Development of a suitable animal model of AIDS is much needed in AIDS research to study infection and pathogenesis as well as to evaluate methods of prevention and treatment of HIV infection. Small animals such as rodents are attractive candidates for AIDS research due to the availability of various inbred and genetically engineered strains, extensive knowledge or their immune system, especially in mice, and the relative ease of breeding and maintaining animal colonies. Transgenic small animal models carrying entire HIV genome or selected genes have been instrumental to understand functions of HIV genes in vivo and their role in HIV pathogenesis. The type of cells in which HIV genes are expressed seems to be an import prerequisite for the study of HIV gene functions in transgenic mice. Mice constitutively expressing the entire HIV-1 genome or HIV-1 nef gene in CD4 + T cells and in the cells of macrophage/dendritic lineage develop an AIDS-like disease very similar to AIDS disease in humans. Similarly, expression of Nef in adult mice, using inducible system, results in the AIDS-like disease. This disease is characterized by thymic atrophy, impaired thymocyte maturation, loss of CD4+ T cells, increased activation and turnover of T cells, which can occur in the absence of lymphypenia, and non-lymphoid organ disease involving the lungs and kidneys. Susceptibility of adult mice to the pathological effects of Nef suggests that the AIDS-like disease in the constitutively expressing Nef Tg mice is not due to developmental defects caused by early expression of Nef. This model highlights the important role of Nef in HIV-1 pathogenesis. The high similarity in the disease in these Tg mice with human AIDS strongly suggest that these mice are a relevant model to study AIDS. This study further evidence that mouse cells can support functions of Nef and these Tg mice represent a unique model to study Nef functions in vivo in the context of the primary immune system. Moreover, the inducible Nef Tg model has given us the ability to control the level and time of expression of Nef which was impossible to do in the previously reported constitutive Nef Tg mouse models. These mice will be useful to study immune reconstitution since Nef expression can be turned off after withdrawal from dox.

L’immunosuppression optimale après greffe d’organe solide est une balance délicate et propre à chaque individu entre le risque de rejet et les risques liés à une surexposition au traitement immunosuppresseur. L’évaluation de la fonction résiduelle des lymphocytes T après stimulation par un mitogène (pharmacodynamie effective) devrait permettre de mesurer l’effet direct des médicaments immunosuppresseurs sur leur cible. Nous avons étudié différents paramètres de pharmacodynamie effective chez 34 receveurs pédiatriques de greffe d’organes solides traités par tacrolimus et mycophénolate. Les tests proposés dans ce travail sont adaptés au milieu pédiatrique et à une réalisation en temps réel. La quantification du CD25 parmi les CD4 activés par l’OKT3 permet de distinguer deux groupes de patients selon leur degré d’immunosuppression. L’âge médian est plus bas et la concentration plasmatique médiane en MPA plus élevée dans le groupe de patients plus fortement immunosupprimés. L’étude des paramètres immunologiques pouvant influencer la réponse (sécrétion des interleukines, proportion des sous-populations lymphocytaires CD4, CD8, T naïfs et Trég) ainsi que l’étude du pouvoir de restauration de la fonction lymphocytaire par l’Il-2, la guanosine ou la xanthosine, ne permettent pas de mieux comprendre les variabilités interindividuelles observées. Ces résultats devront être confirmés sur une cohorte plus grande de patients afin de juger de leur intérêt en pratique clinique. / Optimal immunosuppression following solid organ transplantation is unique to each individual and requires a balance between risks of rejection and overexposure to immunosuppressive therapy. The evaluation of residual function of T lymphocytes after mitogen stimulation (effective pharmacodynamic monitoring) should allow measurement of the direct effect of immunosuppressive drugs on their target. We studied various parameters of effective pharmacodynamic monitoring in 34 paediatric patients receiving solid organ transplants and treated with tacrolimus and mycophenolate (MPA). The tests proposed in this work are adapted to the paediatric setting in real time. Quantification of CD25 among CD4 cells activated by OKT3 can differentiate two groups of patients according to their degree of immunosuppression. Median values for age and MPA plasma concentration are lower and higher, respectively, in the patient group most heavily immunosuppressed. Neither study of the parameters that may influence the response (secretion of interleukins, proportion of lymphocyte subpopulations CD4, CD8, naive and regulatory T cells) nor study of the restoration of basal cell function brought about by Il2, guanosine or xanthosine, helped to explain the observed inter-individual variability. These results should be confirmed in a larger cohort of patients in order to test their relevance in clinical practice.

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.

Background Colorectal cancer (CRC) is the second most common malignancy in developed countries. The mortality is high, with nearly half of patients dying from the disease. The primary treatment of CRC is surgery, and decisions about additional treatment with chemotherapy are based mainly on tumor stage. Novel prognostic markers that identify patients at high risk of recurrence and cancer-related death are needed. The development of CRC has been described in terms of two different pathways; the microsatellite instability (MSI) and chromosomal instability (microsatellite stable, MSS) pathway. More recently, the CpG island methylator phenotype (CIMP), characterized by frequent DNA hypermethylation, has been described as an alternative pathway of tumorigenesis. The event of DNA methylation is dependent on one-carbon metabolism, in which folate and vitamin B12 have essential functions. The purpose of this thesis was to study CIMP in CRC. The specific aims were to investigate the potential role of components of one-carbon metabolism as risk factors for this subgroup of tumors, and the prognostic importance of CIMP status, taking into consideration important confounding factors, such as MSI and tumor-infiltrating T cells. Methods CRC cases and referents included in the Northern Sweden Health and Disease Study (NSHDS, 226 cases and 437 referents) and CRC cases in the Colorectal Cancer in Umeå Study (CRUMS, n=490) were studied. Prediagnostic plasma concentrations of folate and vitamin B12 were analyzed in NSHDS. In both study groups, CIMP status was determined in archival tumor tissue by real-time quantitative PCR using an eight-gene panel (CDKN2A, MLH1, CACNA1G, NEUROG1, RUNX3, SOCS1, IGF2 and CRABP1). MSI screening status and the density of tumor-infiltrating T cells were determined by immunohistochemistry.  Results An inverse association was found between plasma concentrations of vitamin B12 and rectal, but not colon, cancer risk. We also found a reduced risk of CIMP-high and CIMP-low CRC in study subjects with the lowest levels of plasma folate. We found that patients with CIMP-low tumors in both NSHDS and CRUMS had a poorer prognosis compared with CIMP-negative, regardless of MSI screening status. We also found that MSS CIMP-high patients had a poorer prognosis compared with MSS CIMP-negative. The density of tumor-infiltrating T cells and CIMP status were both found to be independent predictors of CRC patient prognosis. A particularly poor prognosis was found in patients with CIMP-low tumors poorly infiltrated by T cells. In addition, the density of T cells appeared to be more important than MSI screening status for predicting CRC patient prognosis. Conclusion Rather than being one disease, CRC is a heterogeneous set of diseases with respect to clinico-pathological and molecular characteristics. We found that the association between risk and plasma concentration of vitamin B12 and folate depends on tumor site and CIMP status, respectively. Patient prognosis was found to be different depending on CIMP and MSI screening status, and the density of tumor-infiltrating T cells.

Hypoxia is a common feature of solid tumors and one of the hallmarks of tumor microenvironment. Tumor hypoxia plays an important role in angiogenesis, malignant progression, metastatic development, chemo-radio resistance and favours immune evasion by the emergence of tumor variants with increased survival and anti-apoptotic potential. There is very little work done on the impact of tumor hypoxia on the regulation of tumor susceptibility to the lysis induced by cytotoxic antitumor response. Therefore, we asked whether hypoxia confers tumor resistance to cytotoxic T lymphocyte (CTL)-mediated killing. We demonstrated that exposure of target cells to hypoxia has an inhibitory effect on the CTL-mediated autologous target cell lysis. Such inhibition was not associated with an alteration of CTL reactivity and tumor target recognition. We also showed that the concomitant hypoxic induction of Signal transducer and activator of transcription 3 (STAT3) phosphorylation on tyrosine 705 residue (pSTAT3) and hypoxia inducible factor 1 alpha (HIF-1α) is functionally linked to the alteration of Non small cell lung carcinoma (NSCLC) target susceptibility to CTL-mediated killing. We also showed that hypoxia-induced resistance of lung tumor to CTL-mediated lysis was associated with autophagy induction in target cells. Inhibition of autophagy resulted in impairment of pSTAT3 (via inhibition Src kinase) and restoration of hypoxic tumor cell susceptibility to CTL-mediated lysis. Moreover, in vivo inhibition of autophagy by hydroxychloroquine (HCQ) in B16F10 tumor bearing mice and mice vaccinated with TRP2 peptide dramatically increased tumor growth inhibition. Collectively, the current study establishes a novel functional link between hypoxia-induced autophagy and the regulation of antigen specific T cell lysis and points to a major role of autophagy in the control of in vivo tumor growth.Finally, as resistance of tumor targets to killer cells is likely to be regulated by multiple factors, we further aimed to identify the microRNA's regulated by hypoxia in NSCLC and melanoma and their putative involvement in the regulation of tumor susceptibility to antigen-specific CTL-mediated killing. MicroRNA-210 (miR-210) was significantly induced in a HIF-1α dependent manner in NSCLC and melanoma cells and miR-210 was expressed in hypoxic zones of human NSCLC tissues. Moreover, we demonstrated that hypoxia-induced miR-210 regulates tumor cell susceptibility to CTL-mediated lysis in part by suppressing PTPN, HOXA1 and TP53I11 expression indicating that miR-210 plays a potential role in the regulation of anti-tumor immune response.

Infection is a leading cause of death in hemodialysis patients, partly due to dysfunctional immunity. Frequent dialysis therapy improves patient outcomes and quality of life. We hypothesize that extended home hemodialysis (EHHD) also improves immune function compared to conventional in-hospital hemodialysis (CHD); therefore, we designed a prospective matching-cohort clinical study to assess serum inflammatory markers and the functional capacity of monocyte-derived dendritic cells (MDDCs) and T-lymphocytes. Serum CRP was decreased in EHHD patients suggesting that extended dialysis may decrease inflammatory solute/cytokine levels. Compared to controls, MDDCs from hemodialysis patients had similar endocytic capacity, expression of co-stimulatory molecules, and T-cell activation capacity. However, CHD was associated with the highest expression of CD83 and CD40. Activated T-cells in CHD patients also produced significantly more immunosuppressive IL-10 compared to EHHD patients and controls. Therefore, EHHD may improve immune function by decreasing inflammation, MDDC pre-activation, and synthesis of immunosuppressive cytokines.

Throughout history, human populations have continuously been challenged by new and emerging infectious diseases. For the past 26 years, sub-Saharan Africa and other countries around the world have been dealing with a pandemic caused by a relatively new pathogen called the human immunodeficiency virus (HIV). Although antiretroviral (ARV) therapies effectively reduce morbidity and mortality rates, the long term use in those who have access to treatment inevitably leads to drug-related toxicity and resistance. Even with a strong commitment from governments to expand and finance prevention and treatment programs, transmission rates continue to outpace the benefits of these efforts. Therefore to effectively eradicate the disease, research is focusing on the design of protective and therapeutic vaccines. The first major step in designing these alternative therapies is to define correlates of immune protection. / The research presented in this thesis focuses on characterizing the quantitative and qualitative features of T cell immune responses in individuals who spontaneously control viral replication and exhibit a benign course of disease while remaining off ARV therapy. A comprehensive analysis of HIV-specific IFN-gamma secreting immune responses revealed that neither the breadth nor the magnitude of responses directed against the entire HIV proteome accurately predicts the viral load or rate of CD4 decline. Subsequent analyses showed that the preferential targeting of Gag was associated with reduced rates of CD4 decline and was later confirmed in a cohort of individuals in primary infection whereby the relative breadth and magnitude of Gag p24 was inversely correlated with viral load set point. / The maintenance of polyfunctional immune responses in HIV-infected subjects with a benign course of disease prompted us to develop a method that could comprehensively assess the breadth, magnitude and specificity of three functionally distinct subsets of HIV-specific lymphocytes (single IFN-gamma, single IL-2 and dual IFN-gamma/IL-2 secretors). Survey of immune responses in chronically infected individuals revealed that only the breadth and magnitude of dual IFN-gamma/IL-2 secreting lymphocytes correlated with reduced viral loads and increased CD4 counts suggesting that secretion of IFN-gamma alone was a poor correlate of protection. We also showed that the contribution of polyfunctional lymphocytes to the total response was greater for epitopes restricted by major histocompatibility complex (MHC) class I alleles associated with slow disease progression compared to those restricted by alleles associated with rapid or neutral rates of HIV disease progression. / Taken together, this work supports the view that immune monitoring of infected and vaccinated individuals should include methodologies capable of detecting both IFN-gamma and IL-2 secretion from responding T lymphocytes. The studies presented here have furthered our understanding of what constitutes protection from disease progression emphasizing that both specificity and polyfunctionality are features of effective control of viral replication.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 1, 2010). Vita. Thesis advisor: Habib Zaghouani. "May 2008" Includes bibliographical references.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 181-228.