Patients suffering from transsexualism (TS) who apply for sex reassignment surgery (SRS) go through a complex evaluation process before being accepted for treatment. In general, the results from SRS are satisfying. However, further knowledge is needed to clearly delineate transsexualism from other related gender identity disorders (GID) and to improve the selection of candidates for SRS. Personality has for a long time been considered as the key concept for that purpose but systematic studies using reliable instruments are lacking. The present study aims at improving the assessment procedure, validating the concept of transsexualism and studying the outcome of SRS and important prognostic factors. Two methodological studies deal with the development and validation of two self-report instruments based on DSM-III-R: SCID screen covering Axis II personality disorders/traits and Global Assessment of Functioning (GAF-scale, Axis V). SCID screen diagnoses of personality disorders (PD) were compared with diagnoses from independent structured interviews by means of the SCID-II. The overall kappa in identifying a PD was 0.78 varying from 0.34 to 0.81 for the specific PDs when cut-off was adjusted. When applied to a group of GID-patients SCID screen diagnoses agreed well with clinical diagnoses (kappa 0.77). Self-report of the GAF also proved to be a reliable (overall Pearson r=0.62) and useful method and the study lends further support to the validity of Axis V. In three papers a group of 19 transsexuals was studied by means of a) SCID screen to examine their personality in a dimensional and traditional categorical way, b) the GAF-scale to study psychosocial functioning, c) Structural Analysis of Social Behavior (SASB) to examine self-image and d) Defense Mechanism Test (DMT) to analyze psychological defense structures from a psychodynamic perspective. Patients with atypical gender identity disorders (GIDAANT) and patients with borderline personality disorders as well as healthy subjects were used as contrast groups. Among the transsexuals 10 out of 19 had an additional axis I disorder and 37% had at least one PD, predominantly within cluster B. When analyzed dimensionally according to SCID screen, frequent subthreshold personality pathology was found and biological women fulfilled more axis II criteria than men. TS had less axis I and II pathology compared with GIDAANT and psychiatric patients. According to SASB, TS had a positive self-image with both self-control and spontaneous self and predominating self-love. They appeared significantly more healthy on self-image measures than GIDAANT patients. The DMT revealed a different pattern; TS patients were more disturbed in several areas than patients with borderline personality disorder. TS showed no ”emotional investment” and poorer reality orientation in contrast to both healthy controls and the borderline group but shared a similar pregenital pathology with the borderline patients. Finally, five-year outcome was studied among the transsexuals from a multidimensional approach (e.g. work, interpersonal relations, partnership, subjective opinion) and related to index- measurements on DSM-III-R, SCID screen, GAF, SASB and DMT. Based on combined outcome variables, 68% of the subjects were judged to have improved and 16% had an unsatisfactory outcome. One single case regretted the sex change. SCID screen pathology and SASB disturbances emerged as significant predictors for negative outcome, as well as male biological sex and lack of partnership. It was concluded, that although outcome is in general very favorable, the instruments under investigation, in particular SCID screen and SASB, revealed valuable prognostic information and they are suggested to become part of the future routine assessment of candidates for SRS. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1995, härtill 6 uppsatser.</p> / digitalisering@umu

Interest in cellular immunotherapy has increased with the recognition of the pivotal role that dendritic cells (DC) play in the adaptive immune system. The preparation of DC to present tumour antigens and subsequent induction of tumour specific T cells have been widely documented. This thesis studied the ability of cord blood (CB) stem cells to differentiate into functional CD34+DC, followed by the optimisation of electroporation of RNA into these cells. Total RNA derived from a leukaemic cell line and a primary human leukaemic sample was electroporated into CD34+DC DC and we were able to generate anti-leukaemic cytotoxic T lymphocytes (CTL). The CTL specifically targeted leukaemia but not normal cells. While the in vitro data showed promising results of the CTL specificity, a NOD-SCID model of human ALL was established to allow the CTL to be tested in vivo. We established a reproducible model of human ALL in NOD-SCID mouse using four primary human ALL samples. The adoptively transferred anti-leukaemic CTL into the ALL bearing NOD-SCID mice showed that ALL engraftment was significantly delayed. However, the addition of total RNA loaded CD34+DC DC did not enhance the in vivo CTL effect. Lastly, by dissecting the CTL response, we found that the polyclonal CTL were targeting survivin, HM1.24 and CT-7 antigens. The CTL clones generated from these polyclonal CTL showed high specificity for leukaemia but not normal cells. In conclusion, these preliminary data support the use of total RNA electroporated CD34+DC as a means of inducing anti-leukaemic CTL, and have demonstrated the efficacy of the CTL in a NOD-SCID model of ALL. This study has also provided insight into the polyclonal CTL response and future studies will likely continue along this path.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 62 pages. Includes Vita. Includes bibliographical references.

La maladie de Fabry est une maladie lysosomale présentant une grande hétérogénéité phénotypique et génotypique. Elle est causée par des mutations au niveau du gène GLA, situé sur le chromosome X, entrainant un déficit de l'enzyme alpha-galactosidase A. Celui-ci mène à une accumulation de globotriaosylcéramide (Gb3), de globotriaosylsphingosine (lyso-Gb3) et de galabiosylcéramide (Ga2) et leurs isoformes et analogues respectifs qui sont utilisés comme biomarqueurs pour la maladie de Fabry. Il est possible de les quantifier dans les liquides biologiques tels que le plasma et l’urine des patients. Le principal traitement consiste en une thérapie d’enzyme de remplacement (TER) qui n’est pas efficace pour tous les patients, car des complications peuvent survenir suite à une réponse auto-immune contre l’enzyme infusée. Le développement d’autres thérapies est au coeur de la recherche actuelle. La thérapie génique à l’aide d’un vecteur viral pour cette maladie lysosomale est en développement au Canada. Un modèle de souris NOD/SCID/Fabry (NSF) a été généré pour le développement du vecteur viral, la compréhension de la distribution des biomarqueurs au niveau des tissus de différents organes et l’évaluation des effets du traitement. Ce modèle est une souris avec une inactivation complète du gène GLA ainsi que l’absence de système immunitaire. Les objectifs du projet de maîtrise étaient donc de: 1) développer et valider une méthode pour l’homogénéisation des tissus, l’extraction et le dosage du Gb3 et ses isoformes/analogues dans les tissus de différents organes de souris Fabry et souris contrôles à l’aide de la chromatographie liquide ultra performance couplée à la spectrométrie de masse en tandem (UPLC-MS/MS); 2) comparer les profils de distribution des biomarqueurs dans différents organes pour des souris atteintes de la maladie de Fabry et des souris contrôles. L’analyse des souris NSF a permis d’établir un profil de biomarqueurs comportant jusqu’à 22 isoformes pour chaque organe et de mettre en évidence des différences significatives entre la distribution du Gb3 dans lesdits organes. La quantité la plus élevée de Gb3 se retrouve au niveau de la rate, suivis du petit intestin, des reins, des poumons, du coeur, du foie et du cerveau. Les isoformes formés d’un acide gras sans insaturation sont les plus abondants dans l’ensemble des organes. Un transfert technologique sera effectué pour l’analyse des biomarqueurs dans des échantillons de plasma de patients Fabry ayant reçu la thérapie génique. En conclusion, la méthode mise au point est sensible et offre un outil efficace pour l’analyse des tissus de différents organes de souris par la production d’un profil de biomarqueurs, ce qui peut aussi mener à de meilleurs outils pour monitorer les patients atteints de la maladie de Fabry.

The immune system is responsible for defending a host animal from a wide variety of threats. Manipulation of the immune system can result in beneficial outcomes such as immunity to pathogens, or deleterious outcomes such as autoimmunity. Advances in our understanding of how the immune system develops and functions have benefited greatly from studies in animals, particularly in mice where the genetics are well known and a multitude of reagents are readily available for experimental use. Although much has been learned from animal experimentation, it must be cautioned that animals are not humans. Unforeseen outcomes and complications often arise when translating research obtained in animal models to treatment of human patients. A small animal model in which the human immune system can be established and manipulated experimentally in vivo would be valuable for the study of human immune responses in infectious diseases, transplantation, and autoimmunity, and ultimately for translation of these findings to the human patient. Contribution to this model is the overall goal of this thesis. The severe combined immunodeficiency (Prkdcscid, termed scid) mutation was discovered in 1983 in the C.B-17 strain of mice. Scid mice lack functional T and B lymphocytes and are unable to mount immune responses or reject allogeneic or xenogeneIc grafts. However, C.B-17-scid mice do develop normal or even elevated innate immune function. Based on the ability of scid mice to accept xenografts, human peripheral blood mononuclear cells have been injected to generate "Hu-PBL-scid mice." These Hu-PBL-scid mice have been proposed as an in vivo model of the human immune system. Although this model was described over 12 years ago, there are a number of obstacles that impede its ability to recapitulate human immunity. The Hu-PBL-scid mouse model established using C.B-17-scid mice as recipients is hindered by 1) low levels of human leukocyte engraftment, 2) engraftment of predominately memory/activated human T cells with specificity directed against host murine MHC antigens, 3) rapid transition of engrafted human lymphocytes to a functionally anergic state, and 4) a paucity of knowledge providing an understanding of the mechanisms that underlie engraftment and function of the human lymphocytes in the immunodeficient hosts. This thesis was initiated at the time the NOD-scid mouse became available for establishment of Hu-PBL-scid mice, and this model has subsequently been termed "Hu-PBL-NOD-scid." The NOD-scid mouse was designed as an improved recipient for human PBL based on its innate immune characteristics. Defects in innate immunity in wild-type NOD/Lt mice include reduced NK cell activity, defects in macrophage development and function, and a lack of hemolytic complement. NOD-scid mice retained these characteristics, and engraft human cells at higher levels compared to C.B-17-scid mice. However, the ratio of CD4+ to CD8+ T cells in Hu-PBL-NOD-scid mice remained skewed towards the CD8+ population, similar to that of the Hu-PBL-C.B-17-scid mouse. My thesis was based on the overall hypothesis that additional genetic manipulation of the recipient strain would enhance further the engraftment and function of human cells. Based on the engraftment results obtained in NOD-scid mice, we performed these genetic manipulations using the NOD-scid strain as the reference. We first hypothesized that removal of MHC expression would lower human anti-mouse xenoreactivity and enhance engraftment of naïve T cells, a cell population not readily detectable in Hu-PBL-C.B-17-scid or Hu-PBL-NOD-scid mice. Towards this goal, the NOD-scid mouse expressing a targeted mutation for beta-2 microglobulin (B2mnull) mouse was created by Dr. Leonard Shultz at the Jackson Laboratory. Because B2m is required for expression of MHC class I and for development of functional NK cells, we predicted that NOD-scid-B2mnull mice would first exhibit a normalized CD4:CD8 T cell ratio resulting from reduced CD8 engraftment due to decreased human anti-mouse MHC class I reactivity. Since MHC class I molecule expression is required for the development of NK cells, we further predicted that there would be a reduction of NK cell activity, permitting enhanced engraftment. Data presented in this thesis demonstrates that human PBL engraft in NOD-scid-B2mnull mice at levels higher than NOD-scid mice, and with an increased CD4:CD8 T cell ratio. The mechanism(s) responsible for the increased engraftment of human cells in these mice became a major focus of this thesis. This thesis is composed of three Specific Aims. Specific Aim 1 was to determine the mechanism(s) underlying human cell engraftment and function in Hu-PBL-scid mice. These data are contained in Part 1 of the Results. Specific Aim 2 was to elucidate the costimulation interactions between human T cells and murine host APCs that control the level of engraftment and activation state of the human lymphocytes. These data are contained in Part 2 of the Results. Specific Aim 3 was to utilize these fundamental observations to initiate studies into the induction of primary human immune responses in Hu-PBL-scid mice. Although the goal of these latter studies was not attained, the data from the experiments performed is provided in Part 3 of the Results, and is discussed relative to future directions for this arm of the project. In Part 1 of the Results, we utilized in vitro and in vivo techniques to study the underlying mechanisms regulating human cell engraftment and function in scid mice. We observed that the absence of mouse MHC class I antigens in NOD-scid-B2mnull mice does not reduce the stimulatory capacity of APCs toward human T cells in vitro. We further demonstrated that naïve human T cells persist for at least 2 weeks in the peritoneal cavity of Hu-PBL-NOD-scid and Hu-PBL-NOD-scid-B2mnull mice. However, in both strains of recipients, the human cells progress to an anergic phenotype as documented by cell surface molecule expression and by functional activity. We further documented that the increased engraftment of human CD4+ T cells observed in NOD-scid-B2mnull mice is due predominantly to the ablation of host NK cell activity. Increased engraftment of human CD4+ cells in NOD-scid-B2mnull mice can be recapitulated in NOD-scid mice by antibody-mediated depletion of residual host NK cells. Finally, we demonstrated that expression of MHC class II molecules by recipient mice facilitated stimulation and engraftment of human cells. However, mouse MHC class II expression is not required for human cell engraftment into scid mice. In Part 2 of the Results, we addressed the cellular and costimulatory interactions of engrafted human cells in scid mice. Costimulatory interactions between T cells and APC are now known to be critical for the proper activation and functioning of cells in the immune system. Understanding the role of costimulatory interactions between human T cells, human APCs, and mouse APCs became a major focus of this thesis. We demonstrated that human CD4+ T cells are required for the engraftment of human CD8+ T cells. The mechanism by which human CD4+ cells mediate this "helper" activity requires expression of CD154. Antibody-mediated blockade of CD154 in vivo abrogates human cell engraftment in scid mice. The role of host APCs in the engraftment of human lymphocytes was demonstrated by blocking host CD40 with antibody. Preventing human T cell CD154 interaction with host CD40 on murine APCs blocked human cell engraftment in scid mice, demonstrating the importance of "trans-costimulation" in human T cell activation. This "trans-costimulation" appeared to be mediated by B7 expression on mouse APCs. We further demonstrated that in vivo depletion of human CD8+ T cells in Hu-PBL-NOD-scid mice leads to increased levels of human CD4+ T cells, elevated human immunoglobulin in the serum, and increased incidence of EBV-related lymphoproliferative disorders. These observations suggested that human CD8+ T cells are able to regulate human CD4+ T cell help and provide "surveillance" activity for EBV-related lymphoproliferative disorders. In Part 3 of the Results, we used the Hu-PBL-scid mouse to initiate experiments designed to generate primary human immune responses in vivo. These experiments were based on our observation that few if any human APCs survive in scid mice, and on reports that dendritic cells (DC) are required for activation of naïve T cells and initiation of a primary immune response. We used recombinant viruses expressing HIV-1 proteins that are being developed as potential vaccines for HIV as our immunizing reagent. For APCs, we used DCs from NOD-scid mice expressing human MHC class II molecules as the source of APCs presenting antigen to the engrafted human T cells in the scid mice. Our attempts to induce a primary immune response using DC from human MHC-transgenic NOD-scid mice were unsuccessful, as were direct immunization protocols. The results section, however, does highlight deficiencies that could be approached experimentally in future studies. In summary, the results presented in this thesis advance our understanding of the fundamental mechanisms controlling human cell engraftment in scid mice. This information supports the long-term goal of establishing a functional human immune system in a small animal model. We have identified many of the cell interactions and factors that regulate human cell engraftment and function in scid mice, and we have provided insights into host characteristics that will provide optimized engraftment of naïve human T cells. The studies led to the novel observations of the regulation of human CD4+ T cells by human CD8+ T cells, B cell activation, and progression of latent EBV infection to lymphoproliferative disorders in vivo. These studies further provide new information regarding "trans-costimulation", a previously unrecognized mechanism of T cell activation. These results provide data on the fundamental mechanisms that underlie obstacles to the goal of achieving engraftment of a functional human immune system in scid mice.

No description available.

Utilization of tolerogenic dendritic cells (tolDCs) as a cell-based therapy represents a promising strategy in treatment of autoimmune diseases including type 1 diabetes (T1D). Numerous protocols have been established to generate tolDCs ex vivo and their therapeutic effect has been demonstrated in animal models of autoimmune diseases. In this thesis we compared three different variants of such protocols which are based on the combined treatment of bone marrow- derived DCs with vitamin D and dexamethasone applied at different time points of their maturation towards tolDCs. We assessed the efficiency of these protocols in regards of their effect on the expression of co-stimulatory molecules CD40, CD80, CD86, and MHC II and the chemokine receptor CCR7 on the surface of tolDCs. Then, we evaluated the migration pattern of antigen unloaded tolDCs in vivo as well as their effect on the induction of immune responses and cell proliferation of lymph node cells. This was achieved by labelling of tolDCs with membrane dye PKH26 and by following their migration path by flow cytometry after intraperitoneal (i.p) or subcutaneous (s.c.) injection into either left or right side of the body. On day 1, 3, 5, 7, and 9, the presence of PKH26+ tolDCs was examined in spleen, pancreatic, mesenteric, inguinal and axillary...

In der humanen Haut spielen antimikrobielle Peptide (AP) bei Entzündungsgeschehen bakteriellen und nicht-bakteriellen Ursprungs eine bedeutende Rolle. Neben einer konstitutiven Expression AP können Zytokine deren vermehrte oder abgeschwächte Expression bewirken. In dieser Arbeit wurden die AP humanes β-Defensin (HBD) -2, HBD-3 und Psoriasin (PSO) in Bezug auf deren Expression in gesunder Haut und deren Modulation durch Zytokine in vivo anhand des xenogenen NOD-SCID-Maus-Transplantationsmodells untersucht. Nach erfolgreicher Transplantation von humaner Haut auf NOD-SCID Mäuse wurden die Zytokine TNF-α, IFN-γ und IL-13 in unterschiedlicher Dosierung einzeln und in Kombination intradermal appliziert. Für TNF-α konnte eine erhöhte Expression von HBD-2, HBD-3 und PSO auf RNA-Ebene mittels in-situ-Hybridisierung und Protein-Ebene mittels immunhistochemischer Nachweismethoden gezeigt werden. Eine erhöhte Expression nach Injektion von IFN-γ ließ sich für HBD-3 auf RNA-Ebene und Protein-Ebene und für HBD-2 auf RNA-Ebene erfolgreich belegen. PSO zeigte auf Protein-Ebene nach Modulation mit IFN-γ eine bei höherer Dosierung leicht abnehmende Expression. Eine Änderung der Expression durch IL-13 ließ sich nicht eindeutig belegen. In dieser Arbeit konnte die in der Literatur in vitro beschriebene Modulationsfähigkeit der untersuchten AP durch die verwendeten Zytokine in vivo belegt werden.

The chimeric Alb-uPA SCID mouse that has been transplanted with human hepatocytes is a model to facilitate in vivo study of HCV. We explored further development of the model by using repopulation with immortalized human hepatocytes (IHH) in place of primary human hepatocyte (PHH) transplantation to support HCV infection. In vitro HCV studies typically utilize a human hepatoma cell line (Huh7) and rely on transfection with transcribed genomic RNA derived from a unique HCV strain (JFH1). Unfortunately, this system has not been successful in support of infection with serum-derived HCV (HCVser). IHH may offer an alternative since their differentiation status remains close to that of PHH. IHH transfected with HCV RNA (H77 or JFH1) or infected with HCVser showed stable intracellular and supernatant HCV RNA by real-time RT-PCR. IHH showed intracellular HCV NS3 proteins. HCV transfected or infected IHH secrete infectious HCVcc for in vivo and vitro. / Experimental Surgery

Long segment tracheal obstruction is a potentially fatal condition arising from primary malignancy, congenital tracheal stenosis, or benign strictures. Shorter lesions are amenable to primary surgical repair, but anastomotic tension remains a limiting factor. Attention has therefore turned towards tissue engineered interposition grafts, consisting of cartilage, vascular, and airway epithelial cell layers. For clinical applications, it is essential to develop tools for noninvasive monitoring of in vivo graft development. This investigation therefore sough to optimize a tracheal patch and tube model suitable for SCID mouse implantation, and to demonstrate simultaneous tracking of human chondrocytes and epithelial cells labeled with contrasting intracellular magnetic resonance labels. Cartilage labeled with iron oxide nanoparticles was successfully distinguished from unlabeled control tissue both in vitro and in vivo. Gadolinium dendrimer labeling of adjacent airway epithelial cells, however, did not initially permit enhanced visualization. Further model optimization is required for simultaneous cell tracking.