Cellular mechanisms involved in neurodegeneration and neuroprotection are continuing to be explored, and this paper focuses on some novel discoveries that give further insight into these processes. Oligodendrocytes and activated astroglia are likely generators of the pro-inflammatory cytokines, such as the tumor necrosis factor family and interleukin family, and these glial support cells express adhesion receptors (e.g., VCAM) and release intercellular adhesion molecules (ICAM) that have a major role in neuronal apoptosis. Even brief exposure to some substances, in ontogeny and sometimes in adulthood, can have lasting effects on behaviors because of their prominent toxicity (e.g., NMDA receptor antagonists) or because they sensitize receptors (e.g., dopamine D2 agonists), possibly permanently, and thereby alter behavior for the lifespan. Cell cycle genes which may be derived from microglia, are the most-recent entry into the neuroprotection schema. Neuroprotection afforded by some common substances (e.g., melatonin) and uncommon substances [e.g., nicotine, green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG), trolox], ordinarily thought to be simple radical scavengers, now are thought to invoke previously unsuspected cellular mechanisms in the process of neuroprotection. Although Alzheimer's disease (AD) has features of a continuous spectrum of neural and functional decline, in vivo PET imaging and and functional magnetic resonance imaging, indicate that AD can be staged into an early phase treatable by inhibitors of β and γ secretase; and a late phase which may be more amenable to treatment by drugs that prevent or reverse tau phosphorylation. Neural transplantation, thought to be the last hope for neurally injured patients (e.g., Parkinsonians), may be displaced by non-neural tissue transplants (e.g., human umbilical cord blood; Sertoli cells) which seem to provide similar neurotrophic support and improved behavior-without posing the major ethical dilemma of removing tissue from aborted fetuses. The objective of this paper is to invite added research into the newly discovered (or postulated) novel mechanisms; and to stimulate discovery of additional mechanisms attending neurodegeneration and neuroprotection.

During differentiation stem cells are exposed to a range of microenvironmental chemical and physical cues. In this study, human multipotent progenitor cells (hMLPCs) were differentiated from umbilical cord into oligodendrocytes and astrocytes. Chemical cues were represented by a novel defined differentiation medium containing the neurotransmitter norepinephrine (NE). In traditional 2 dimensional (2D) conditions, the hMLPCs differentiated into oligodendrocyte precursors, but did not progress further. However, in a constructed 3 dimensional (3D) environment, the hMLPCs differentiated into committed oligodendrocytes that expressed MBP. When co-cultured with rat embryonic hippocampal neurons (EHNs), hMLPCs developed in astrocytes or oligodendrocytes, based on presence of growth factors in the differentiation medium. In co-culture, physical cues provided by axons were essential for complete differentiation of both astrocytes and oligodendrocytes. This study presents a novel method of obtaining glia from human MLPCs that could eliminate many of the difficulties associated with their differentiation from embryonic stem cells. In addition, it reveals the complex interplay between physical cues and biomolecules on stem cell differentiation.

Les affections articulaires touchant le cartilage, telles que les lésions focales et l’arthrose, correspondent aux principales causes de baisse de performance et d’arrêt prématuré de la carrière sportive du cheval. Ainsi, le traitement des affections du cartilage représente un enjeu vétérinaire majeur dans le monde équin, du fait des importantes pertes financières qu’elles occasionnent à la filière. Les faibles capacités de réparation intrinsèque du cartilage, ainsi que l’absence de thérapie à long terme des dommages cartilagineux, nécessitent le recours à des thérapies de nouvelles générations telle que l’ingénierie tissulaire du cartilage. Dans ce cadre, notre étude s’est attachée à comparer différents types cellulaires pour la génération de cartilage in vitro, afin d’envisager une implantation pour traiter les atteintes cartilagineuses chez le cheval. Une technique initialement développée chez l’Homme, la transplantation de chondrocytes autologues, représente toujours un « gold standard » en ingénierie tissulaire du cartilage. Dans ce travail de thèse, après avoir développé une nouvelle génération de substitut cartilagineux de haute qualité biologique, à partir de chondrocytes articulaires équins, des limites techniques et biologiques inhérentes au type cellulaire persistent. Ainsi, nos travaux se sont tournés vers la recherche de types cellulaires alternatifs. Les cellules souches/stromales mésenchymateuses (CSM) néonatales issues de cordon ombilical telles que les CSM de sang placentaire (CSM-SPL) et les CSM de gelée de Wharton (CSM-GW) pourraient représenter un avantage thérapeutique du fait de leur isolement non-invasif, de leur forte prolifération cellulaire et de leur capacité de différenciation en chondrocyte. Il est néanmoins indispensable de définir le meilleur candidat thérapeutique, parmi ces deux sources cellulaires, pour l’obtention d’un substitut cartilagineux de qualité biologique optimale. Ces résultats de thèse ont montré d’importantes différences dans le processus de chondrogenèse de ces deux sources de CSM néonatales et plaident en faveur de l’utilisation des CSM-SPL dans le cadre d’une stratégie thérapeutique d’ingénierie tissulaire du cartilage équin. Ces travaux ont permis une meilleure compréhension de la biologie du chondrocyte et des CSM. De surcroît, ces travaux permettent d’envisager de futurs essais cliniques chez le cheval, afin de traiter les affections articulaires de ce modèle gros animal. / Articular cartilage disorders, such as focal defects and osteoarthritis, are the main causes of decreased performance or early retirement of sport- and racehorses. Thus, cartilage disorders represent a major veterinary issue in the equine industry, due to significant financial losses. Poor intrinsic cartilage repair properties and the absence of long- term therapy for cartilage defects lead to the development and use of new generation therapies such as autologous chondrocytes implantation. In this context, our study aimed to compare different cell types for the in vitro cartilage generation, in order to implant the biological substitute to treat cartilage defects in the horse. A therapeutic strategy initially developed in human medicine, the autologous chondrocytes transplantation, always represents a "gold standard" in cartilage tissue engineering. In the present study, after developing a new generation of cartilaginous substitute of high biological quality, composed of equine articular chondrocytes, technical and biological limits inherent to the cell type persist. Thus, we have used alternative cell types such as neonatal mesenchymal stem/stromal cells (MSCs) from umbilical cord, such as umbilical cord blood MSC (UCB-MSCs) and umbilical cord matrix or Wharton jelly MSCs (UCM- MSCs). These MSCs sources could represent a therapeutic advantage due to their non-invasive isolation, their high cell proliferation and their ability to differentiate into chondrocytes. Nevertheless, it is essential to define the best therapeutic candidate between these two MSCs sources, to obtain an optimal quality for the neocartilaginous substitute. Our data highlighted important differences in the chondrogenesis process of these two neonatal MSCs sources, allowing us to consider UCB-MSCs as the best therapeutic candidate for equine cartilage tissue engineering. This work allows a better understanding of the chondrocyte and MSCs biology. Moreover, this work leads the way to setting-up future clinical trials in the horse, in order to treat articular defects of this large animal model.

Master of Science / Department of Anatomy and Physiology / Masaaki Tamura / Previous studies have shown that both human and rat umbilical cord matrix mesenchymal stem cells (UCMSC) possess the ability to control the growth of breast carcinoma cells. Comparative analysis of two types of UCMSC suggest that rat UCMSC-dependent growth regulation is significantly stronger than that of human UCMSC. Accordingly, the present study was designed to clarify their different tumoricidal abilities by analyzing gene expression profiles in two types of UCMSC. Gene expression profiles were studied by microarray analysis using Illumina HumanRef-8-V2 and RatRef-12 BeadChip for the respective UCMSC. The gene expression profiles were compared to untreated naïve UCMSC and those co-cultured with species-matched breast carcinoma cells; human UCMSC vs. MDA-231 human carcinoma cells and rat UCMSC vs. Mat B III rat carcinoma cells. The following selection criteria were used for the screening of candidate genes associated with UCMSC-dependent tumoricidal ability; 1) gene expression difference should be at least 1.5 fold between naive UCMSC and those co-cultured with breast carcinoma cells; 2) they must encode secretory proteins and 3) cell growth regulation-related proteins. These analyses screened 17 common genes from human and rat UCMSC. The comparison between the two sets of gene expression profiles identified that two tumor suppressor genes, adipose-differentiation related protein (ADRP) and follistatin (FST), were specifically up-regulated in rat UCMSC, but down-regulated in human UCMSC when they were co-cultured with the corresponding species’ breast carcinoma cells. The suppression of either protein by the addition of a specific neutralizing antibody in co-culture of rat UCMSC with Mat B III cells significantly abrogated UCMSC ability to attenuate the growth of carcinoma cells. Over-expression of both genes by adenovirus vector in human UCMSC enhanced their 4 ability to suppress the growth of MDA-231 cells. In the breast carcinoma lung metastasis model generated with MDA-231 cells, systemic treatment with FST-over-expressing human UCMSC significantly attenuated the tumor burden. These results suggest that both ADRP and FST may play important roles in exhibiting stronger tumoricidal ability in rat UCMSC than human UCMSC and imply that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression.

Master of Science / Department of Anatomy and Physiology / Mark L. Weiss / Wharton's jelly is a non-controversial source of mesenchymal stromal cells. Isolation of the cells is non-invasive and painless. The cells have been shown to have a wide array of therapeutic applications. They have improved symptoms when transplanted in a variety of animal disease models, can be used in tissue engineering applications to grow living tissue ex vivo for transplantation, and can be used as drug delivery vehicles in cancer therapy. The cells have also been shown to be non-immunogenic and immune suppressive. This thesis focuses on optimizing isolation protocols, culture protocols, cryopreservation, and characterization of cells in different growth conditions. Results from the experiments indicate that isolation of cells by enzyme digestion yields cells consistently, a freezing mixture containing 90% FBS and 10% DMSO confers maximum viability, and the expression of mesenchymal stromal cell consensus markers does not change with passage and cryopreservation. The results of the experiments also show that cells grow at a higher rate in 5% oxygen culture conditions compared to 21% oxygen culture conditions, serum does not have an effect on growth of the cells, serum and oxygen do not have effects on the expression of mesenchymal stromal cell consensus markers and the cells are stable without nuclear abnormalities when grown in 5% oxygen and serum free conditions for six passages after first establishing in serum conditions.

Master of Science / Department of Animal Sciences and Industry / Duane L. Davis / Intestinal absorption of colostral leukocytes (CL), peripheral blood mononuclear cells (PBMC), and porcine umbilical cord matrix stem cells (PUC) was analyzed in neonatal pigs. Maternal CL have previously been demonstrated in pigs, and maternal PBMC have been observed in calves to enter neonatal circulation after ingestion. PUC are primitive stem cells that are easily isolated from Wharton's jelly of the porcine umbilical cord. These cells do not have an immunogenic effect on the host upon initial transplantation. The general characteristics of PUC may allow them to serve as a delivery system to the neonate. Cellular migration through the duodenum, jejunum, and ileum was assessed using confocal microscopy. In vitro experiments utilized an organ explant culture system to determine the trafficking of labeled cells. Small-intestine tissue was collected from stillborn and sacrificed neonates. All three cell types (CL, PBMC, and PUC) were detected below the luminal surface, after 72 h of culture with media, and regardless of whether explants were from stillborns or live-born pigs. In vivo trafficking was assessed using neonatal pigs that were fed PBMC isolated from their mother or PUC from an unrelated pig. The effect of prior exposure to 25% acellular colostrum (AC) in medium was evaluated for both cell types. Piglets were euthanized 8 h or 24 h post feeding and sections of the small intestine collected. Both PBMC and PUC were found in all intestinal samples. Exposure to AC had no detected effect on the ability of either cell type to attach and migrate into the tissue. Labeled PUC were detected on the surface of the epithelium and in the lamina propria 8 h post treatment. PBMC were observed on the surface of the epithelium, in the lamina propria, and superficial submucosa 8 h following ingestion. In neonates sacrificed 24 h post treatment, both PUC and PBMC were observed on the surface of the epithelium, in the lamina propria, superficial submucosa, and deep submucosa of the small intestine. PUC and PBMC were noted at the apex, intermediate between the apex and the base, or at the base of the villus.

The engraftment of haemopoietic stem/progenitor cells (HSPCs) from umbilical cord blood (UCB) into adult recipients, although advantageous in terms of sourcing units, the decreased need to match donor and recipient and reduced risk of graft versus host disease (GvHD), is delayed compared to grafts using HSPCs from mobilised peripheral blood (MPB) or bone marrow (BM). One reason for this is the limited number of HSPCs (CD34+/CD133+ cells) in a unit of UCB compared to MPB or BM. The CXCR4-CXCL12 axis is widely recognised as a key player in the bone marrow homing, retention, and engraftment of HSPCs. The aim of this thesis was to investigate whether the engraftment of HSPCs from UCB into the bone marrow could be improved. Firstly, a novel in vitro 3D time-lapse chemotaxis assay to assess the homing capacity of human UCB CD133+ HSPCs, towards the chemokine CXCL12 was developed. One advantage of this assay was that it distinguished cell chemotaxis from chemokinesis and allowed these parameters to be quantified. Human UCB CD133+ HSPC chemotaxis towards CXCL12 was inhibited by the CXCR4 antagonist, AMD3100. Importantly, the presence of CXCL12 or AMD3100 had no affect on cell chemokinesis. To complement the in vitro chemotaxis assay, a short term in vivo homing assay in NSG mice was successfully established. The effect of siRNA silencing of the CXCR4 co-receptor, CD164, which is also expressed on CD133+ HSPCs, on cell migratory and homing ability was investigated. CD164 knock-down using siRNA in human UCB CD133+ HSPCs did not demonstrate an effect on homing to NSG bone marrow in vivo or chemotaxis to CXCL12 in vitro. However, homing to NSG mouse spleen was significantly reduced in cells silenced for CD164. Following this, an 8 day HSPC expansion system using nanofibre scaffolds (Nanex) and differing cytokines was investigated. These serum and feeder free conditions yielded a significant expansion of cells that retained CD133+CD34+ expression and their in vitro chemotactic ability to CXCL12. Time constraints did not permit the engrafting ability of these cells to be analysed in an in vivo HSC reconstitution assay that was initiated. However these studies will provide the basis to support future related research in this laboratory.

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.

La transplantation de sang de cordon ombilical (TSCO) est utilisée pour traiter les enfants atteints de maladies hématologiques en l’absence de donneurs apparentés compatibles. Elle est associée avec des risques plus élevés d’échec de greffe et d’infections opportunistes dans les premiers mois qui suivent la transplantation en comparaison avec une greffe de moelle osseuse. Par contre, la TSCO comporte un risque plus faible de maladie du greffon contre l’hôte et une incidence comparable de rechute de leucémie. Ces quatre complications impliquent directement les lymphocytes T. Dans le but de mieux comprendre le schéma particulier des évènements qui suivent la TSCO et d’améliorer le pronostic des patients, nous avons étudié le potentiel fonctionnel, la persistance et la reconstitution antivirale des lymphocytes T au sein d’un groupe d’enfants transplantés de sang de cordon ombilical (SCO). Étant donné que le SCO contient une majorité de lymphocytes T naïfs, nous avons étudié les lymphocytes T spécifiques au HLA-A2:Melan-A26-35 A27L; seul répertoire naïf et abondant caractérisé chez l’homme. Nous avons observé que les lymphocytes T du SCO se différencient en populations effectrices, s’oligoclonalisent, produisent de l’IFN-γ et lysent spécifiquement leur cible suite à la stimulation. Néanmoins, ces cellules produisent moins d’IFN-γ et sont moins bifonctionnelles que leurs homologues issus du sang périphérique d’adultes. Chez les patients, les lymphocytes T du SCO s’épuisent après la TSCO : ils s’oligoclonalisent dramatiquement, sont principalement en différenciation terminale, et une importante fréquence exprime PD-1 (« programmed death-1 ») dans les 3 à 6 premiers mois post-greffe. Très peu de patients sont capables de développer des réponses antivirales durant cette période et la fréquence de lymphocytes T qui expriment PD-1 semble aussi avoir un impact sur le risque subséquent de faire une rechute de leucémie. La deuxième vague de lymphocytes T émergeant à 6 mois post-TSCO mène à une population fonctionnelle et diversifiée. En conclusion, la fonctionnalité des lymphocytes T présents dans les 3 à 6 premiers mois post-TSCO doit être rétablie pour améliorer les risques d’infections opportunistes et de rechute de leucémie. / Umbilical cord blood (UCB) is increasingly used as a source of hematopoietic progenitor cells to treat a variety of disorders in children. UCB transplantation (UCBT) is associated with a reduced incidence of graft-versus-host disease, a robust graft-versus-leukemia effect, more frequent graft failures and a higher incidence of opportunistic infections (OI) compared to bone marrow transplantation; four processes in which donor-derived T lymphocytes are known to be predominantly involved. UCB T cells are mostly naïve. To examine the differential functionality of UCB T cells, CD8+ T cells specific for the melanoma-associated HLA-A2-restricted Melan-A26-35 A27L peptide were isolated from UCB and UCBT recipients, as it represents an abundant preimmune repertoire in human. Following in vitro stimulation, UCB T cells proliferated, oligoclonalized, acquired cell surface markers reflective of effector/memory differentiation, expressed cytolytic activity and produced IFN-γ. While functional properties of UCB T cells resembled their counterparts in adult peripheral blood, they were more likely to reach terminal differentiation following stimulation, produced less IFN-γ and were less frequently bifunctional (IFN-γ and cytolysis). Following UCBT, T cells became exhausted: they oligoclonalized dramatically, exhibited a terminal differentiation phenotype and a high frequency also expressed PD-1 (“ programmed death 1 ”) in the first 3-6 months post-UCBT. Moreover, very few patients developed an antiviral response during this period. Finally, the frequency of PD-1+ CD8+ T cells was significantly higher in subjects who subsequently experienced leukemic relapse. A second wave of T cells emerging at 6 months post-UCBT was observed and characterized by an increase in the repertoire diversity till 1 year, the development of a naïve population with polyfunctional potential and the progressive reconstitution of antiviral responses. This study reports to the biological properties of UCB-derived CD8+ T cells and provides a rationale for the characteristics of UCBT in terms of immune reconstitution and OI. These results also suggest that the first wave of CD8+ T cells in the first 3-6 months post-UCBT should be targeted in priority to improve both OI and leukemic relapse risks.

The increasing incidence of children suffering from allergic diseases could be caused by sensitization of immature immune system during the intrauterine development. Several important scientific papers have demonstrated the ability of cord blood cells to respond by elevated proliferation activity after stimulation by common allergens. Following these findings, present study follows the production of cytokines which play a role in the pro- and anti-allergenic tuning of the immune system. Umbilical cord blood cells were stimulated with polyclonal activators (phytohaemagglutinin) and common allergens (ovalbumin, timothy grass, birch, mite). Subsequently, cytokine production was monitored using selected methods that reflect different stages of cell activation - at the level of mRNA by quantitative real time PCR (qRT-PCR), by flow cytometry detection of the presence of intracellular cytokines in different cell subpopulations and by ELISA measurement of cytokines in CBMC culture supernatants. The results obtained point to a very weak ability of these common allergens (timothy grass, birch, mite, ovalbumin) to stimulate CBMC to produce cytokines observed by all of these methodological procedures. Although we did not observe significant differences in CBMC cytokine production (IL-2, IL-4, IL-10, IL-12,...