O Magnésio é o segundo cátion intracelular mais comum e desempenha importante papel na modulação de funções de transporte e receptores, atividades enzimáticas, metabolismo energético, síntese de proteínas e ácidos nucleicos e proteção de membranas biológicas. Apesar de sua importância, o conhecimento de sua homeostase não é completo, principalmente por dificuldade de acesso a seus estoques intracelulares e da ausência de métodos laboratoriais confiáveis para medida da fração iônica. O desenvolvimento recente de um eletrodo íon-seletivo permitiu a determinação das concentrações de Mg iônico(Mgi), em pequenas amostras de sangue, o que possibilitou a realização de estudos para determinação desta fração no período neonatal. A presença de alguns distúrbios, como o Crescimento Intra-uterino Restrito(CIUR) e a Asfixia Perinatal, poderiam potencialmente levar a desvios da homeostase do Mg, ainda não totalmente esclarecidos. O objetivo deste estudo foi descrever, em Recém-nascidos de termo(RNT) sem CIUR, os níveis de Mgi e total (MgT) em sangue de cordão umbilical, 3o e 7o dias de vida e comparar os valores obtidos entre os RNT, com e sem CIUR e asfixia perinatal. Realizou-se um estudo prospectivo, no qual foram incluídos 95 RNT, divididos em dois grupos de estudo: Grupo I - sem CIUR(50 RN - 52,6%) e Grupo II - com CIUR(45 RN - 47,4%). A presença de CIUR foi determinada por um peso de nascimento abaixo do percentil 10 para a curva de Ramos(1983), associado a uma relação P/P50 < 0,85. Cada um desses grupos foi subdividido em 2 subgrupos : Grupo Ia - 30 RN (31,6%), sem CIUR e sem asfixia perinatal; Grupo Ib - 20 RN(21,0%), sem CIUR e com asfixia perinatal; Grupo IIa - 40 RN(42,1%), com CIUR e sem asfixia perinatal; Grupo IIb - 5 RN(5,3%), com CIUR e asfixia perinatal. A presença de asfixia perinatal foi indicada por um Apgar de 5o minuto < 6 associada a presença de um dos seguintes critérios: pH de sangue de cordão umbilical < 7,2 , disfunção de um ou mais órgãos, sequelas neurológicas no período neonatal imediato. Foram realizadas determinações de Mgi, Cálcio iônico(Cai), Uréia(U), pH, MgT, Fósforo(P) e Creatinina(Cr), em sangue de cordão umbilical, no 3o e no 7o dias de vida. Verificou-se que nos RNT sem CIUR(Grupo Ia), as concentrações médias de MgT, ao nascimento, foram menores do que as de RN com CIUR e elevaram-se, de forma significante, até o 7o dia de vida, enquanto as de Mgi mantiveram-se. As concentrações de Mgi neste grupo, foram significativamente menores do que as de RN com CIUR(Grupo IIa) durante a 1a semana de vida e do que as de RN com asfixia perinatal(Grupo Ib) no 3o e 7o dias de vida. Concluiu-se que, em RNT sem CIUR, há um aumento dos níveis de MgT durante a 1a semana de vida, sem alteração das concentrações de Mgi. A presença de CIUR, bem como a asfixia perinatal, podem influenciar as concentrações neonatais de Mg, através de seus efeitos de modulação da homeostase deste íon, durante os períodos fetal e neonatal / Magnesium is the second most abundant intracellular cation and plays an important role in regulation of transporting and receptors functions, enzymatic activities, energy metabolism, protein and nucleic acid synthesis and biologic membranes protection. In spite of this, the knowledge of its homeostasis is still limited, mainly due to inacessibility of its intracellular stores and the absence of a reliable methodology to measuring the ionized fraction. The recent development of an ion-selective electrode has allowed not only the determination of ionized magnesium(iMg) concentrations in a small blood sample volume, but also an increasing number of researches as to this fraction in neonatal period. The presence of some disorders,i.e. like Intrauterine Growth Restriction (IUGR) and Perinatal Asphyxia, could lead to an unclear imbalance of magnesium homeostasis, in a way not yet clear. The aim of this study was to describe, in term newborns without IUGR, iMg and Total Mg (TMg) concentrations in umbilical cord blood, third and seventh days of life and to compare the results among term newborns with and without IUGR and perinatal asphyxia. Ninety-five term newborn infants were enrolled in a prospective study and were divided into two study groups: Group I : without IUGR(50RN - 52.6%) and Group II - with IUGR(45RN - 47.4%). Intrauterine growth restriction was defined as a birth weight below the 10th percentil for Ramos Curve(1983) besides to a birth weight ratio <0,85. Each one of these groups were divided in two subgroups: Group Ia :30 RN (31,6%), without IUGR or perinatal asphyxia; Group Ib : 20 RN (21,0%), without IUGR, with perinatal asphyxia ; Group IIa : 40 RN (42,1%), with IUGR, without perinatal asphyxia; Group IIb: 5 RN(5,3%), with perinatal asphyxia and IUGR. Perinatal asphyxia was defined as a 5 minutes Apgar score < 6 besides to one of the following: umbilical cord blood pH < 7,2, disfunction of one or more organs, neonatal neurologic manifestations. iMg, TMg, ionized calcium, urea, pH, phosphorus and creatinine concentrations were determined in umbilical cord blood, third and seventh days of life. We observed that in term newborns without IUGR (Group Ia), TMg concentrations increased significantly during the first week of life, while iMg concentrations remained unchanged. iMg levels in this group, were significantly lower than in the group with IUGR (Group IIa) from birth to 7th day of life and than in the group without IUGR, with perinatal asphyxia (Group Ib) in the third and seventh days of life. We concluded that in term newborns without IUGR, TMg levels increased during the first week of life, while iMg levels remained unchanged. The presence of IUGR, as well as, perinatal asphyxia, may influence neonatal levels of magnesium, through their effect on the modulation of this ion homeostasis, during fetal and neonatal periods

INTRODUÇÃO: Células-tronco mesenquimais da geleia de Wharton (GW-CTM) exibem a capacidade de modular a resposta das células T e geralmente esses efeitos imunomoduladores são anti-inflamatórios. Devido ao seu potencial imunossupressor, as CTM emergiram recentemente como uma ferramenta promissora para terapia celular. No entanto, essas células têm uma vida útil limitada in vitro, com redução progressiva da sua capacidade de autorrenovação e parada irreversível do ciclo celular. O resultado deste processo é a perda da funcionalidade das CTM, o que limita a sua utilização para fins terapêuticos. Pouco se sabe sobre a variabilidade individual das amostras de GW-CTM e como isso poderia afetar sua expansão in vitro, seu potencial imunomodulador e o processo de envelhecimento. Neste estudo, avaliamos o perfil de citocinas imunomoduladoras e a capacidade das GW-CTM inibir a proliferação de células T durante o processo de senescência replicativa para determinar se a resposta esperada é afetada. MÉTODOS: GW-CTM foram cultivadas até a senescência replicativa ser atingida; as amostras foram coletadas numa fase inicial (passagem 5), numa etapa intermediária (passagem 15) e na senescência replicativa (passagem geralmente entre 20 e 25) para análise do perfil basal de moléculas imunomoduladoras. GW-CTM foram cocultivadas com amostras de duas células mononucleares de sangue periférico (PBMC), obtidas de doadores de plaquetas saudáveis. PBMC foi estimulado com fitohemaglutinina (PHA) durante 72 horas e cocultivado com três amostras de GW-CTM diferentes para medir a supressão da proliferação das células T. Os experimentos foram realizados utilizando as passagens P5 e P10. As análises foram feitas por PCR em tempo real, western blot e citometria de fluxo. RESULTADOS: Nossos resultados mostram que a expressão gênica e a secreção de moléculas imunomoduladoras variam entre amostras de GW-CTM, sem padrão específico. Em cocultura, todas as GW-CTM foram capazes de inibir a proliferação de células T CD3+ ativadas por mitogéno, embora em diferentes graus. E cada PBMC respondeu à cocultura com um nível diferente de inibição. Além disso, o perfil imunomodulador de todas as GW-CTM foi essencialmente mantido, mesmo após várias passagens. CONCLUSÃO: Nossos dados indicam que cada GW-CTM exibe um comportamento único, diferindo nos padrões de expressão e secreção de citocinas e capacidade imunomoduladora. Essa variabilidade intrínseca entre amostras pode influenciar a eficácia de GW-CTM quando utilizadas em terapia celular / INTRODUCTION: Wharton\'s jelly mesenchymal stem cells (WJ-MSC) exhibit the ability to modulate T cell responses and these immunomodulatory effects are usually anti-inflammatory. Due to their immunosuppressive potential, MSC have recently emerged as a promising tool for cell therapy. However, MSC have a limited lifespan in vitro, with a progressive reduction in their capacity for selfrenewal leading to irreversible arrest of cell division. The result of this process is the loss of stem cell functionality, which limits its use for therapeutic purposes. Information on the variability of individual cell samples impacting upon in vitro expansion, immunomodulatory potential, and aging processes is still lacking. In this study, we evaluated the immunomodulatory cytokine profile and capacity to inhibit T cell proliferation of WJ-MSC progressing to replicative senescence to determine if the expected response is affected. METHODS: WJ-MSC were cultured until replicative senescence was reached and the samples were collected at an early stage (passage 5), at an intermediate stage (passage 15), and in replicative senescence (passage generally between 20 and 25) to analyze the basal profile of immunomodulatory molecules. WJ-MSC were co-cultured with samples from the same two peripheral blood mononuclear cells (PBMC), obtained from healthy platelet donors. PBMC were stimulated with phytohemagglutinin (PHA) for 72 hours and tested against 3 different WJ-MSC to measure suppression of T cell proliferation. The experiments were performed using passages P5 and P10. Analyses were done by real-time PCR, western blot, and flow cytometry. RESULTS: Our results show that gene expression and secretion of immunomodulatory molecules varied among WJ-MSC samples with no specific pattern discernible. In co-culture all WJ-MSC were capable of inhibiting mitogen-activated CD3+ T cell proliferation, although to different extents and each PBMC responded with its unique level of inhibition. In addition, the immunomodulatory profile of each WJ-MSC sample was essentially maintained even after several passages. CONCLUSION: Our results indicate that each WJMSC displays a unique behavior, differing in patterns of cytokine mRNA expression and immunomodulatory capacity. The intrinsic variability between samples may influence the effectiveness of WJ-MSC when employed therapeutically

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.

Η σύγχρονη παραγωγή φαρμακευτικών και άλλων χημικών ουσιών και ο αναγκαίος τοξικολογικός τους έλεγχος επιφέρει την χρήση ενός μεγάλου αριθμού πειραματοζώων με αποτέλεσμα την αύξηση του κόστους αλλά και την έγερση ζητημάτων που σχετίζονται με την ασφάλεια και την βιοηθική. Στην βάση αυτού του προβληματισμού η ανάπτυξη νέων in vitro δοκιμασιών κυτταρο-τοξικότητας με δυνατότητα ακριβέστερης πρόβλεψης των αρχικών δόσεων οξείας από του στόματος τοξικότητας, προβάλλει ως αναγκαιότητα στις μέρες. Μέχρι τώρα έχουν χρησιμοποιηθεί σε in vitro δοκιμασίες μετασχηματισμένα, αθανατοποιημένα ή πρωτογενή κύτταρα καθώς και εμβρυικά βλαστικά κύτταρα (hESCs). Επίσης πρόσφατα χρησιμοποιήθηκαν μεσεγχυματικά βλαστικά κύτταρα από τον μυελό των οστών (BM-hMSCs). Τα κύτταρα αυτά ωστόσο παρουσιάζουν μειονεκτήματα που σχετίζονται με την δυσκολία απομόνωσης τους, την ετερογένεια τους, αλλά και τον πρόωρο φαινότυπο γήρανσης κατά την καλλιέργεια τους. Σε αυτή την μελέτη διερευνάται για πρώτη φορά η χρήση των βλαστικών κυττάρων της γέλης του Wharton (WJSCs) του ομφαλίου λώρου σε in vitro δοκιμασία κυτταροτοξικότητας. Τα κύτταρα αυτά παρουσιάζουν σημαντικά πλεονεκτήματα σε σχέση με άλλα μεσενχυματικά κύτταρα καθώς: απομονώνονται εύκολα, καλλιεργούνται εκτεταμένα με διατήρηση των βλαστικών τους ιδιοτήτων, δεν εγείρουν ηθικά ζητήματα για τη χρήσή τους, παρουσιάζουν γενετική και φαινοτυπική σταθερότητα και ήπιο ανοσολογικό προφίλ. Στην παρούσα μελέτη εξετάστηκαν παράλληλα και συγκριτικά με τα κύτταρα της γέλης του Wharton, οι κυτταρικές σειρές HepG2 (ηπατικού καρκινώματος) και NIH 3T3 (ινοβλάστες ποντικού) αλλά και τα μεσεγχυματικά κύτταρα λίπους AD-hSCs. Όπως προτείνεται από το πρωτόκολλο της ICCVAM δοκιμάστηκαν 12 ουσίες αναφοράς ενώ η μέτρηση της επιβίωσης των κυττάρων έγινε με την δοκιμασία MTS και NRU. Τα αποτελέσματα μας δείχνουν ότι τα κύτταρα της γέλης του Wharton μπορούν να αποτελέσουν αξιόπιστο και ελπιδοφόρο μοντέλο για δοκιμασίες in vitro τοξικότητας. Το μοντέλο αυτό μπορεί να λειτουργήσει συμπληρωματικά, ή ακόμα και να ξεπεράσει, ήδη επικυρωμένα μοντέλα κυτταροτοξικότητας . / The modern production of pharmaceuticals, other chemicals and their required toxicological controls results in the use of a large number of laboratory animals leading in increased costs as well as raising questions considering safety and bioethics. Alternatively, in vitro cytotoxicity assays are highlighted with the ability of a more accurate prediction of the starting dose of oral acute toxicity. Occasionally several cell lines have been used including transformed and immortalized cells or primary cells and embryonic stem cells (hESCs). For the same purpose adult mesenchymal stem cells derived from the bone marrow (BM-hMSCs) have been recently used but they exhibit difficulties in their isolation, heterogeneity, and premature senescence phenotype during their sub-cultivation. In this study for the first time we investigated the use of mesenchymal stem cells (WJSCs) isolated from fetal umbilical cord, in particular from the Wharton’s Jelly. These cells exhibit the advantage of easily being isolated and cultured in large quantities without ethical issues, genetic and phenotypic stability and subimmunological profile. Two different cell lines HepG2 (liver carcinoma) and NIH 3T3 (mouse fibroblasts) and mesenchymal adipose-derived stem cells AD-hSCs have been used and compared with the WJSCs in parallel. 12 substances have been tested for their cytotoxicity effect on cell survival using the MTS assay as suggested by ICCVAM. Our results indicate that this model is a reliable and promising approach for in vitro cytotoxicity tests on human cells and it can complement or even overpass validated cytotoxicity models.

Orientador: Rogerio Martins Amorim / Resumo: As células-tronco mesenquimais (CTM) apresentam propriedades antiinflamatórias, imunomoduladoras, neuroprotetoras e neurorregenerativas. Pela capacidade regenerativa limitada do tecido neural, o potencial terapêutico das células-tronco tem sido estudado em várias enfermidades do sistema nervoso central (SNC). Sendo assim, o objetivo deste trabalho foi avaliar a viabilidade, segurança e a resposta a transplantes intratecais sucessivos, de CTM provenientes de diversas fontes em equinos. Para isso, foram avaliados o potencial de transdiferenciação, terapêutico e o co-cultivo com líquido cefalorraquidiano (LCR) de CTM provenientes da medula óssea, tecido adiposo e cordão umbilical. Posteriormente, as CTM foram utilizadas para o transplante por via intratecal (IT) pela cisterna lombo-sacra (LS), atlanto-occipital (AO) e entre as vértebras C1-C2 em animais sadios (GS), totalizando 3 aplicações com intervalo de 30 dias. Adicionalmente as CTM foram rastreadas por cintigrafia após os transplantes pelas via AO e LS. Finalmente, as CTM foram utilizadas para o transplante IT entre as vértebras C1-C2 em animais com incoordenação motora decorrente de Mieloencefalite Equina por Protozoário (MEP) – grupo doente (GD), sob o mesmo protocolo. Todos os animais (GS e GD) foram avaliados por exame clínico e neurológico, análise hematológica e do LCR antes e após os transplantes. Os resultados demonstraram que as CTM provenientes de diversas fontes apresentaram efeitos positivos na presença de LCR,... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Objetivo: Determinar a influência da presença de cafeína no sangue de cordão umbilical na ocorrência de apneia. Métodos: Estudo de coorte prospectivo de recém-nascidos pretermos com peso de nascimento menor de 2.000 g. Os critérios de exclusão foram: mães que receberam opióides , ventilação mecânica durante os primeiros 4 dias de vida, malformação congênita cerebral e cardíaca maiores, asfixia perinatal, hemorragia peri-intraventricular severa, exsanguíneotransfusão antes do quarto dia de vida e uso de metilxantina antes da extubação. Os recém-nascidos foram divididos em: com e sem cafeína detectável no sangue de cordão umbilical e acompanhados nos primeiros quatro dias de vida para a ocorrência de apneia. Resultados: 87 com e 40 sem cafeína detectável no sangue de cordão umbilical foram estudados. A mediana da concentração de cafeína dos 87 pacientes com cafeína detectável no sangue de cordão umbilical foi 2,3 µg/ml (0,2-9,4 µg/ml). Não houve associação entre a ocorrência de apneia e a presença de cafeína no sangue de cordão umbilical. Recém-nascidos com cafeína detectável no cordão umbilical tiveram apnéia mais tarde (66.3 horas) do que aqueles com níveis indetectáveis (54.2 horas). Conclusão: a detecção de níveis de cafeína no sangue de cordão umbilical não diminuiu a ocorrência de apneia da prematuridade. Nós sugerimos que novos estudos com a administração de altas doses de cafeína para mães antes do parto prematuro, como estratégia para prevenir a apneia da prematuridade, devam ser realizados. / Objective: To determine the influence of presence of caffeine in umbilical cord blood on apnea occurrence. Methods: A prospective cohort study with preterm newborns with birth weight less than 2,000 g was undertaken. Exclusion criteria were: mothers that received opioids, mechanical ventilation during the first 4 days of life, cerebral and major cardiac malformations, perinatal asphyxia, severe periintraventricular hemorrhage, exchange transfusion before the fourth day of life, and those that received methylxantine prior to extubation. Neonates were divided in: with detectable and undetectable caffeine in umbilical cord blood. Newborns were followed for the first 4 days for occurrence of apnea spells. Results: 87 with and 40 without detectable caffeine in umbilical cord blood were studied. The median caffeine concentration of the 87 patients with detectable caffeine in umbilical blood was 2.3 µg/ml (0.2-9.4 µg/ml). There was no association between occurrence of apnea spells and presence of caffeine in umbilical cord blood. Neonates with detectable caffeine in umbilical blood had apnea later (66.3 ± 4.14 hours) than those with undetectable levels (54.2 ± 6.26 hours). Conclusion: The detected levels of caffeine in umbilical cord blood did not decrease the occurrence of apnea of prematurity. We suggest that further studies on administration of high dose of caffeine to mothers prior to a preterm delivery as a preventive measure for apnea of prematurity deserve to be conducted.

Introdução: A célula NK possui uma importante função no sistema imune inato de defesa primária contra vírus e patógenos e também realiza a imunovigilâcia tumoral. Muitos estudos clínicos tem avaliado o uso dessas células na imunoterapia adotiva. A expansão e a ativação da célula NK requer sinais e estímulos para manter a sua sobrevivência. Atualmente existem muitos protocolos para a expansão e ativação da célula NK, porém não existe uma definição do melhor método para uso clínico. Objetivo: O estudo tem como objetivo avaliar a melhor forma para expansão das células NK isoladas de células mononucleares do sangue de cordão umbilical e placentário.Método: Foram avaliadas cinco diferentes condições para expansão de células NK de mononucleares isoladas do sangue do cordão umbilical e placentário. Foram testados protocolos utilizando as interleucinas (IL), IL-2, IL-3, IL-15; com ou sem a presença do co-cultivo com células-tronco mesenquimais do cordão umbilical (CTM-CU) e, também o co-cultivo com células apresentadoras de antígeno artificiais ligadas a IL-21 à membrana (mbIL21 APC). Resultados: Os protocolos utilizando co-cultivo com APC mbIL21 foram superiores aos demais quanto à capacidade de expansão de células NK (CD3-, CD56+, CD16+). O protocolo de co-cultura de APC, CTM-CU e estímulo com IL-2 apresentaram um aumento significativo de NK (CD3-, CD56+, CD16+) quando comparado ao protocolo de APC/IL-2 sem CTM-CU (p<0,05). Conclusão: A expansão ex vivo de células NK na presença das APC e CTM-CU apresentaram uma proporção estatisticamente superior de célula NK CD16+ quando comparada com condições de cultivo com apenas a APC, tendo essas células NK potencial para utilização na imunoterapia adotiva associada com anticorpos monoclonais ou anticorpos bi-específicos. / Background: Natural killer (NK) cells play a major role in innate immunity, especially against viral pathogens, and are also a part of the immune surveillance of tumors. Several clinical trials have evaluated the use of these cells for adoptive cell immunotherapy. Ex vivo expansion of NK cells, however, is a complex process which requires multiple cell signals to ensure cell survival, proliferation, and activation. There are many protocols used for NK cell expansion and activation, however, there is a lack of evidence regarding which method is the most effective for clinical grade NK cells expansion. Objective: The main purpose of this study is to evaluate an optimal protocol for the ex vivo expansion of NK cells isolated from umbilical cord blood mononuclear cells (CB-MNC). Methods: Five different conditions for the expansion of umbilical cord-derived NK cells were evaluated. Each protocol was a different combination of interleukins (IL-2, IL-3, and IL-15) with or without the presence of feeder cells or artificial antigen presenting cells (aAPCs). Feeder cells utilized were umbilical cord-derived mesenchymal stem cells (UC-MSC), and aAPCs were membrane-bound IL-21 artificial APCs (mbIL21 aAPCs). Results: Protocols employing mbIL21 aAPCs demonstrated greater expansion of natural killer cells (CD3- CD56+) than the other protocols. The protocol employing aAPCs, IL-2 and UC-MSC feeder cells had a statistically significant higher proportion of CD16+ NK cells when compared to the protocol without the MSC feeder cells, but there was no significant difference in the expansion of total natural killer cells concerning these two protocols. Conclusion: Ex vivo expansion of NK cells in the presence of aAPCs and UC-MSC feeder cells yielded a significant higher proportion of CD16+ NK when compared to the aAPCs only culture condition, and could be a better product for NK adoptive immunotherapy in conjunction with monoclonal or bi-specific antibodies.

Objetivo: Determinar a influência da presença de cafeína no sangue de cordão umbilical na ocorrência de apneia. Métodos: Estudo de coorte prospectivo de recém-nascidos pretermos com peso de nascimento menor de 2.000 g. Os critérios de exclusão foram: mães que receberam opióides , ventilação mecânica durante os primeiros 4 dias de vida, malformação congênita cerebral e cardíaca maiores, asfixia perinatal, hemorragia peri-intraventricular severa, exsanguíneotransfusão antes do quarto dia de vida e uso de metilxantina antes da extubação. Os recém-nascidos foram divididos em: com e sem cafeína detectável no sangue de cordão umbilical e acompanhados nos primeiros quatro dias de vida para a ocorrência de apneia. Resultados: 87 com e 40 sem cafeína detectável no sangue de cordão umbilical foram estudados. A mediana da concentração de cafeína dos 87 pacientes com cafeína detectável no sangue de cordão umbilical foi 2,3 µg/ml (0,2-9,4 µg/ml). Não houve associação entre a ocorrência de apneia e a presença de cafeína no sangue de cordão umbilical. Recém-nascidos com cafeína detectável no cordão umbilical tiveram apnéia mais tarde (66.3 horas) do que aqueles com níveis indetectáveis (54.2 horas). Conclusão: a detecção de níveis de cafeína no sangue de cordão umbilical não diminuiu a ocorrência de apneia da prematuridade. Nós sugerimos que novos estudos com a administração de altas doses de cafeína para mães antes do parto prematuro, como estratégia para prevenir a apneia da prematuridade, devam ser realizados. / Objective: To determine the influence of presence of caffeine in umbilical cord blood on apnea occurrence. Methods: A prospective cohort study with preterm newborns with birth weight less than 2,000 g was undertaken. Exclusion criteria were: mothers that received opioids, mechanical ventilation during the first 4 days of life, cerebral and major cardiac malformations, perinatal asphyxia, severe periintraventricular hemorrhage, exchange transfusion before the fourth day of life, and those that received methylxantine prior to extubation. Neonates were divided in: with detectable and undetectable caffeine in umbilical cord blood. Newborns were followed for the first 4 days for occurrence of apnea spells. Results: 87 with and 40 without detectable caffeine in umbilical cord blood were studied. The median caffeine concentration of the 87 patients with detectable caffeine in umbilical blood was 2.3 µg/ml (0.2-9.4 µg/ml). There was no association between occurrence of apnea spells and presence of caffeine in umbilical cord blood. Neonates with detectable caffeine in umbilical blood had apnea later (66.3 ± 4.14 hours) than those with undetectable levels (54.2 ± 6.26 hours). Conclusion: The detected levels of caffeine in umbilical cord blood did not decrease the occurrence of apnea of prematurity. We suggest that further studies on administration of high dose of caffeine to mothers prior to a preterm delivery as a preventive measure for apnea of prematurity deserve to be conducted.

Orientador: Fernanda da Cruz Landim / Resumo: As células tronco mesenquimais (CTMs) são indicadas para a terapia alogênica pois seus riscos de rejeição são significativamente reduzidos, uma vez que não são células apresentadoras de antígenos. O objetivo deste trabalho foi investigar a influência da criopreservação por longo tempo e das concentrações de SFB (10 e 20%) na: viabilidade, proliferação e potencial de diferenciação de CTMs. Para isso CTMs-TA e CTMs-GW criopreservadas por 43 meses foram submetidas ao processo de caracterização através da citometria de fluxo, teste de proliferação e diferenciação (por imunocitoquímica e expressão gênica). Também foi avaliada a influência de duas concentrações de SFB sobre a expressão gênica de células induzidas e não induzidas à diferenciação adipogênica e osteogênica, sendo observada também a influência, do processo de indução de diferenciação sobre a expressão gênica de CTMS-TA e CTMs-GW. As CTM criopreservadas por longo tempo mantiveram sua viabilidade após a reconstituição e também o potencial de diferenciação osteogênica in vitro confirmada no RT-qPCR. No teste de proliferação realizado com as CTMs-TA e CTM-GW com 10 e 20% de SFB, observamos que as CTMs-TA podem ser cultivadas com qualquer uma das concentrações, porém nas CTMs-GW identificamos que a proliferação é maior quando são cultivadas com 20% de SFB. Através da imunocitoquímica observamos que a concentração de SFB não influencia a diferenciação induzida em nenhuma das duas fontes estudadas. Já a análise da diferenciaç... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mesenchymal stem cells (MSCs) are recomended for allogeneic therapy because their risks of rejection are significantly reduced since they are not antigen presenting cells. The objective of this work was to investigate the influence of long-term cryopreservation and FBS concentrations (10 and 20%) on viability, proliferation and differentiation potential of MSCs. For this, MSCs from adipose tissue (-AT) and CTMs from Wharton jelly (-WJ) cryopreserved for 43 months were submitted to the characterization process through flow cytometry, proliferation and differentiation test (by immunocytochemistry and gene expression). The influence of two concentrations of FBS on gene expression of cells induced and non-induced for adipogenic and osteogenic differentiation was also evaluated. Long-term cryopreserved MSCs maintained their viability after reconstitution and also the potential for in vitro osteogenic differentiation confirmed in RT-qPCR. In the proliferation test performed with MSC-AT and MSC-WG with 10 and 20% FBS, we observed that the MSC-AT can be grown at any of the concentrations, but in the MSC-WG proliferation is greater when cultivated with 20% FBS. Through immunocytochemistry we observed that the concentration of FBS does not influence the induced differentiation in either sources of cells studied. The analysis of the spontaneous differentiation, by immunocytochemistry, showed greater accumulation of fat droplets in MSC-AT and MSC-WG cultured with 20% FBS, indicating the ... (Complete abstract click electronic access below) / Doutor

Resumo:o presente trabalho teve por objetivo isolar células mesenquimais da geléia de Wharton do cordão umbilical canino e avaliar, in vitro, o efeito do plasma rico em plaquetas (PRP) e da matriz óssea desmineralizada (MOD) na diferenciação osteogênica destas células na ausência de meio osteoindutor. As células foram isoladas e caracterizadas como células mesenquimais indiferenciadas com fenótipo semelhante a de fibroblastos e fenótipo característico confirmado pela citometria de fluxo com CD44+, CD105+, CD271+, CD34- e CD45-; além da habilidade de diferenciação em adipócitos, condrócitos e osteócitos. Com exceção do grupo controle (células mesenquimais), em todos os grupos, G-PRP (células mesenquimais e PRP), G-MOD (células mesenquimais e MOD) e G-PRP+MOD (células mesenquimais e PRP + MOD), houve diferenciação osteoblástica com produção de matriz óssea mineralizada, confirmada pela coloração vermelho de Alizarina. A dosagem de fosfatase alcalina (FA) aumentou aos 14 dias em todos os grupos, com redução temporal subsequente. Proteínas ósseas foram expressas na reação em cadeia da polimerase em tempo real e puderam confirmar a diferenciação óssea em todos os grupos. A osteopontina teve sua maior expressão no G-PRP aos sete dias, seguido do grupo G-PRP+MOD... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract:This study aimed to isolate MSCs from canine Wharton's jelly umbilical cord and evaluate in vitro the effect of PRP and the MOD in osteogenic differentiation of these cells in the absence of osteogenic medium. Cells were isolated and characterized as undifferentiated mesenchymal cells with a phenotype fibroblasts-like and characteristic phenotype of confirmed by flow cytometry with CD44 +, CD105 +, CD271 +, CD34-and CD45-, plus the ability to differentiate into adipocytes, chondrocytes and osteocytes. Except for the control group (MSCs), in all groups, G-PRP (PRP and MSCs), G-DBM (DBM and MSCs) and G-PRP + DBM (MSCs and PRP + DBM) was differentiation osteoblastic with production of mineralized bone matrix confirmed by Alizarin red staining. The measurement of alkaline phosphatase (AP) increased up to 14 days in all groups. Bone proteins were expressed in the polymerase chain reaction in real time and were able to confirm the differentiation bone in all groups. Osteopontin had its greatest expression in G- PRP to seven days followed by G-PRP + DBM, at other times the G-PRP + DBM had higher expression than the other groups. The osteocalcin and Runx2 were expressed more intensely in the G-PRP + DBM at the end of the experiment. It can be concluded that the PRP and DBM are capable of promoting osteogenesis mesenchymal cells in Wharton's jelly of the umbilical cord with canine, with advantage in the presence of DBM / Orientador: Alexandre Lima de Andrade / Banca: Rita Cássia Menegati Dornelles / Banca: Márcio Mateus Beloti / Doutor