Objetivos: Avaliar o potencial terapêutico e a segurança do uso intravítreo de (FMMO) contendo células CD34+ em pacientes portadores de degeneração macular relacionada com a idade na forma atrófica. Casuística e Métodos: Foram avaliados 10 pacientes com degeneração macular relacionada à idade (DMRI) seca e acuidade visual no pior olho <=20/100. Foi obtido aspirado da medula óssea de todos os pacientes, e após o processamento do material no hemocentro, foi injetado 0,1 ml da suspensão de FMMO intravítreo no olho com pior acuidade. Os pacientes foram avaliados no baseline, 1, 3, 6, 9 e 12 meses após a injeção. Todos realizaram medida da melhor acuidade visual corrigida (MAVC), microperimetria, eletrorretinografia multifocal (ERGmf), autofluorescência, Tomografia de coerência óptica (OCT) e responderam o questionário VFQ-25 em todos seguimentos. Também foi realizada angiografia fluoresceínica antes da injeção, seis e doze meses após. Resultados: Todos os pacientes completaram o seguimento de seis meses, e seis finalizaram os doze meses. Antes da injeção, a média da MAVC foi de 1,18 logMAR (20/320-1); variando de 20/125 a 20/640-2. Aos doze meses, a média foi de 1,0 logMAR (20/200), com melhora significativa em todos os meses de seguimento. A média do limiar de sensibilidade na microperimetria mostrou melhora significativa a partir do sexto mês (p=0,009). Ao se dividirem os pacientes com área de atrofia maior e menor, por meio da medida da hipoautofluorescência, observou-se que a melhora foi significativa apenas no grupo de menor atrofia. Não houve diferença significativa na ERGmf. A angiofluoresceinografia não apresentou crescimento de neovasos ou tumores. O questionário de qualidade de vida mostrou diferença significativa na saúde mental (p=0,003) e na visão de cores (p=0,005) e forte tendência à significância na análise que abordou a visão geral (p=0,05) e dependência (p=0,067). Houve declínio em relação à saúde geral (p=0,77). Conclusões: Os resultados indicam que o uso de FMMO intravítreo em pacientes com DMRI é seguro e está associado à melhora da acuidade visual e microperimetria. Pacientes com área menor de atrofia têm melhor resposta. Não é possível afirmar, mas acredita-se no resgate funcional das células em sofrimento, que ainda não se degeneraram. / Objectives: To assess the therapeutic potential and safety of the intraviteral use of a bone marrow mononuclear fraction (BMMF) containing CD 34+ cells in patients with the atrophic form of age-related macular degeneration (AMD). Casuistic and Methods: The study was conducted on 10 patients with the atrophic form of ARMD with worse eye visual acuity <=20/100. Bone marrow was aspirated from each patient under local anesthesia. After processing and separation of mononuclear cells, 0.1 ml of the suspension was injected intravitreally into the eye of lower acuity. The patients were evaluated at baseline and 1, 3, 6, 9 and 12 months after the injection. All were submitted to measurement of best corrected visual acuity (BCVA), microperimetry, multifocal electroretinography (ERGmf), autofluorescence, and optical coherence tomography (OCT) and all responded to the VFQ-25 questionnaire at all follow-up visits. Fluorescein angiography was performed before the injection and six and 12 months later. Results: All patients completed the six month follow-up and six completed the 12 month follow-up after the injection. Before the injection, mean BCVA was 1.18 logMAR (20/320-1), ranging from 20/125 to 20/640-2 and at 12 months it was 1.0 logMAR (20/200), with a significant improvement over all followup months. Mean perimetric threshold sensitivity improved significantly starting at the sixth month (p=0.009). When the patients with a larger area of atrophy measured by hypoautofluorescence were considered separately, a significant improvement was observed only in the group with lower atrophy. There was no significant difference in electroretinography. Angiofluoresceinography did not reveal neovessel or tumor growth. The quality of life questionnaire showed a significant difference in mental health (p=0.003) and color vision (p=0.005), and a strong tendency in the analysis of general vision (p=0.05) and dependence (p=0.067). There was a decline in general health (p=0.77). Conclusions: The results indicate that the use of intravitreal BMMF in patients with AMD is safe and is associated with improved visual acuity and microperimetry. Patients with a smaller atrophy area obtained a better response. The paracrine effect of these cells may explain the functional improvement observed in the present study; however, a larger series should be study to confirm these clinical findings.

Amplification of hematopoietic stem cells (HSCs) from human cord blood has applications for a variety of cell therapy protocols. The purpose of this thesis (performed in collaboration with ViaCell, Inc.) was to analyze differential gene expression (especially related to cytokines) during the process of human HSC amplification in vitro. When applied to markers previously shown to be specific for HSC's and/or progenitor cells, the analysis validates ViaCell's cellular product. Total cellular RNA was isolated from cord blood samples at various stages of amplification and used to synthesize cDNAs as probes for hybridization arrays. mRNA candidates increased in cell populations enriched for stem cells were first identified using hybridization arrays, then confirmed by RT-PCR. Restriction mapping confirmed RT-PCR amplicons. The results identified several developmentally interesting cytokines (CD117, Jagged-2, Manic Fringe, and Notch) upregulated in stem cell enriched fractions. Analysis of one candidate previously shown to be a marker for HSCs and progenitors, CD117, was extended using Western blots to show a CD117-related protein upregulation. The observed upregulations did not contain many inflammatory cytokines, which could hinder survival of HSC grafts. The future hope for the non-CD117 candidates is as potential growth modifiers for stem cell samples isolated by clonogenic amplification.

Les vecteurs lentiviraux (LV) sont des outils efficaces de transfert de gène, largement utilisés en thérapie génique, en particulier pour la transduction ex vivo de cellules souches et progénitrices hématopoïétiques (CSPH). Afin d’étudier simultanément la fusion et la transduction dans les CSPH avec les LV, nous avons adapté une méthode basée sur latechnologie du transfert d’énergie entre deux molécules fluorescentes (FRET). Pour mettre en place cette technique, des LV capables d’incorporer spécifiquement une enzyme, la bétalactamase (BLAM-LV) et de coder une forme tronquée du récepteur au facteur de croissance nerveuse (DELTA-NGFR), sont produits. Nos résultats montrent que les LV sont soumis à une restriction post-entrée forte dans les cellules hématopoïétiques, que ce soit dans des lymphocytes T immortalisés ou bien des CSH CD34+. Nous montrons également que cette inhibition post-entrée peut être partiellement saturée après une forte augmentation de la multiplicité d’infection ou en présence d’additifs de culture, comme la Vectofusin-1® ou laRetronectin®. De plus, nous avons montré lors de la transduction de CSPH avec des vecteurs BLAM-LV que la Vectofusin-1® agit sur l’étape d’entrée en augmentant l’adhésion et la fusion entre les membranes virale et cellulaire. Cette technique représente donc un nouvel outil sensible et efficace pour étudier de façon concomitante l’étape de fusion et le niveau de transduction dans les cellules cibles. A terme, ce travail permettra une meilleure compréhension de la biologie des LV mais pourra également conduire à l’élaboration de protocoles de transduction lentivirale plus efficaces. / Lentiviral vectors (LV) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking to precisely evaluate parameters that control the efficiency of transduction in relation with the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer (FRET)-based HIV-1 fusion assay to measure the entry of non-replicative recombinant LV in various cell types, including primary human hematopoietic stem and progenitor cells, and to quantify the level of transduction of he same initially-infected cells. The assay utilizes recombinant LV containing betalactamase (BLAM)-Vpr chimeric proteins (BLAM-LV) and encoding a truncated form of thelow affinity nerve growth factor receptor (DELTA-NGFR). This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing for instance the extent of lentiviral post-entry restrictions occuring in cells of hematopoietic origin. The assay also shows that transduction enhancers like Vectofusin®-1 or Retronectin® can partially relieve this post-entry block but their effects differ in the way to promote LV entry. Furthermore, our results show that Vectofusin®-1 acts at the entry step by promoting the adhesion and the fusion between lentiviral and cellular membranes. In conclusion, one such assay should be useful to study hematopoietic post-entry restrictions directed against LV and should allow improvements in various LV-based gene therapy protocols.

Regulation of hematopoiesis through the finite control of hematopoietic stem cell (HSC) quiescence and proliferation is critical to the health of the organism since disturbances in blood production can lead to clinical malignancies such as anemia or leukemia. Therefore, elucidating the processes that govern HSCs is vital to advance our understanding of hematological diseases. Interestingly, HSCs can be regulated through a variety of ways. Extrinsic cues from the niche provide signals that govern HSC quiescence, proliferation, self-renewal, and differentiation. These external signals are converted to internal messages through the use of signal transduction pathways that relay information from the cytoplasm to the nucleus. While many pathways contribute to HSC regulation, the PI3K/AKT/mTOR-pathway is especially pertinent because it has been implicated in cell survival, metabolism, proliferation, and death. Many groups have identified key players within PI3K/AKT/mTOR-signaling that regulate HSC quiescence; however, these studies are hindered by the redundancy of multiple isoforms and compensatory signaling mechanisms by other members within the pathway. PDK1 is considered to be a master regulator of PI3K-signaling because it is directly activated by PI3Ks and can govern activity of a variety of substrates within the PI3K-pathway. Because of this, it is an excellent candidate to fully delineate how PDK1-mediated PI3K-signaling functions to maintain HSC quiescence. In the current study, conditional deletion of PDK1 in HSCs was achieved through the use of a hematopoietic specific Vav-Cre line. The loss of PDK1 in HSCs led to reduced numbers and an inability to provide radioprotection in primary BMTs. Furthermore, PDK1-deficient HSCs exhibit impaired quiescence and increased cycling. Strikingly, PDK1-mutant HSCs have markedly high levels of reactive oxygen species (ROS), which led to increased proliferation, DNA damage, and apoptosis in progenitor cells. Administration of a ROS scavenger, N-acetyl cysteine (NAC) partially rescued the increased proliferation and differentiation of phenotype Pdk1Hem-KO cells both in vitro and in vivo, suggesting that abnormal HSC activity in PDK1-deficient cells was in part due to increased ROS. Furthermore, mechanistic studies identified a remarkable decrease in the levels of nuclear HIF1α in HSCs. Immunoblots and phosflow studies demonstrated reduced activation of p-p70S6K, a well defined positive regulator, and increased GSK3β, a key negative regulator of the HIF1α protein. These data suggested that ROS levels are unrestrained since HIF1α is not present in Pdk1Hem-KO HSCs to activate transcription of genes that moderate oxidative stress. In addition, Pdk1Hem-KO HSCs also show increased levels of Hif3α and IPAS mRNA, which are believed to inhibit the transcriptional activation of HIF1α. In essence, the results from these experiments are the first to implicate PDK1 as a critical regulator of HSC quiescence and as a moderator of PI3K-signaling to alleviate oxidative stress. In addition, this study is the first to suggest that HIF3α and IPAS may play a role in HSCs.

Hematopoietic stem cells facilitate lifelong production of a diverse repertoire of functional mature blood cells. They are a critical biological reservoir that enable organisms to endure physiological challenges such as inflammation, disease, and age. The functional maintenance of hematopoietic stem cells depends not only on intrinsic cell pathways, but also on extrinsic cues that guide core behaviors like homing and self-renewal. Careful study of these extrinsic regulatory networks can deepen our appreciation of fundamental stem cell biology and motivate therapeutic approaches to treat hematologic disease. Here I show how derangement of the bone marrow regulatory environment perturbs normal hematopoiesis, and demonstrate the dependence of hematopoietic stem cells on a circulating endocrine factor.

"February 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 171-209). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Understanding the factors that regulate the growth and differentiation of haemopoietic stem cells (HSC) remains a major challenge. In this study, the proliferation and differentiation of CD34+ cells from normal donors and chronic myeloid leukaemia (CML) patients was compared. The proliferation and entry of CML cells into the cell cycle was decreased relative to cells from normal donors, and greater heterogeneity in the phenotype of CML cells at the initiation of culture was observed. Analysis of phenotype concomitant with cell division also demonstrated that the differentiation of normal CD34+ cells was consistent between donors, while marked variability was observed in the differentiation of CD34+ cells from CML patients. This included expression of CD13, CD33, CD38 and HLA-DR, which were linked to cell division in normal but not CML cells. The tyrosine kinase inhibitor, imatinib, is a novel drug displaying promising results in the treatment of CML by specifically inhibiting the growth of leukaemic cells. To examine whether myelosuppression observed in patients treated with imatinib may arise from inhibition of normal haemopoiesis, imatinib was added to colony assays established using cells from normal bone marrow. Suppression of monocyte/macrophage growth, but not that of eosinophils or neutrophils, was observed at therapeutic concentrations of imatinib. Inhibition of monocytic differentiation to macrophages was also observed and was associated with decreased functional capacity such as altered antigen uptake, production of proinflammatory cytokines and stimulation of responder cells. The specific suppression of monocyte/macrophage differentiation and function was not due to blockade of tyrosine kinases known to be inhibited by imatinib and was consistent with an inhibition of the M-CSF/c-fms signalling pathway. This hypothesis was tested using a cell line that was dependent on M-CSF for growth and survival. Cell proliferation and phosphorylation of c-fms were inhibited at an IC50 of 1.9μM and 1.4μM imatinib respectively and this was not attributable to decreased c-fms expression. These important findings therefore identify c-fms as a further target of imatinib, and suggest that imatinib should be considered for treatment of diseases where c-fms is implicated. This includes breast and ovarian cancer and inflammatory conditions such as rheumatoid arthritis. Potential side effects resulting from imatinib treatment must also be considered. / Thesis (Ph.D.)--School of Medicine, 2004.

Several systems were examined for the production and delivery of recombinant vaccines for fish. C. crescentus was employed to produce a fragment of the IHNV glycoprotein. When administered by injection to 0.5 gram rainbow trout (Oncorhynchus mykiss), one of the fusion proteins (184 amino acids of the IHNV glycoprotein fused to 242 amino acids of the C-terminus of the Caulobacter crescentus) protected the fish against lethal challenge with IHNV. Attenuated strains of Yersinia ruckeri were generated using allelic exchange mutagenesis. These strains were characterized in terms of in vitro growth characteristics and invasiveness. Attenuated E. coli and Y. ruckeri were exploited to deliver plasmid DNA to fish cells in vitro; attenuated Y. ruckeri bacteria were examined in vivo as bivalent vaccine delivery vehicles, either through the expression of a fragment of the IHNV glycoprotein or by carrying a plasmid DNA vaccine encoding the complete IHNV glycoprotein. A cell wall deficient strain (11.29��dap) protected rainbow trout against lethal challenge with pathogenic Y. ruckeri. Gene transfer to fish was not detected by luciferase reporter gene assays. No clear protection from IHNV challenge was observed. / Graduation date: 2002

We show that the prion protein (PrP) is expressed on the surface of bone marrow cell populations enriched in long-term repopulating hematopoietic stem cells. Affinity purification of the PrP-positive and PrP-negative fractions from these populations, followed by competitive reconstitution assays, show that all long-term repopulating hematopoietic stem cells express PrP. Hematopoietic stem cells from PrP null bone marrow exhibit impaired self-renewal in serial competitive transplantation experiments, and premature exhaustion when exposed to cell cycle-specific myelotoxic injury. Therefore, PrP is a novel marker for hematopoietic stem cells and regulates their self-renewal. / Singapore-MIT Alliance (SMA)

CD4+CD25+FoxP3+ regulatory T cells (Treg) possess the capacity to modulate both adaptive and innate immunity. Due to their suppressive nature, Treg cells have been studied and tested in a variety of scenarios in an attempt to ameliorate undesired immune responses. While graft versus host disease (GVHD) has in fact emerged as the first clinical application for human Treg cells (Riley et al. 2009), equally important are issues concerning hematopoietic engraftment and immune reconstitution. Currently, little is known about the effect(s) that regulatory T cells may exert outside the immune system in this context. Based on cytokine effector molecules they can produce we hypothesized that Treg cells could regulate hematopoietic phenomena. The studies portrayed in this dissertation demonstrate that Treg cells can differentially affect the colony forming activity of myeloid and erythroid progenitor cells. In-vitro as well as in-vivo findings demonstrate the ability of Tregs to inhibit and augment the differentiation of primitive and intermediate myeloid (interleukin (IL)-3 driven) and late erythroid (erythropoietin driven) hematopoietic progenitor cells, respectively. The inhibitory and enhancing affects appeared to be mediated by independent pathways, the former requiring cell-cell contact, major histocompatibility complex (MHC) class II expression on marrow cells and involving transforming growth factor beta (TGF-beta), whereas the latter required interleukin (IL)-9 and was not contact dependent. Strikingly, we observed that in addition to regulating hematopoietic activity in normal primary BM cells, Tregs were also capable of suppressing colony forming activity by the myelogenous leukemia cell line NFS-60. Furthermore, studies involving endogenous Treg manipulations in-situ (i.e. depletion of these cells) resulted in elevated overall myeloid colony activity (CFU-IL3) and diminished colony numbers of erythroid precursors (CFU-E) in recipients following BMT. Consistent with these results, it was found that upon co-transplant with limiting numbers of bone marrow cells, exogenously added Treg cells exert in-vivo regulation of myeloid and erythroid CFU activity during the initial weeks post-transplantation. This regulation of hematopoietic activity by freshly generated Tregs upon transplantation led to the elaboration of a second hypothesis; following lethal total body irradiation (TBI) the host microenvironment facilitates regulatory T cell activation/effector function. Substantial evidence has accumulated in support of this hypothesis, for example we demonstrate up-regulation of surface molecules such as GARP and CD150/SLAM, which have been previously reported as indicators of Treg activation following TCR signaling and co-stimulation, occurs in donor (reporter) Treg populations. Acquisition of an activated phenotype and hence of effector/modulatory function is consistent with the previous in-vivo observations, indicating that both recipient and donor Treg cells can influence hematopoietic progenitor cell activity post-transplant. Finally, the present studies may be of great relevance in the emerging field of Treg cell based immunotherapy for prevention and/or treatment of HSCT complications.