin concentrations were statistically significantly reduced; P = 0.0017 (4 mg/kg), P = 0.0004 (10 mg/kg), P = 0.0002 (25 mg/kg) in the treated groups as compared to the controls. No statistically significant differences were evident in the concentrations of mean
corpuscular hemoglobin between controls and the treated groups. White blood cell counts of the treated groups were not statistically significantly different from those of the controls throughout the study period. No statistically significant differences were found in the differential white cells and platelet counts between treated groups and the controls. Discussions: The results of this study showed that administration of oleanolic acid had no effects on the general wellbeing, bodyweights, body temperature, respiratory and heart rates, and blood pressure of vervet monkeys. A statistically significant increase in the aspartate aminotransferase activity of the group treated with 25 mg/kg oleanolic acid, together with the increase in the alanine aminotransferase levels during the same time period, might indicate oleanolic acid-induced hypersensitivity, and accordingly hepatocellular alteration. However, since serum concentrations of these enzymes returned to baseline levels, as well as the absence of variations over time in other parameters of the hepatic function, particularly alkaline phosphatase activity, it is likely that there was no underlying subacute liver disease. Serum renal function parameters also appeared to be within normal physiological limits. No pronounced changes were observed in the hematological parameters of monkeys that received oleanolic acid.
Conclusion: This study's results, suggest that oleanolic acid does not produce cumulative liver enzyme alterations, and has no detrimental effects on the renal, hematopoetic and cardiovascular systems of vervet monkeys.
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Genetic Associations with Survival Outcomes after Matched Unrelated Donor Allogeneic Hematopoietic Stem Cell TransplantationKaraesmen, Ezgi 21 September 2020 (has links)
No description available.
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O transplante de células tronco hematopoéticas alogênico e autogênico na leucemia mielóide aguda em primeira remissão completa: análise de 62 pacientes / The allogeneic and autologous hematopoietic stem cell transplantation in acute myeloid leukemia in first complete remission: analyses of 62 patientsBueno, Nadjanara Dorna 03 April 2008 (has links)
O transplante de células tronco hematopoéticas alogênico e autogênico na leucemia mielóide aguda em primeira remissão completa: analise de 62 pacientes. Os pacientes foram submetidos a transplante de células tronco hematopoéticas alogênico e autogênico. Ao final do estudo estavam vivos no alogênico 43,3% e no autogênico 62,5%. Consolidação intensiva teve melhor sobrevida no alogênico. Os pacientes com DECH aguda grau II tiveram melhor sobrevida. Dois pacientes com DECH crônica extensa morreram. Óbito por infecção ocorreu com maior freqüência no alogênico seguido de recidiva. No autogênico a recidiva foi a principal causa de óbito. Morte por toxicidade ocorreu em 47% dos pacientes que foram a óbito no alogênico e em 8,3% no autogênico. Na analise múltipla de Cox a consolidação intensiva e DECH crônica, tiveram significância. / The allogeneic and autologous hematopoietic stem cell transplantation in acute myeloid leukemia in first complete remission: analyses of 62 patients. The patients were submitted to allogeneic and autologous hematopoietic stem cell transplantation. The end of the study were kept alive in allogeneic 43,3% and in autologous 65,2%. Patient in allogeneic who were consolidated had better survival. Patients with acute GVHD grade II had better survival. Two patients with chronic GVHD in intense, died. Infection was the most frequent dead cause in allogeneic following relapse. In autologous the relapse was the principal cause of death. Toxicity occurred in 47% of patients who died in allogeneic and 8,3% in autologous. In cox multiple analyses intensive consolidation and chronic GVHD had significance.
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O transplante de células tronco hematopoéticas alogênico e autogênico na leucemia mielóide aguda em primeira remissão completa: análise de 62 pacientes / The allogeneic and autologous hematopoietic stem cell transplantation in acute myeloid leukemia in first complete remission: analyses of 62 patientsNadjanara Dorna Bueno 03 April 2008 (has links)
O transplante de células tronco hematopoéticas alogênico e autogênico na leucemia mielóide aguda em primeira remissão completa: analise de 62 pacientes. Os pacientes foram submetidos a transplante de células tronco hematopoéticas alogênico e autogênico. Ao final do estudo estavam vivos no alogênico 43,3% e no autogênico 62,5%. Consolidação intensiva teve melhor sobrevida no alogênico. Os pacientes com DECH aguda grau II tiveram melhor sobrevida. Dois pacientes com DECH crônica extensa morreram. Óbito por infecção ocorreu com maior freqüência no alogênico seguido de recidiva. No autogênico a recidiva foi a principal causa de óbito. Morte por toxicidade ocorreu em 47% dos pacientes que foram a óbito no alogênico e em 8,3% no autogênico. Na analise múltipla de Cox a consolidação intensiva e DECH crônica, tiveram significância. / The allogeneic and autologous hematopoietic stem cell transplantation in acute myeloid leukemia in first complete remission: analyses of 62 patients. The patients were submitted to allogeneic and autologous hematopoietic stem cell transplantation. The end of the study were kept alive in allogeneic 43,3% and in autologous 65,2%. Patient in allogeneic who were consolidated had better survival. Patients with acute GVHD grade II had better survival. Two patients with chronic GVHD in intense, died. Infection was the most frequent dead cause in allogeneic following relapse. In autologous the relapse was the principal cause of death. Toxicity occurred in 47% of patients who died in allogeneic and 8,3% in autologous. In cox multiple analyses intensive consolidation and chronic GVHD had significance.
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