Introdução: Células tronco mesenquimais (CTM) são uma população residente nos tecidos adultos de origem mesodérmica, com funções regenerativas de manutenção da integridade tecidual, com destaque no desempenho imunomodulador. Esse aspecto levou as CTM a tornarem-se ferramentas terapêuticas valiosas da pesquisa à assistência ao paciente em doenças autoimunes e de cunho inflamatório. Além disso, CTM podem ser isoladas de materiais tidos como descarte de procedimentos, como dentes decíduos, filtros de transplante de medula óssea e gordura. Nesse panorama, torna-se necessário estabelecer o efeito que a origem tecidual tem na eficiência imunoreguladora e na possível aplicabilidade clínica destas células. Objetivo: Comparar o potencial imunomodulador de células mesenquimais isoladas a partir de filtros descartados após a infusão de medula óssea, de lipoaspirado e de polpa de dentes decíduos. Métodos: Foi realizada a comparação da capacidade proliferativa de CTMs, cultivadas na presença de lisado plaquetário, das diversas fontes através do cálculo de population doubling das CTM em co-cultura com linfócitos T isolados em coluna magnética e com células mononucleares de sangue periférico, estimuladas com fitohemaglutinina; e determinado por citometria de fluxo o efeito das CTM das diversas fontes sobre as subpopulações linfocitárias. Resultados: CTM das três fontes foram capazes de inibir a proliferação de linfócitos e CTM de tecido adiposo foram mais eficientes em induzir o fenótipo de células T reguladoras e na diminuição de células T citotóxicas. Conclusão: comparadas à CTM isoladas de medula óssea e de polpa dentária, as CTM originadas de tecido adiposo exibem efeito imunomodulador mais acentuado. / Background: Mesenchymla stromal cells (MSC) reside in most adult tissue of mesenchymal origen, with a broad functions envolving cell repopulation and maintenence of tissue homeostasis, trough immunemmodulatory action. MSC are valuable terapêutic instruments applied from research to autoimune and inflamatory diseases. MSC can be isolated from diverse discarted biological matherials, like lipoaspirate, exfoliated deciduous teeeth and boné marrow ransplant filters. There so it´s necessary to stablish how source can impact MSC efficiency and possible clinical aplications. Objective: Compare immunomodulatory potential of adipose MSC and dental pulp MSC to boné marrow MSC. Methods: MSC from three selected sources were cocultured with phytohemaglutinin stimulated and magnetically isolated T cells and peripheral blood mononuclear cells; immunephenotype of cocultivated lymphocytes were also conducted. Results: MSC from all analyzed sources were capable to inhibit lymphocyte proliferation. Adipose MSC were capable to induce Treg phenotype and decrease T CD8+ limphocytes. Conclusion: Cell culture and therapy with MSC present many paradigms and we address to some of those to elucidate the possible most efficient source.

Introdução: Células tronco mesenquimais (CTM) são uma população residente nos tecidos adultos de origem mesodérmica, com funções regenerativas de manutenção da integridade tecidual, com destaque no desempenho imunomodulador. Esse aspecto levou as CTM a tornarem-se ferramentas terapêuticas valiosas da pesquisa à assistência ao paciente em doenças autoimunes e de cunho inflamatório. Além disso, CTM podem ser isoladas de materiais tidos como descarte de procedimentos, como dentes decíduos, filtros de transplante de medula óssea e gordura. Nesse panorama, torna-se necessário estabelecer o efeito que a origem tecidual tem na eficiência imunoreguladora e na possível aplicabilidade clínica destas células. Objetivo: Comparar o potencial imunomodulador de células mesenquimais isoladas a partir de filtros descartados após a infusão de medula óssea, de lipoaspirado e de polpa de dentes decíduos. Métodos: Foi realizada a comparação da capacidade proliferativa de CTMs, cultivadas na presença de lisado plaquetário, das diversas fontes através do cálculo de population doubling das CTM em co-cultura com linfócitos T isolados em coluna magnética e com células mononucleares de sangue periférico, estimuladas com fitohemaglutinina; e determinado por citometria de fluxo o efeito das CTM das diversas fontes sobre as subpopulações linfocitárias. Resultados: CTM das três fontes foram capazes de inibir a proliferação de linfócitos e CTM de tecido adiposo foram mais eficientes em induzir o fenótipo de células T reguladoras e na diminuição de células T citotóxicas. Conclusão: comparadas à CTM isoladas de medula óssea e de polpa dentária, as CTM originadas de tecido adiposo exibem efeito imunomodulador mais acentuado. / Background: Mesenchymla stromal cells (MSC) reside in most adult tissue of mesenchymal origen, with a broad functions envolving cell repopulation and maintenence of tissue homeostasis, trough immunemmodulatory action. MSC are valuable terapêutic instruments applied from research to autoimune and inflamatory diseases. MSC can be isolated from diverse discarted biological matherials, like lipoaspirate, exfoliated deciduous teeeth and boné marrow ransplant filters. There so it´s necessary to stablish how source can impact MSC efficiency and possible clinical aplications. Objective: Compare immunomodulatory potential of adipose MSC and dental pulp MSC to boné marrow MSC. Methods: MSC from three selected sources were cocultured with phytohemaglutinin stimulated and magnetically isolated T cells and peripheral blood mononuclear cells; immunephenotype of cocultivated lymphocytes were also conducted. Results: MSC from all analyzed sources were capable to inhibit lymphocyte proliferation. Adipose MSC were capable to induce Treg phenotype and decrease T CD8+ limphocytes. Conclusion: Cell culture and therapy with MSC present many paradigms and we address to some of those to elucidate the possible most efficient source.

Introdução: Células-troco mesenquimais (CTM) vêm mostrando seus benefícios na doença do enxerto-versus-hospedeiro (DECH), observada no transplante de células tronco hematopoéticas (TCTH), existem três questões em aberto: (1) Expansão de CTM em meio de cultura suplementado com soro fetal bovino (SFB), pelo o risco de xenorreação; (2) Otimização de condições de cultura para a obtenção, em tempo hábil, de um numero que permita de 4 a 6 infusões de 2x106cells/kg do receptor; (3) Obter células do doador de medula óssea, evitando assim a utilização de um terceiro doador. Objetivos: Este estudo foi desenhado para comparar o lisado de plaquetas (LP) e o SFB na expansão de CTM, a densidade de plaqueamento das células e os dias entre cada passagem, e para investigar se as células nucleadas totais obtidas da bolsa e filtro do TCTH, podem ser utilizadas para expansão de CTM para utilização clínica. Métodos: Células residuais foram removidas do filtro e da bolsa utilizados para o TCTH, plaqueadas e depois da primeira passagem foram cultivadas em diferentes concentrações com SFB ou LP e observado o número de dias que levaram para chegar a 80% de confluência. Em seguida, as culturas com as mesmas densidades de plaqueamento foram suplementadas com LP ou SFB e depois de sete dias contou-se o número de células para analisar o quanto elas cresceram nesse período. Resultados: A proliferação de CTM, na presença de LP e SFB foi em média 11,88 e 2,5 vezes, respectivamente, num período de 7 dias. A concentração mais elevada de células usando LP demorou menos tempo para atingir a confluência, em comparação com os três inferiores. Este estudo sugere que o LP é a melhor escolha como suplemento para expandir CTM, e permite a proliferação de um número suficiente de CTM de doadores para uso clínico. / Introduction: Mesenchymal stromal cells (MSC) have shown their benefits in graft-versus-host disease (GVHD), with three unsettled matters:(1) MSCs expansion in medium with Fetal Calf Serum (FCS) and its risk of xenoreaction; (2) The number of cells indicated for therapy is 2x106cells/Kg with the need to optimize expansion, number and time wise; and (3) the utilization of third party donors. Aims: This study was designed to compare the platelet lysate (LP) and FCS on the expansion of MSC, the optimal cell plating density and days between each pass, and to investigate if donor total nucleated cells (TNC) obtained from the washouts of hematopoietic stem cell transplantation (HSCT) explants can be expanded to be used at clinical grade. Methods: TNC were removed, plated and after the first passage were cultivated in different concentrations with FCS or PL and the number of days reach 80% of confluence was observed. Next, cultures with the same plating density were fed either with PL or FCS and after seven days counted to analyze how much they have grown in that period. Results: The proliferation of mesenchymal stromal cells in the presence of PL and SFB was averaged 11.88 and 2.5 times, respectively, in a period of 7 days. The highest concentration of plating cells using PL, took less time to reach confluence as compared with the three lower ones. This study suggests that the PL is the best choice as a supplement to expand MSC, and allows the proliferation of a sufficient number of donors MSC at P2 for clinical use.

Pferde als Pflanzenfresser benötigen für die Verdauungsvorgänge im Magen-Darm-Kanal eine Vielzahl von Mikroorganismen. In der Pathogenese der equinen Kolikerkrankungen spielen die aus einem Teil dieser Bakterien stammenden Endotoxine (Lipopolysaccharide, LPS) eine wichtige Rolle. Das Caecum und das Colon ascendens scheinen der Ort einer pathologischen Endotoxinabsorption beim Pferd zu sein. Mit Hilfe von Limulus-Amoebozyten-Lysat-Tests (chromogenes Substrat, Endpunkt Methode) wurden die Endotoxinkonzentrationen bei 52 gesunden Pferden und 105 an Magen-Darm-Kolik erkrankten Pferde bestimmt. Durch wiederholte Messungen wurde die Entwicklung der Endotoxinkonzentration bei Kolikpferden im Krankheitsverlauf untersucht. Im Plasma aller gesunden Pferde wurden Endotoxine nachgewiesen, mit einem Mittelwert von = 5,90 pg/ml ± 2,78 pg/ml. Bei 90,5% der Pferden mit Kolik lag die Endotoxinkonzentration in der ersten Probe nach Einlieferung in die Klinik über 10 pg/ml. Kolikformen mit grundsätzlich hohen Endotoxinkonzentrationen konnten herausgefunden werden. In dieser Untersuchung waren das die Hernia foraminis omentalis mit einem LPS-Mittelwert von 91,57 pg/ml, die Dünndarmstrangulation durch Lipoma pendulans mit einem LPS-Mittelwert von 89,32 pg/ml und die Torsio coli totalis 360° mit einem LPS-Mittelwert von 88,21 pg/ml.:INHALTSVERZEICHNIS Seite ABKÜRZUNGEN .................................................................................................... 12 1. EINLEITUNG ............................................................................................... 14 2. SCHRIFTTUM .............................................................................................. 16 2.1. Herkunft und Aufbau der Endotoxine ........................................ 16 2.1.1. Struktur der Zellwand der gram-negativen Bakterien .............. 16 2.1.2. Chemische Struktur der Endotoxine ............................................ 17 2.1.2.1. Das O-spezifische Polysaccharid .................................................. 18 2.1.2.2. Das Kern-Oligosaccharid ............................................................... 18 2.1.2.3. Das Lipoid-A .................................................................................... 19 2.2. Biologische Wirkung der Endotoxine .......................................... 22 2.2.1. Bindung der LPS und Aktivierung der Zellen ........................... 23 Endotoxinrezeptoren im Plasma ...................................... 24 Endotoxinrezeptoren auf der Zellmembran .................... 24 2.2.2. Freisetzung und biologische Wirkung der Vermittlermoleküle ........................................................................ 25 2.2.2.1. Lipide .............................................................................................. 25 Prostaglandine (PG) .......................................................... 26 Leukotriene (LT) ................................................................. 29 2.2.2.2. Proteine ........................................................................................... 29 Tumor-Nekrose-Faktor und Interleukin-1 ..................... 30 Interleukin-8 ........................................................................ 32 Akute-Phase-Antwort und Interleukin-6 ........................ 33 2.2.2.3. Andere endogene Mediatoren ...................................................... 35 2.2.3. Experimentelle Erfahrungen .......................................................... 37 2.3. Die Darmmikroflora des Pferdes als Ausgangspunkt für die Entstehung einer Endotoxämie ........................................ 38 2.4. Darmkanal/Blut-Schranke für Endotoxine; Rolle der Leber .... 41 2.5. Endotoxine und Krankheiten des Pferdes .................................. 42 2.5.1. Hämodynamische und hämostatische Anomalien .................... 43 2.5.1.1. Der Schock ........................................................................................ 43 2.5.1.2. Disseminierte intravasale Gerinnung (DIG) ................................ 44 Labor-Nachweisverfahren: ................................................. 46 Thrombozyten .......................................................... 46 Antithrombin III ....................................................... 46 Fibrin/Fibrinogen-Degradationsprodukte .......... 48 Fibrinogen ................................................................. 48 Andere hämostatische Parameter .......................... 49 2.5.2. Magen-Darm-Erkrankungen des Pferdes ................................... 50 Begriffsbestimmung: Kolik ................................................ 50 2.5.2.1. Endotoxämie bei Magen-Darm-Koliken ..................................... 51 2.5.2.2. Begleiterkrankungen, die sich durch die Wirkung der Endotoxine aus Magen-Darm-Koliken entwickeln ................... 54 2.5.2.3. Kolitis, Typhlokolitis ..................................................................... 56 Kolitis .................................................................................... 56 Typhlokolitis ....................................................................... 58 Klinisches Bild ......................................................... 59 Pathomorphologische Befunde ............................. 60 Ätiologie und Pathogenese .................................... 62 2.5.2.4. Laktat-Azidose und Anion gap bei Pferden mit Magen-Darm-Kolik ......................................................................... 66 2.6. Therapeutische Möglichkeiten zur Bekämpfung der Endotoxämie .................................................................................... 68 2.6.1. Hemmung der Zytokininduktion durch Lipoid-A-Teilstrukturen ................................................................ 69 2.6.2. Anwendung von entzündungshemmenden Präparaten .......... 70 2.6.3. Infusionstherapie ............................................................................ 72 2.6.4. Eine alternative Möglichkeit zur Vorbeugung der Endotoxinwirkung durch Fütterungsmaßnahmen .................... 73 2.6.5. Antiendotoxische Immuntherapie ............................................... 73 2.6.6. Endotoxinneutralisierendes Protein ............................................ 76 3. EIGENE UNTERSUCHUNGEN ................................................... 77 3.1. Material ............................................................................................. 77 3.2. Methodik .......................................................................................... 79 3.2.1. Angewandte Materialien ............................................................... 79 3.2.2. Gewinnung der Proben .................................................................. 81 3.2.3. Endotoxinbestimmung .................................................................. 82 3.2.3.1. Analysator ........................................................................................ 82 3.2.3.2. Reagenzien ....................................................................................... 82 3.2.3.3. Herstellung der Reaktionsansätze ................................................ 83 3.2.3.4. Analyse ............................................................................................. 83 Reaktionsprinzip ................................................................. 83 Standardisierung ................................................................. 84 Probenbehandlung .............................................................. 84 Beschickung der Mikroküvette ......................................... 85 Erstellung der Standardkurve ........................................... 86 3.2.4. Fibrinogenbestimmung .................................................................. 88 3.2.4.1. Reagenzien ....................................................................................... 88 3.2.4.2. Analyse ............................................................................................. 89 Reaktionsprinzip ................................................................. 89 Standard ................................................................................ 89 Plasmaproben ....................................................................... 90 3.2.5. Erklärung zum Index Endotoxin/Fibrinogen ............................ 90 3.2.6. Bestimmung der Antithrombin III-Aktivität .............................. 91 Reaktionsprinzip ................................................................. 92 3.2.7. Anion gap-Bestimmung ................................................................ 92 3.2.8. Enzymaktivitätsbestimmung ........................................................ 92 3.2.9. Statistische Auswertung ................................................................ 93 Erklärung zu Pearson Korrelationskoeffizienten und der Zeichen der Differenzsignifikanz ...................... 94 Erklärung der Elemente des Plot-Box-Diagrammes ...... 94 3.3. Ergebnisse ........................................................................................ 96 3.3.1. Vergleich der Ergebnisse der klinisch gesunden Pferde und der Pferde mit Kolik .................................................. 96 3.3.2. Untersuchungsergebnisse der Kolikpatienten ........................... 99 3.3.2.1. Ergebnisse der Endotoxinbestimmung ..................................... 101 3.3.2.2. Ergebnisse der Fibrinogenbestimmung ..................................... 104 3.3.2.3. Vergleich der Parameter Endotoxin – Fibrinogen; Index Endotoxin/Fibrinogen ...................................................... 106 3.3.2.4. Ergebnisse der Voruntersuchungen der AT-III-Aktivität im Plasma .......................................................... 109 3.3.2.5. Ergebnisse der Anion gap-Bestimmung .................................... 110 3.3.2.6. Ergebnisse der Enzymaktivitätsbestimmung ........................... 112 3.3.2.7. Vergleich der Ergebnisse von konservativ und chirurgisch behandelten Patienten ............................................. 114 3.3.2.8. Vergleich der Ergebnisse von überlebenden und verendeten Patienten ............................................................ 117 3.3.3. Korrelationsanalyse der Meßparameter .................................... 119 4. DISKUSSION ............................................................................................ 122 4.1. Endotoxine ..................................................................................... 122 Aufgabe und Methodik .................................................... 122 Endotoxine bei gesunden Pferden .................................. 123 Endotoxine bei Pferden mit Kolik .................................. 124 4.2. Endotoxämie und Magen-Darm-Koliken ................................. 125 4.3. Endotoxine und Fieber ................................................................. 127 4.4. Begleiterkrankungen der Magen-Darm-Koliken ..................... 128 Disseminierte intravasale Gerinnung ............................. 128 Hufrehe ............................................................................... 129 Typhlokolitis; Salmonelleninfektion ............................... 130 4.5. Anion gap ....................................................................................... 131 4.6. Schlußwort ..................................................................................... 131 5. ZUSAMMENFASSUNG .......................................................................... 133 6. SUMMARY ................................................................................................ 135 7. LITERATURVERZEICHNIS .................................................................... 137 8. ANHANG ................................................................................................... 160 / Endotoxaemia in colic illnesses in horses; Quantitative analysis and clinical relevance Horses as herbivores require a multitude of micro-organisms for the digestive processes in the gastrointestinal tract. The endotoxins (lipopolysaccharides, LPS) originating from a part of the bacteria play an important role in the pathogenesis of equine colic illnesses. The caecum and the colon ascendens appear to be the site of a pathological absorption of endotoxins in horses. With the aid of limulus-amoebocyte-lysate tests (chromogeneous substrate, end-point method) the endotoxin concentrations were analysed in 52 healthy horses and 105 horses suffering from gastrointestinal colic. The development of the endotoxin concentration in the case of horses suffering from colic was investigated through repeated measurements throughout the course of the illness. Endotoxins were identified in the plasma of all healthy horses at a mean value of = 5.90 pg/ml ± 2.78 pg/ml. In 90.5% of the horses with colic, the concentration of endotoxins in the first sample subsequent to admission to the clinic was over 10 pg/ml. It was possible to determine specific forms of colic accompanied by fundamentally high concentrations of endotoxins. In this investigation these were omental foramen hernia with a mean LPS value of 91.57 pg/ml, small intestinal strangulation by lipoma pendulans with a mean LPS value of 89.32 pg/ml and colon torsion 360° with a mean LPS value of 88.21 pg/ml.:INHALTSVERZEICHNIS Seite ABKÜRZUNGEN .................................................................................................... 12 1. EINLEITUNG ............................................................................................... 14 2. SCHRIFTTUM .............................................................................................. 16 2.1. Herkunft und Aufbau der Endotoxine ........................................ 16 2.1.1. Struktur der Zellwand der gram-negativen Bakterien .............. 16 2.1.2. Chemische Struktur der Endotoxine ............................................ 17 2.1.2.1. Das O-spezifische Polysaccharid .................................................. 18 2.1.2.2. Das Kern-Oligosaccharid ............................................................... 18 2.1.2.3. Das Lipoid-A .................................................................................... 19 2.2. Biologische Wirkung der Endotoxine .......................................... 22 2.2.1. Bindung der LPS und Aktivierung der Zellen ........................... 23 Endotoxinrezeptoren im Plasma ...................................... 24 Endotoxinrezeptoren auf der Zellmembran .................... 24 2.2.2. Freisetzung und biologische Wirkung der Vermittlermoleküle ........................................................................ 25 2.2.2.1. Lipide .............................................................................................. 25 Prostaglandine (PG) .......................................................... 26 Leukotriene (LT) ................................................................. 29 2.2.2.2. Proteine ........................................................................................... 29 Tumor-Nekrose-Faktor und Interleukin-1 ..................... 30 Interleukin-8 ........................................................................ 32 Akute-Phase-Antwort und Interleukin-6 ........................ 33 2.2.2.3. Andere endogene Mediatoren ...................................................... 35 2.2.3. Experimentelle Erfahrungen .......................................................... 37 2.3. Die Darmmikroflora des Pferdes als Ausgangspunkt für die Entstehung einer Endotoxämie ........................................ 38 2.4. Darmkanal/Blut-Schranke für Endotoxine; Rolle der Leber .... 41 2.5. Endotoxine und Krankheiten des Pferdes .................................. 42 2.5.1. Hämodynamische und hämostatische Anomalien .................... 43 2.5.1.1. Der Schock ........................................................................................ 43 2.5.1.2. Disseminierte intravasale Gerinnung (DIG) ................................ 44 Labor-Nachweisverfahren: ................................................. 46 Thrombozyten .......................................................... 46 Antithrombin III ....................................................... 46 Fibrin/Fibrinogen-Degradationsprodukte .......... 48 Fibrinogen ................................................................. 48 Andere hämostatische Parameter .......................... 49 2.5.2. Magen-Darm-Erkrankungen des Pferdes ................................... 50 Begriffsbestimmung: Kolik ................................................ 50 2.5.2.1. Endotoxämie bei Magen-Darm-Koliken ..................................... 51 2.5.2.2. Begleiterkrankungen, die sich durch die Wirkung der Endotoxine aus Magen-Darm-Koliken entwickeln ................... 54 2.5.2.3. Kolitis, Typhlokolitis ..................................................................... 56 Kolitis .................................................................................... 56 Typhlokolitis ....................................................................... 58 Klinisches Bild ......................................................... 59 Pathomorphologische Befunde ............................. 60 Ätiologie und Pathogenese .................................... 62 2.5.2.4. Laktat-Azidose und Anion gap bei Pferden mit Magen-Darm-Kolik ......................................................................... 66 2.6. Therapeutische Möglichkeiten zur Bekämpfung der Endotoxämie .................................................................................... 68 2.6.1. Hemmung der Zytokininduktion durch Lipoid-A-Teilstrukturen ................................................................ 69 2.6.2. Anwendung von entzündungshemmenden Präparaten .......... 70 2.6.3. Infusionstherapie ............................................................................ 72 2.6.4. Eine alternative Möglichkeit zur Vorbeugung der Endotoxinwirkung durch Fütterungsmaßnahmen .................... 73 2.6.5. Antiendotoxische Immuntherapie ............................................... 73 2.6.6. Endotoxinneutralisierendes Protein ............................................ 76 3. EIGENE UNTERSUCHUNGEN ................................................... 77 3.1. Material ............................................................................................. 77 3.2. Methodik .......................................................................................... 79 3.2.1. Angewandte Materialien ............................................................... 79 3.2.2. Gewinnung der Proben .................................................................. 81 3.2.3. Endotoxinbestimmung .................................................................. 82 3.2.3.1. Analysator ........................................................................................ 82 3.2.3.2. Reagenzien ....................................................................................... 82 3.2.3.3. Herstellung der Reaktionsansätze ................................................ 83 3.2.3.4. Analyse ............................................................................................. 83 Reaktionsprinzip ................................................................. 83 Standardisierung ................................................................. 84 Probenbehandlung .............................................................. 84 Beschickung der Mikroküvette ......................................... 85 Erstellung der Standardkurve ........................................... 86 3.2.4. Fibrinogenbestimmung .................................................................. 88 3.2.4.1. Reagenzien ....................................................................................... 88 3.2.4.2. Analyse ............................................................................................. 89 Reaktionsprinzip ................................................................. 89 Standard ................................................................................ 89 Plasmaproben ....................................................................... 90 3.2.5. Erklärung zum Index Endotoxin/Fibrinogen ............................ 90 3.2.6. Bestimmung der Antithrombin III-Aktivität .............................. 91 Reaktionsprinzip ................................................................. 92 3.2.7. Anion gap-Bestimmung ................................................................ 92 3.2.8. Enzymaktivitätsbestimmung ........................................................ 92 3.2.9. Statistische Auswertung ................................................................ 93 Erklärung zu Pearson Korrelationskoeffizienten und der Zeichen der Differenzsignifikanz ...................... 94 Erklärung der Elemente des Plot-Box-Diagrammes ...... 94 3.3. Ergebnisse ........................................................................................ 96 3.3.1. Vergleich der Ergebnisse der klinisch gesunden Pferde und der Pferde mit Kolik .................................................. 96 3.3.2. Untersuchungsergebnisse der Kolikpatienten ........................... 99 3.3.2.1. Ergebnisse der Endotoxinbestimmung ..................................... 101 3.3.2.2. Ergebnisse der Fibrinogenbestimmung ..................................... 104 3.3.2.3. Vergleich der Parameter Endotoxin – Fibrinogen; Index Endotoxin/Fibrinogen ...................................................... 106 3.3.2.4. Ergebnisse der Voruntersuchungen der AT-III-Aktivität im Plasma .......................................................... 109 3.3.2.5. Ergebnisse der Anion gap-Bestimmung .................................... 110 3.3.2.6. Ergebnisse der Enzymaktivitätsbestimmung ........................... 112 3.3.2.7. Vergleich der Ergebnisse von konservativ und chirurgisch behandelten Patienten ............................................. 114 3.3.2.8. Vergleich der Ergebnisse von überlebenden und verendeten Patienten ............................................................ 117 3.3.3. Korrelationsanalyse der Meßparameter .................................... 119 4. DISKUSSION ............................................................................................ 122 4.1. Endotoxine ..................................................................................... 122 Aufgabe und Methodik .................................................... 122 Endotoxine bei gesunden Pferden .................................. 123 Endotoxine bei Pferden mit Kolik .................................. 124 4.2. Endotoxämie und Magen-Darm-Koliken ................................. 125 4.3. Endotoxine und Fieber ................................................................. 127 4.4. Begleiterkrankungen der Magen-Darm-Koliken ..................... 128 Disseminierte intravasale Gerinnung ............................. 128 Hufrehe ............................................................................... 129 Typhlokolitis; Salmonelleninfektion ............................... 130 4.5. Anion gap ....................................................................................... 131 4.6. Schlußwort ..................................................................................... 131 5. ZUSAMMENFASSUNG .......................................................................... 133 6. SUMMARY ................................................................................................ 135 7. LITERATURVERZEICHNIS .................................................................... 137 8. ANHANG ................................................................................................... 160

Os procedimentos para uso clínico de células-tronco derivadas de tecido adiposo (CT-TA) exigem grandes quantidades de células, por isso, em geral os protocolos envolvem a expansão e cultura celular in vitro. No entanto, as metodologias utilizadas rotineiramente para o cultivo de CT-TA envolvem a utilização de componentes xenobióticos, como a colagenase e o soro fetal bovino (SFB), que representam riscos potencias de reações imunológicas e transmissão de doenças infecciosas. Sendo assim, pretendeu-se no presente estudo analisar diferentes parâmetros metodológicos para isolamento e expansão de CT-TA, na ausência de componentes xenobióticos. Para tanto, as células-tronco foram isoladas por digestão enzimática ou dissociação mecânica e submetidas à expansão na presença de SFB ou lisado de plaquetas humano (LP). Os resultados mostraram que a metodologia de dissociação mecânica representa uma alternativa viável e eficiente para cultivo de CT-TA, e que o emprego de LP como suplemento para o meio de cultura aumentou de forma significativa a proliferação celular. Em função desses resultados, pode-se concluir que é possível a implementação de técnicas de isolamento e expansão de CT-TA, prescindindo-se de componentes xenobióticos. / The procedures for the clinical use of adipose-derived stem cells (ASC) require large amounts of cells, so in general protocols involve culture and cell expansion in vitro.However, the methods routinely used for the culture of ASC involves the use of xenobiotic components, such as collagenase and fetal bovine serum (FBS), that may representing potential risk of immunological reactions and the risk of transmission of infectious diseases. Thus, it was intended in this study to analyze different methodological parameters for the isolation and expansion of ASC in the absence of xenobiotic components. For this, stem cells were isolated by enzymatic digestion and mechanical dissociation and were submitted to expansion in the presence of FBS or human platelet lysate (PL). The results showed that the mechanical dissociation method represents an effective alternative to growing ASC, and that the use of PL as a supplement to the culture medium significantly increased cellular proliferation. In view of these results, we can conclude that it is possible to implement techniques for isolation and expansion of ASC, dispensing xenobiotic components.

Os procedimentos para uso clínico de células-tronco derivadas de tecido adiposo (CT-TA) exigem grandes quantidades de células, por isso, em geral os protocolos envolvem a expansão e cultura celular in vitro. No entanto, as metodologias utilizadas rotineiramente para o cultivo de CT-TA envolvem a utilização de componentes xenobióticos, como a colagenase e o soro fetal bovino (SFB), que representam riscos potencias de reações imunológicas e transmissão de doenças infecciosas. Sendo assim, pretendeu-se no presente estudo analisar diferentes parâmetros metodológicos para isolamento e expansão de CT-TA, na ausência de componentes xenobióticos. Para tanto, as células-tronco foram isoladas por digestão enzimática ou dissociação mecânica e submetidas à expansão na presença de SFB ou lisado de plaquetas humano (LP). Os resultados mostraram que a metodologia de dissociação mecânica representa uma alternativa viável e eficiente para cultivo de CT-TA, e que o emprego de LP como suplemento para o meio de cultura aumentou de forma significativa a proliferação celular. Em função desses resultados, pode-se concluir que é possível a implementação de técnicas de isolamento e expansão de CT-TA, prescindindo-se de componentes xenobióticos. / The procedures for the clinical use of adipose-derived stem cells (ASC) require large amounts of cells, so in general protocols involve culture and cell expansion in vitro.However, the methods routinely used for the culture of ASC involves the use of xenobiotic components, such as collagenase and fetal bovine serum (FBS), that may representing potential risk of immunological reactions and the risk of transmission of infectious diseases. Thus, it was intended in this study to analyze different methodological parameters for the isolation and expansion of ASC in the absence of xenobiotic components. For this, stem cells were isolated by enzymatic digestion and mechanical dissociation and were submitted to expansion in the presence of FBS or human platelet lysate (PL). The results showed that the mechanical dissociation method represents an effective alternative to growing ASC, and that the use of PL as a supplement to the culture medium significantly increased cellular proliferation. In view of these results, we can conclude that it is possible to implement techniques for isolation and expansion of ASC, dispensing xenobiotic components.

Glutathione transferase (GST) A2-2 is the human enzyme most efficient in catalyzing azathioprine activation. Structure-function relationships were sought explaining the higher catalytic efficiency compared to other alpha class GSTs. By screening a DNA shuffling library, five recombined segments were identified that were conserved among the most active mutants. Mutational analysis confirmed the importance of these short segments as their insertion into low-active GSTs introduced higher azathioprine activity. Besides, H-site mutagenesis led to decreased azathioprine activity when the targeted positions belonged to these conserved segments and mainly enhanced activity when other positions were targeted. Hydrophobic residues were preferred in positions 208 and 213. The prodrug azathioprine is today primarily used for maintaining remission in inflammatory bowel disease. Therapy leads to adverse effects for 30 % of the patients and genotyping of the metabolic genes involved can explain some of these incidences. Five genotypes of human A2-2 were characterized and variant A2*E had 3–4-fold higher catalytic efficiency with azathioprine, due to a proline mutated close to the H-site. Faster activation might lead to different metabolite distributions and possibly more adverse effects. Genotyping of GSTs is recommended for further studies. Molecular docking of azathioprine into a modeled structure of A2*E suggested three positions for mutagenesis. The most active mutants had small or polar residues in the mutated positions. Mutant L107G/L108D/F222H displayed a 70-fold improved catalytic efficiency with azathioprine. Determination of its structure by X-ray crystallography showed a widened H-site, suggesting that the transition state could be accommodated in a mode better suited for catalysis. The mutational analysis increased our understanding of the azathioprine activation in alpha class GSTs and highlighted A2*E as one factor possibly behind the adverse drug-effects. A successfully redesigned GST, with 200-fold enhanced catalytic efficiency towards azathioprine compared to the starting point A2*C, might find use in targeted enzyme-prodrug therapies.