As células-tronco mesenquimais (MSCs) são células estromais não-hematopoiéticas que possuem capacidade de diferenciação, sendo capazes, de diferenciar em diversos tecidos. As MSCs residem em vários tecidos vêm sido isoladas de diferentes tecidos, tais como cartilagem, tendão, tecido adiposo, do vaso e sangue umbilical, além de tecidos fetais . O isolamento e caracterização das populações mesenquimais no modelo ovino se faz importante, visto que ele é usado em ensaios pré-clínicos ortopédicos . Nesse estudo foram utilizados 5 amostras de sangue de cordão umbilical e 5 amostras de tecido adiposo peri-renal, provenientes de 10 ovinos fêmeas adultas. As coletas foram realizadas através de cirurgia para que o material coletado fosse o mais asséptico possível. Essas amostras foram submetidas a diferentes protocolos de isolamento, com a finalidade de se testar o mais eficiente. Somente um protocolo de cada tecido mostrou-se eficiente no isolamento da MSCs, porém nenhuma dessas amostras manteve-se viável após a primeira passagem. / Mesenchymal stem cells (MSCs) are non hematopoietic stromal cells that are able to differentiate through several tissues . MCSs home in several tissues and are being isolated from different tissues, such cartilage, tendon, adipose tissue, vessels and umbilical blood, and also from fetal tissues . The isolation and characterization of mesenchymal cells in sheep are important, because it is used in orthopedic pre-clinical trials . In this study were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. These samples were tested in different protocols to evaluate the more efficient. Just one protocol from each source showed significant results in isolation, although none of the samples survived trough the first passage.

La surveillance du bien-être fœtal pendant le travail repose essentiellement sur l’enregistrement du rythme cardiaque fœtal (RCF). Celui-ci, même continu pendant le travail, ne permet pas d’évaluer parfaitement l’oxygénation du fœtus ni le risque d’asphyxie néonatale. En effet, cet outil est imparfait et son évaluation subjective avec une importante variabilité d’interprétation inter et intra opérateur. Des examens dits de seconde ligne sont utilisés en pratique courante pour caractériser l’état fœtal : le prélèvement de sang fœtal au scalp pour l’étude de l’équilibre acido-basique du fœtus (pH ou lactates) ou la pose d’électrode au scalp pour étudier l’ECG fœtal (analyse du segment ST). Ces techniques sont néanmoins invasives et sont soumises à des contraintes techniques. Il y a donc un intérêt à développer des moyens d’évaluation du bien être fœtal à la fois objectifs et non invasifs afin de diminuer la survenue d’une asphyxie périnatale. En effet, celle-ci touche 3 à 8 nouveaux nés pour 1000 naissances. La mortalité en période post-natale est de 25 à 50% des cas et ceux qui survivent développeront des troubles sévères (épilepsie, retard neuro-cognitif et comportemental, paralysie cérébrale…). Au cours de l’accouchement, l’asphyxie périnatale se caractérise par une diminution du pH artériel ombilical. Cette mesure du pH sanguin est donc la mesure de référence pour déterminer la sévérité de l’asphyxie.Une des voies étudiées pour améliorer le dépistage des fœtus à risque d’acidose est l’analyse des modifications du système nerveux autonome (SNA) par analyse de la variabilité du rythme cardiaque fœtal. En effet, la fréquence cardiaque fœtale est en permanence sous l’influence du système nerveux autonome et sa variabilité (VFC) est un reflet de la balance sympathique / parasympathique. Le CHU de Lille a développé une nouvelle méthode d’analyse continue de la VFC ayant montré son efficacité chez l’adulte et chez le nouveau né pour l’évaluation du SNA. L’objectif de ce travail de Thèse est d’adapter cette technologie à l’analyse du SNA fœtal pour obtenir un nouvel indice appelé Fetal Stress Index (FSI) et d’évaluer sa pertinence en situation d’acidose.Cette preuve de concept a été effectuée de manière expérimentale chez le fœtus de brebis. Elle s’est réalisée en 2 temps. Nous avons tout d’abord évalué la performance du FSI par rapport aux méthodes classiques d’analyse de la VFC en termes d’aptitude à détecter les variations du SNA. Après injection d’Atropine, parasympatholytique, ou de Propranolol, sympatholytique, nous avons montré que le FSI était une méthode efficace et spécifique d’évaluation des variations du tonus parasympathique du SNA. Cette étude a également montrée la supériorité du FSI par rapport aux méthodes classiques d’analyse de la VFC en termes de sensibilité et de spécificité. Dans un second temps, nous avons évalué ce nouvel indice comme facteur prédictif de l’état acido basique du fœtus dans 2 modèles expérimentaux d’occlusion cordonale. Dans le premier modèle, l’acidose était obtenue par une occlusion continue du cordon avec une réduction de partielle du débit ombilical. Dans le second, nous réalisions des occlusions totales répétées à intervalles réguliers afin de mimer les contractions utérines lors du travail. Dans les 2 études, nous avons observé une hausse du FSI en cas d’acidose avec une corrélation significative entre le FSI et le pH, mais aussi entre le FSI et les lactates dans le second modèle.En conclusion, le FSI constitue un bon reflet de l’activité parasympathique fœtale. Cet indice permet d’étudier les variations du SNA fœtal avec une meilleure sensibilité et une meilleure spécificité que les méthodes usuelles d’analyse de la VFC et semble bien corrélé à l’état acido basique fœtal. Il s’agit donc d’un indice prometteur qu’il sera intéressant d’incorporer dans une analyse multi paramétrique du rythme cardiaque fœtal. / The monitoring of fetal well being during labor is essentially based on fetal heart rate (FHR) analysis. The recording of FHR, even continuously during labor, does not fully assess fetal oxygenation or neonatal risk of asphyxia. Indeed, this tool is imperfect and subjective with an important inter and intra-operator variability. Second-line examinations to characterize the fetal state are currently used in routine practice, i.e. scalp fetal blood sampling to study the fetal acid-base balance (pH or lactates) or scalp electrode placement to study the fetal ECG (ST segment analysis). These techniques are nevertheless invasive and subject to technical constraints. There is therefore an interest in developing both objective and non-invasive means of evaluating fetal wellbeing to reduce neonatal encephalopathy. Indeed, its prevalence is about 3 to 8 per 1000 births. Post natal mortality is about 25 to 50% and survivors will hav severe diseases (epilepsy, neurologic impairment, cerebral palsy…).One of the possibilities studied to better identify fetuses at risk for acidosis is the analysis of changes in the autonomic nervous system (ANS) in response to hypoxia.Indeed, the regulation of heart rate is dependent on the ANS and thus, its variability is a reflection of the sympathetic / parasympathetic balance. Analysis of heart rate variability (HRV) is a recognized non-invasive tool that is used to assess ANS regulation. The CHU Lille has developed a new continuous tool for the analysis of HRV, which demonstrated its efficacity in adults and neonates to evaluate the ANS. The objective of this thesis was to develop its index, called Fetal Stress Index (FSI), in the fetus and to evaluate it in conditions of acidosis.The study was experimental in a sheep model chronically instrumented and was in 2 steps. First, we evaluate the performance of our method compared to commonly used HRV analysis, regarding the ability to detect the variation of variations of the ANS. After injection of atropine, to inhibit parasympathetic tone, or propranolol to block sympathetic activity, we shown that our method appeared to be effective in detecting parasympathetic inhibition and, moreover, was superior to classical analysis of HRV in terms of sensibility and specificity.In a second time, we evaluated this new index as a predictive factor of the fetal acid-base state in 2 experimental models of fetal hypoxia by occlusion of the cord. In the first one, acidosis was obtained through a partial occlusion of the umbilical cord and in the second one, though repetitive complete occlusion as uterine contractions during labor. In those two studies, we observed a raise of our index in case of acidosis with a correlation beetween FSI and pH and also FSI and lactates in the second model.In conclusion, the FSI reflects fetal parasympathetic activity, has a better detection than others usual methods, and seems well correlated to fetal acid-base status. It is a promising index and it will be interesting to incorporate it in a multi parametric analysis of fetal heart rate to predict acidosis.

Les cellules dendritiques plasmacytoïdes (pDCs) sont considérées comme quantitativement et qualitativement supérieures aux autres types cellulaires pour la synthèse des interférons (IFNs) de type I lors d’une infection virale. Plusieurs observations viennent supporter cette désignation. Tout d’abord, elles expriment un éventail très large des sous-types d’IFN-alpha en comparaison aux autres types cellulaires. Par ailleurs, elles possèdent la capacité de détecter la présence des virus via leurs TLR7 et TLR9, reconnaissant respectivement l’ARN ou l’ADN d’origine virale. Enfin, elles expriment de manière constitutive dans leur cytoplasme le facteur de transcription IRF-7 qui permet une synthèse rapide et robuste des IFNs de type I en réponse à l’infection. <p>Dans un précédent travail, il a été montré que les pDCs néonatales présentent un défaut majeur de synthèse d’IFN-alpha en réponse aux CpG ODNs, ligands du TLR9. Nous avons ensuite étendu notre étude des pDCs néonatales en les stimulant avec le R-848, ligand du TLR7, mais également en présence de virus tels que HCMV et HSV. Dans ces conditions également, la synthèse de l’IFN-alpha est déficiente dans les pDCs du nouveau-né. Nous avons également observé une déficience de production de l’IFN-beta suite à une stimulation via les ligands TLR7 et TLR9, tant au niveau protéique que de l’expression de l’ARN messager. Par ailleurs, la synthèse des cytokines/chimiokines inflammatoires par les pDCs du sang de cordon ainsi que leur maturation, fonctions dépendantes du facteur NF-kappaB, sont également diminuées en comparaison aux pDCs adultes, suite à une stimulation en présence du CpG ODN ou du R-848.<p>L’ensemble de ces données nous a amené à étudier de manière plus précise les voies de signalisation des pDCs néonatales suite à leur activation. Tout d’abord, nous avons observé que les taux d’expression des TLR7 et 9 tout comme le taux basal d’IRF-7 sont équivalents dans les pDCs néonatales et les pDCs adultes. Ensuite, grâce à la technique d’ImageStream (Amnis corporation), nous avons pu quantifier la translocation nucléaire des facteurs de transcription IRF-7 et de NF-kappaB dans les pDCs activées. Nous avons ainsi pu observer que la translocation de NF-kappaB est comparable dans les pDCs adultes et néonatales en réponse aux ligands TLR7 ou TLR9. Par contre, elle est déficiente lors d’une stimulation par HSV. La translocation du facteur IRF-7, quant à elle, est significativement déficiente en réponse au CpG ODN et au virus HSV dans les pDCs néonatales. <p>Nous proposons que le défaut de translocation d’IRF-7 mis en évidence dans les pDCs néonatales pourrait en partie expliquer la déficience de synthèse des IFNs de type I de ces cellules et fournir une base moléculaire à la plus grande susceptibilité du nouveau-né vis-à-vis des infections virales.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The polychlorinated biphenyls (PCBs) are synthetic aromatic chemicals mixtures industrially used since the 30 s decade. Due to its physics characters, as electric current and high temperature tolerance, it has been applied mainly as dielectrics fluids in capacitors and transformers. They toxic effect was evidenced from realized studies, and its extinguish became a challenge. Its prohibition started in United States, in 1976, but only in 1981 in Brazil, although its use in old electrics equipments are still yet consent, until its replacing. The environmental contamination mainly comes out due an inappropriate discard of old electrics equipments, which affect the soil and water. These compounds have a fat great attractive force, becoming accumulated in human and animals adipose tissues. The mainly reason of human contamination is the contaminated foods ingestion, in special, the animal foods source. Due its toxicity and great stability, the objective of this work was to determine the PCBs levels in umbilical cord serum collected from 148 donors that had your children in the Santa Maria University Hospital (HUSM) in 2006, checking the connect with demographic and custom life of donors and the infants information, obtained through questionnaire. The PCBs residues were determinate by gas chromatography with electron-capture detection (GC-μECD), after acid digestion. The confirmation was carried out on a gas chromatography coupled with mass spectrometer (GC/MS). The mean values detected were 0,18 ng mL to PCB 28, 1,71 ng mL to PCB 52, 0,82 ng mL to PCB 153, 2,45 ng mL to PCB 138 and 1,49 ng mL to PCB 180. The most frequent was the PCB 180, detected in 63,5% of samples, followed by PCBs 180 (55,4%), 52 (54,7%), 153 (51,4%) and 28 (19,9%). The PCBs 153 and 180 concentrations were negatively correlation with donors height, while the PCB 52 obtained positive correlation. The PCBs 138 and 180 concentrations were correlation with low development of infants, negatively affecting your length, while the PCB 28 obtained express difference on infants that had weight and head circumference higher. In respect to alimentary habits, express difference was found in PCB 153 concentration in function of fruits consumption: showed a concentration higher in the consumers class of this food. The PCB 153 higher concentration in the class donors that required a forceps delivery significantly disagreed that the others. The donors that lived out of Santa Maria city showed the PCBs 153 and 180 concentrations higher, obtained express difference that the class who lived in Santa Maria city, while the donors that had suffered abortion had higher PCB 52 concentrations, statistically differing for the no abortion class. The birth weights class of infants had higher PCB 52 concentration too, with express difference of normal birth weights class. With regard to malformation presence on infants, significant correlation were found with PCBs 28, 52 e 180 concentrations, that showed higher values in the malformation class. This research results showing that the PCBs presence in the umbilical cord serum, at significant levels, may negatively influence on the infants formation and development, resulting infants with low birth weight, small length and with some kind of malformation presence. As well as the infant, the mother also takes the consequences with PCBs significant levels in the umbilical cord serum, as abortion and difficulty on delivery moment. Literature references were not found about the influence of PCBs on infants malformation, what carry out to deduce that it s the first research about it. / Os bifenilos policlorados (PCBs) são misturas de compostos químicos aromáticos sintéticos utilizados industrialmente desde a década de 30. Devido às suas propriedades físicas, tais como resistência a altas temperaturas e correntes elétricas, foram utilizados principalmente como fluidos dielétricos em capacitores e transformadores. A partir de estudos realizados constatou-se o seu efeito tóxico, tornando-se um desafio abolir o seu uso. Em 1976, nos Estados Unidos, deu-se o início a sua proibição, o que ocorreu no Brasil apenas em 1981, embora ainda seja permitida a utilização dos equipamentos eletro-eletrônicos em uso, até a sua substituição. A contaminação ambiental ocorre principalmente devido ao descarte inadequado de equipamentos eletro-eletrônicos antigos, contaminando a água e o solo. Estes compostos possuem grande afinidade pela gordura, acumulando-se nos tecidos adiposos do homem e dos animais. A principal causa da contaminação humana é a ingestão de alimentos contaminados, em especial, os alimentos de origem animal. Em função da toxicidade e grande persistência destes compostos, o objetivo do presente trabalho foi determinar os níveis de PCBs em soro de cordão umbilical coletado de 148 doadoras que tiveram seus filhos no Hospital Universitário de Santa Maria (HUSM) no ano de 2006, verificando sua associação com os dados demográficos e hábitos de vida das doadoras e os dados dos recém-nascidos, obtidos através de um questionário. A determinação dos resíduos de PCBs foi realizada por cromatógrafo gasoso equipado com micro detector de captura de elétrons (GC-μECD) de Ni63, após extração através da técnica de hidrólise ácida. A confirmação foi realizada por cromatógrafo gasoso acoplado à espectrometria de massas (GC-MS). Foram encontrados valores médios de 0,18 ng mL-1 para o PCB 28, de 1,71 ng mL-1 para o PCB 52, de 0,82 ng mL-1 para o PCB 153, de 2,45 ng mL-1 para o PCB 138 e de 1,49 ng mL-1 para o PCB 180. O PCB 138 foi o mais freqüente, sendo detectado em 63,5% das amostras, seguido dos PCBs 180 (55,4%), 52 (54,7%), 153 (51,4%) e 28 (19,9%). As concentrações dos PCBs 153 e 180 foram correlacionadas negativamente com a altura das doadoras, enquanto que o PCB 52 obteve uma correlação positiva. As concentrações dos PCBs 138 e 180 foram correlacionadas com o baixo desenvolvimento dos recém-nascidos, afetando negativamente o seu comprimento, enquanto que o PCB 28 apresentou diferença significativa nos bebês que tiveram maiores peso e perímetro cefálico, os quais obtiveram maiores concentrações. Em relação aos hábitos alimentares, foi constatada diferença significativa na concentração do PCB 153 em função do consumo de frutas: apresentou uma concentração maior no grupo das consumidoras do referido alimento. A alta concentração do PCB 153 no grupo das doadoras que necessitaram de um parto com fórceps diferiu significativamente dos outros grupos de parto. As doadoras residentes fora de Santa Maria tiveram maiores concentrações dos PCBs 153 e 180, diferindo estatisticamente das que residiam em Santa Maria, enquanto que as doadoras que tinham sofrido aborto tiveram concentrações mais altas do PCB 52, diferindo estatisticamente do grupo que não teve aborto. O grupo de recém-nascidos classificado com baixo peso ao nascimento também obteve concentrações mais altas do PCB 52, com diferença significativa do grupo de bebês com o peso normal. Em relação à presença de malformação nos bebês houve diferença significativa com as concentrações dos PCBs 28, 52 e 180, as quais foram detectadas em níveis mais elevados no grupo dos bebês malformados. Os resultados do presente estudo demonstram que a presença de PCBs no soro do cordão umbilical, em níveis significativos, pode influenciar negativamente na formação e no desenvolvimento do recém-nascido, resultando no nascimento de um bebê com baixo peso, menor comprimento e com a presença de algum tipo de malformação. Assim como o bebê, a mãe também pode sofrer conseqüências com níveis significativos de PCBs no soro do cordão umbilical, tais como a ocorrência de aborto e dificuldades no momento do parto. Não foram encontradas referências na literatura sobre a influência de PCBs na malformação de bebês, o que nos leva a deduzir que este seja o primeiro estudo realizado sobre o tema.

The prevalence of allergy is increasing and it is becoming a serious problem not on- ly in medicine, but also in social and economic terms. The most effective way to minimize the development of allergic diseases is preventive measures. In recent years, many studies have attempted to confirm or rebut the hypothesis that early administration of probiotic bacteria to newborns and pregnant women before birth could have preventive effects on the development of allergy. In the Czech Republic, the probiotic strain Escherichia coli O83:K24:H31 (EC O83), being registered with the State Health Institute for Drug Control under the name Colinfant Newborn, has long been used to prevent allergies and paediatri- cians have long been known and used it against various diarrhoea. The aim of this work was to elucidate the effect of EC O83 on CBMC (cord blood mononuclear cells) and to compare the ability of CBMC of healthy mothers (children with a relatively low risk of developing allergic disease) and allergic mothers (children at high risk of developing allergies) to form cytokines in response to EC O83 stimulation. Phytohemagglutinin was used as a positive control, Escherichia coli Nissle 1917 was used as a reference probiotic strain, which is much more known abroad than EC O83. Cytokine production was detected by...

(EN) Reduced microbial stimulation of an immature neonatal immune system can lead to a poor balance adjustment of immune responses, thus contributing to the development of allergic diseases, whose incidence continues to rise. One of the promising precautionary measures seems to be an early preventive administration of probiotic bacteria to pregnant or nursing mothers, or to newborns. Previous works have described a beneficial effect of Escherichia coli O83:K24:H31 (E. coli O83) in the prevention of allergic diseases. In order to contribute to the clarification of E. coli O83 effects on the neonatal immune system, its immune- modulating properties were tested in vitro on umbilical cord blood cells. The ability of E. coli O83 to support the maturation of in vitro-derived dendritic cells from cord blood precursors (moDCs) of the children of healthy (children with a relatively low risk of allergy) and allergic (children at a relatively high risk of developing allergies) mothers was tracked by flow cytometry, qPCR and ELISA. Probiotic bacteria-stimulated moDCs were subsequently cultured with autologous naive CD4+ T lymphocytes and immune response polarization was also characterised by flow cytometry, qPCR, and ELISA. It was evident from the results that E. coli O83 promoted moDCs maturation. The presence of...

The human leukocyte antigen (HLA) is the most polymorphic region in the human genome and accounts for more than 10% of human diversity. This region plays an important role in matching donors and recipients for transplantation. The South African Bone Marrow Registry (SABMR) does not reflect the demographics of the South African population. The large number of polymorphisms resulting from HLA diversity in the Black South African population and their limited representation in the SABMR reduce the chances of finding adequate matches between donors and recipients in this group. Umbilical cord blood is an alternative to bone marrow for the treatment of fatal diseases. Less strict HLA matching is required due to the naive nature of the T cells in cord blood. A public umbilical cord blood bank is a necessity in trying to cater for the diverse population in South Africa. However, the ethnic diversity of the South African population poses a great challenge in constituting a public umbilical cord blood bank that is representative of the entire population. The Roche designed next generation sequencing (NGS) high resolution (HR) HLA typing kit enables sequencing of additional HLA exons and could improve the degree of matching between individuals to ultimately decrease adverse reactions. An extensive study of the literature was performed to establish the demographics, linguistics, and HLA diversity of the South African population to determine how a public cord blood bank should be constituted. In addition, HLA genotyping was performed by 454 NGS on 20 samples that had previously been HLA typed by conventional methods. The 454 NGS technique made use of a Roche designed medium and high resolution HLA typing kit to genotype the samples. It was possible to assign accurate genotypes to 95.5% of the loci of interest for the total number of 20 samples using the MR kit, compared with 98.5% using the HR kit. In conclusion, the present study indicates the extreme HLA diversity in the South African population, and therefore, recommends constituting the first public umbilical cord blood bank in Gauteng on the basis of race or major ethnic groupings. A minimum number of 10 000 cord blood units is needed to initiate the bank. Furthermore, the 454 NGS platform together with the HR HLA typing kit display potential as an alternative method to be used in a public cord blood bank, as well as routine clinical and diagnostic laboratories, to ultimately improve HLA matching between donors and recipients. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Immunology / unrestricted

Multipotente mesenchymale Stromazellen (MSCs) stellen nicht nur beim humanen Patienten, sondern auch in der Veterinärmedizin einen vielversprechenden Therapieansatz in der Behandlung erkrankter muskuloskelettaler Gewebe dar. Ziel der Behandlung ist dabei die Regeneration der betroffenen Strukturen im Vergleich zur Reparation nach konservativer Therapie. Vor allem im Bereich von Sehnenerkrankungen können nach MSC-Applikation vielversprechende Ergebnisse im Hinblick auf niedrigere Rezidivraten beobachtet werden. Dennoch sind noch nicht alle Umstände einer optimalen MSC-Anwendung geklärt. Hierbei sind unter anderem Fragen bezüglich der Herkunft und Gewinnung von MSCs offen, da Unterschiede von MSCs aufgrund ihrer Gewebezugehörigkeit bereits nachgewiesen wurden. Grundlegende umfassende Arbeiten zum Vergleich von equinen MSCs aus verschiedenen Quellen sowie deren mögliche Beeinflussung durch die Isolierung aus dem Gewebe lagen bislang noch nicht vor. Ziel dieser Studie war es daher, equine MSCs aus verschiedenen Quellen zu gewinnen und mögliche Unterschiede in vitro aufzuzeigen. Weiterhin sollten Unterschiede zwischen den Zelleigenschaften nach Anwendung verschiedener Isolationsprotokolle untersucht werden. In der hier vorliegenden Studie wurden MSCs aus Fett- und Sehnengewebe, Knochenmark, Nabelschnurblut und Nabelschnurgewebe von Pferden isoliert und vergleichend charakterisiert. Dabei wurden für die soliden Körpergewebe zwei unterschiedliche Isolationsmethoden, die Digestion und die Explantation, angewendet, um mögliche Einflüsse auf die gewonnen Zellen zu ermitteln. Die untersuchten Kriterien beinhalteten Zellertrag, Proliferation, Differenzierungspotenz und das Migrationsverhalten von MSCs. Hinblickend auf eine Anwendung von MSCs bei Sehnenerkrankungen wurde auch die Expression von Sehnenmarkern verglichen. In der vorliegenden Studie konnte gezeigt werden, dass sich die MSCs aus verschiedenen Quellen hinsichtlich der Zellausbeute und ihres Wachstumspotentials unterschieden. Aus soliden Geweben konnten mittels Digestion im Vergleich zu Körperflüssigkeiten signifikant mehr MSCs isoliert werden (p < 0,001). Dabei erbrachte die Isolation von MSCs mittels Digestionsmethode einen deutlich höheren Zellertrag nach der Passage 0 im Vergleich zur Explantationsmethode (p < 0,05). Im weiteren Verlauf der Kultivierung zeigten MSCs aus Sehnengewebe und Fettgewebe ein signifikant besseres Proliferationsverhalten im Vergleich zu Knochenmark-MSCs und Nabelschnurblut-MSCs. Im Hinblick auf das Differenzierungspotential konnten signifikante Unterschiede zwischen den MSCs aus den verschiedenen Quellen beobachtet werden. MSCs aus Knochenmark zeigten eine sehr gute osteogene Differenzierungsfähigkeit im Vergleich zu MSCs aus den geburtsassoziierten Geweben (p < 0,05). Im Gegensatz dazu zeichneten sich diese MSCs durch eine deutlich bessere chondrogene Differenzierung im Vergleich zu Knochenmark-MSCs aus (p < 0,05). Im Hinblick auf die Isolationsmethode konnten keine Unterschiede im Differenzierungspotential beobachtet werden. Weitere Unterschiede aufgrund der Zellquelle lassen sich in der Genexpression der Sehnenmarker erkennen. MSCs aus Fettgewebe und Sehnengewebe exprimierten Kollagen 1A2 auf höchstem Niveau. Sklexaris hingegen wurde von MSCs aus Nabelschnurblut und Sehnengewebe am höchstem exprimiert. Dabei zeigten MSCs, die mittels Digestionsmethode isoliert worden waren, ein signifikant höheres Expressionslevel von Skleraxis im Vergleich zur Explantationsmethode (p < 0,05). Die Ergebnisse der vorliegenden Studie lassen einen Einfluss der Zellquelle auf die Zellcharakteristika erkennen. MSCs aus Fettgewebe stellen dabei eine vielversprechende Alternative zu Knochenmark-MSCs dar. Allerdings scheint für eine klinische Anwendung von MSCs eine selektive Auswahl der Zellquelle entsprechend der vorliegenden Erkrankung von Vorteil zu sein. Dabei ist eine Isolierung von MSCs aus soliden Geweben mittels Digestionsverfahren zu empfehlen, da hier deutlich höhere Zellzahlen gewonnen werden können. Eine negative Beeinflussung der Zelleigenschaften durch die enzymatische Digestion lässt sich nach den vorliegenden Ergebnissen nicht vermuten. Inwiefern die beobachteten Unterschiede bei in-vivo-Anwendungen von Bedeutung sind, muss jedoch noch umfassend untersucht werden. / Not only in humans but also in veterinary medicine, multipotent mesenchymal stromal cells (MSCs) are a promising treatment option in the therapy of injured musculoskeletal tissues. This is due to the improved tissue regeneration instead of the insufficient reparation following conventional therapies. With regard to an application of MSCs for treatment of tendinopathies in horses, lower rates of reinjury have been reported. However, further investigations to optimize the MSC treatment are still outstanding. Differences in MSCs from different origins have been already reported, but there are still remaining questions about the influence of origin and isolation procedures of MSCs. Fundamental research on equine MSCs derived from different sources and their potential impact due to the isolation process has not been published so far. The aim of this study was to isolate equine MSCs from different sources and to demonstrate potential differences in vitro. Furthermore, differences in cell features following different isolation methods were investigated. In the present study, MSCs from horses were isolated from adipose tissue, tendon tissue, bone marrow, umbilical cord blood and umbilical cord tissue and subsequently subjected to comparative characterization. In case of the solid tissues, two different isolation methods, digestion and explantation, were performed in order to analyze influences on obtained cells. Investigated cell features included cell yield, proliferation, differentiation and migration potential. Furthermore, expression of tendon markers was evaluated with regard to an application of MSCs in tendinopathies. In the present study it was shown that MSCs derived from different sources differ distinctly in cell yield and proliferation potential. In comparison to body fluids, significantly more MSCs could be isolated from solid tissues when using the digestion method (p < 0.001). Furthermore, the cell yield at first cell harvest was distinctly higher when performing the isolation by digestion in comparison to isolation by explantation (p < 0.05). With regard to further cultivation, MSCs derived from tendon tissue and adipose tissue displayed a significantly better proliferation potential compared to MSCs derived from other sources. Considering the differentiation potential, significant differences were obvious between the MSCs derived from different sources. Bone marrow-MSCs showed an excellent osteogenic differentiation capacity in comparison to MSCs derived from umbilical cord blood and tissue (p < 0.05). In contrast, the birth-associated MSCs displayed a distinctly better chondrogenic differentiation than MSCs derived from bone marrow (p < 0.05). No difference in the differentiation potential was noticeable following the different isolation procedures. Furthermore, differences in the gene expression of tendon markers were evident with regard to the cell source. MSCs derived from adipose tissue and tendon tissue expressed collagen 1A2 on the highest level. On the other hand, scleraxis was expressed highest in MSCs derived from umbilical cord blood and tendon tissue. In these cells, MSCs isolated by the digestion method showed a significantly higher expression level of scleraxis in comparison to MSCs isolated by explantation (p < 0.05). Based on the results obtained so far, a relevant impact of the source of MSCs on cell features was evident. MSCs derived from adipose tissue are a promising alternative to bone marrow-MSCs. However, with regard to a clinical application of MSCs, a selection of the MSC source depending on the respective intended use seems to be advantageous. For routine isolation of MSCs from solid tissues, the digestion method could be recommended due to the higher obtainable cell numbers. Furthermore, a negative influence of the enzymatic digestion on the cell features was not detectable. However, to what extent the observed differences in vitro are relevant for in-vivo-applications needs to be further investigated.

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.

La transplantation de sang de cordon ombilical (TSCO) constitue un traitement de choix pour une multitude de pathologies hématologiques malignes et non malignes chez l’enfant et dans certains cas l’adulte. La TSCO est associée à certaines complications, dont une reconstitution immunitaire plus lente et une incidence élevée d’infections opportunistes, notamment celles reliées au cytomégalovirus (CMV) et au virus varicella-zoster (VZV). Dans le cadre de ce travail, nous nous sommes intéressés dans un premier temps à la caractérisation de la reconstitution immunitaire spécifique au CMV et au VZV. Nos résultats ont démontré que la reconstitution de l’immunité cellulaire ne requiert ni un statut séropositif pré-transplantation ni le développement de la maladie. De plus, des reconstitutions spontanées ont été détectées chez certains patients séronégatifs vis-à-vis du CMV ou du VZV. Outre le fait qu’elle se manifeste surtout à partir de 6 mois post-transplantation, ladite reconstitution mérite le qualificatif de « protectrice » en termes de réactivations virales et du développement de signes cliniques lorsqu’une fréquence de 150 cellules produisant l’IFN-γ/million est dépassée. Toutefois, moins de 5% des patients développent une réponse T anti-VZV et anti-CMV au cours 100 premiers jours suivant la TSCO. Il est donc possible que les lymphocytes CD8+ T provenant du SCO, comparativement à leurs homologues provenant de la moelle osseuse (MO), présentent un défaut de fonctionnalité, communément appelé « épuisement clonal ». La caractérisation du répertoire de récepteurs inhibiteurs exprimés par les cellules T CD8+ suivant la TSCO ou la transplantation de moelle osseuse (TMO) a révélé une augmentation significative de la fréquence des cellules exprimant PD-1 tôt suivant la transplantation. Cette population, caractérisée majoritairement par un phénotype effecteur-mémoire (EM), démontre une perte significative de la capacité proliférative et exprime moins d'IFN-γ, d'IL-2, de TNF-α et de CD107a. Une meilleure caractérisation de la reconstitution immunitaire après TSCO permettrait, d'une part de sélectionner des biomarqueurs en vue d’une meilleure gestion des patients à risques de développer des infections virales et/ou de rechuter, et d'autre part d'améliorer leur pronostic. / Umbilical cord blood transplantation (UCBT) is a treatment of choice for a variety of hematological malignancies and non-malignant diseases in children and, in some cases, in adults. UCBT is associated with a slower immune reconstitution and a high incidence of viral infections, especially related to cytomegalovirus (CMV) and the varicella-zoster virus (VZV). As part of this work, we aimed to assess the reconstitution of CMV and VZV-specific T cell responses. Neither pre-transplant serostatus nor disease development is required for development of T cell mediated immunity. Moreover, spontaneous reconstitution detected in some patients who were seronegative for CMV or VZV. Detected especially after 6 months post-transplant, antiviral responses are protective in terms of viral reactivation and development of clinical signs, when a frequency of 150 cells producing d'IFN-γ / million is achieved. However, less than 5% of patients develop an antiviral response during the first 100 following UCBT. Compared to their bone marrow (BM) counterparts, UCB CD8+ T lymphocytes may be functionally impaired, a state commonly called « clonal exhaustion ». Characterization of the inhibitory receptors repertoire expressed by CD8+ T cells following UCBT and BMT showed a significant increase in the frequency of cells expressing PD-1 early after transplantation. This population, mainly characterized by effector phenotype, showed a significant loss of proliferative capacity and produced less IFN-γ, IL-2, TNF-α and CD107a. An improved understanding of the CD8+ T cell compartment following UCBT, as well as biomarkers related to T cell exhaustion will decrease infection, transplant related mortality and correlate with better prognosis