Dissertations / Theses on the topic 'Immunodeficit'

Bone defects resulting from non-unions, fractures, significant revision joint replacements, tumour resection and osteolysis present a clinical problem. While autografts are considered the gold standard, ubiquitous use of this reparative technique is limited by graft supply and site morbidity. Recent progresses in tissue engineering using stem cells, bone enhancing molecules and gene therapy have provided more hypotheses for bone defect treatment. In vivo assessment to test these hypotheses requires animal models to mimic human conditions. Immunodeficient or nude animals have the advantage of hosting materials from human and other xenographic origins without immuno-intolerance or rejection. A thorough understanding of the biology in nude animals is vital for the further advancement of connective tissue healing and regeneration strategies. Nude mice are excellent xenographic hosts for in- vivo characterisation and provide a reproducible animal source. The immune deficiencies of nude compared to normal animals may however, influence bone healing and need to be addressed. This dissertation (a) investigated potential bone defect models in nude mice and nude rats (b) incorporated the selected bone defect model to evaluate the effect of T cell deficiency and age on bone defect healing in nude animals (c) determined the feasibility of a critical size defect (CSD) in nude mice. A distal-femur-condylar-defect (DFCD) model was successfully performed in nude mice and rats. The model was found to have some advantages as a bone defect model: (1) located at a weight-bearing skeletal site (2) no requirements for an internal or external fixator (3) does not obstruct or limit mobility (4) location is not in close proximity to any major organs such as the brain (5) easy identification of surface anatomy (6) defect size is standardised and reproducible (7) does not require lengthy and complicated surgery and (8) cost effective. This dissertation confirmed that bone healing in nude mice is similar to that of normal immunocompetent mice. Absence of T lymphocytes did not delay or inhibit bone repair. Use of older nude mice did not seem to affect the healing rate, in contrast to older normal mice, which showed delay in bone healing in the initial phase. Establishment of critical sized defects in mice at a weight-bearing location was not feasible due to the robust healing of murine. This dissertation recommends that the DFCD model could be utilized for the assessment of xenogenic materials at early time point.

Animal models have been relevant to study the molecular mechanisms of cancer and to develop new antitumor agents. Anyway, the huge divergence in mouse and human evolution made difficult the translation of the gained achievements in preclinical mouse based studies. The generation of clinically relevant murine models requires their humanization both concerning the creation of transgenic models and the generation of humanized mice in which to engraft a functional human immune system, and reproduce the physiological effects and molecular mechanisms of growth and metastasization of human tumors. In particular, the availability of genotypically stable immunodepressed mice able to accept tumor injection and allow human tumor growth and metastasization would be important to develop anti-tumor and anti-metastatic strategies. Recently, Rag2-/-;gammac-/- mice, double knockout for genes involved in lymphocyte differentiation, had been developed (CIEA, Central Institute for Experimental Animals, Kawasaki, Japan). Studies of human sarcoma metastasization in Rag2-/-; gammac-/- mice (lacking B, T and NK functionality) revealed their high metastatic efficiency and allowed the expression of human metastatic phenotypes not detectable in the conventionally used nude murine model. In vitro analysis to investigate the molecular mechanisms involved in the specific pattern of human sarcomas metastasization revealed the importance of liver-produced growth and motility factors, in particular the insulin-like growth factors (IGFs). The involvement of this growth factor was then demonstrated in vivo through inhibition of IGF signalling pathway. Due to the high growth and metastatic propensity of tumor cells, Rag2-/-;gammac-/- mice were used as model to investigate the metastatic behavior of rhabdomyosarcoma cells engineered to improve the differentiation. It has been recently shown that this immunodeficient model can be reconstituted with a human immune system through the injection of human cord blood progenitor cells. The work illustrated in this thesis revealed that the injection of different human progenitor cells (CD34+ or CD133+) showed peculiar engraftment and differentiation abilities. Experiments of cell vaccination were performed to investigate the functionality of the engrafted human immune system and the induction of specific human immune responses. Results from such experiments will allow to collect informations about human immune responses activated during cell vaccination and to define the best reconstitution and experimental conditions to create a humanized model in which to study, in a preclinical setting, immunological antitumor strategies.

The nervous system and immune system contact one another through two-way communication in order to establish and preserve homeostasis. The sympathetic neurotransmitter norepinephrine has an impact on how the immune system responds by affecting regional blood flow and activation of adrenergic receptors on leukocytes. Former studies showed that immune cells are capable of releasing nerve growth factor allowing for the establishment and continuation of sympathetic nerves in targeted tissues. From this gathered information, it was hypothesized that sympathetic nerves would prove to be less frequent in spleens from the immunodeficient R2G2 mouse strain (Envigo) when compared to 129P3/J (129) and C57BL/6 (C57) strains. R2G2 mice are an immunodeficient strain that lacks functional T, B, and natural killer cells. Ten to eleven week aged-matched male mice were measured by body weight, spleen weight, and temperature. Spleens were cut and fixed for histological investigation. Sympathetic nerves were labeled by immunostaining tyrosine hydroxylase (TH). Hematoxylin & eosin (H&E) was used to stain spleen sections in order to evaluate cytoarchitecture. Von Willebrand factor (VWF) was used to immunostain for megakaryocytes. R2G2 mice showed slightly higher temperatures and body weights but yielded a significantly smaller spleen weight (R2G2, 38.20 ± 1.48; 129, 65.08 ± 11.71; C57, 81.33 ± 8.38; P< 0.0001, ANOVA). TH stain revealed sympathetic innervation in all strains but location and morphology differed in R2G2 mice compared to controls. Control spleens had nerves which entered white pulp regions of the spleen and were closely related to leukocytes. Fiber profiles in the controls were filamentous with small acute bends. R2G2 differed by having (TH+) nerve fibers more associated with arteries and less localized in the surrounding parenchyma. The fibers were abnormally swollen and held a more granular shape instead of a filamentous shape. The H&E stain showed clear red and white pulp zones in the control spleens with 129 showing more distinct germinal centers than C57. R2G2 H&E sections showed cytoarchitecture with indistinct pulp areas. VWF staining revealed R2G2 mice had an abundant amount of megakaryocytes versus control mice megakaryocyte counts (R2G2, 11.28 ± 3.87 per 20X field; 129, 1.73 ± 0.70; C57, 1.42 ± 0.13; P< 0.0001, ANOVA) and extramedullary hematopoiesis was highly prominent. This evidence supports that leukocytes secrete neurotrophic factors or are vital to establishing normal growth of TH+ nerves toward the white pulp. Leukocytes may not be required for sympathetic innervation of blood vessels in the spleen, however, lack of leukocytes shows TH+ nerve fibers with abnormal morphology in severely immune threatened mice.

The experiments described in this thesis investigated the growth of bovine cells infected with Theileria sp. macroschizonts in various strains of immunodeficient mice. It was hoped that it would be possible to create a system for the growth of Theileria infected cells in vivo without using bovine hosts. Previous work had shown that T.parva infected cells would establish as subcutaneous tumours in irradiated Swiss and nude mice (Irvin et al, 1977; Veterinary Parasitology 3, p.141-160). Most of the work in this thesis concerned the related parasite, T.annulata. T.anmilata infected cells were found to grow as subcutaneous tumours in irradiated Balb/c, irradiated NIMR and Balb/c nude, and unirradiated C.B-17 scid (severe combined immunodeficiency) mice. Infected cells failed to establish in C57 beige mice, with or without irradiation. The growth and survival of the tumours was dependent on the degree of immunosuppression of the host, and the size of the cell dose administered. In Balb/c mice, T.annulata tumours regressed as mice recovered from irradiation. Analysis of lymphocyte subsets using a fluorescence-activated cell sorter (FACS) showed differential susceptibility of B-cells, T-helper and cytotoxic T-cells to a sublethal dose of ionising radiation (46y). The numbers of these lymphocytes increased more rapidly after irradiation in tumour bearing mice than in those without tumours. In irradiated (46y) Balb/c nude and scid mice, subcutaneous T.annulata tumours failed to regress, despite the development of general haemorrhage and central necrosis. In scid mice, high doses (2xl07) of T.annulata infected cells injected intraperitoneally gave rise to ascites. However, low doses (2xl06 cells) did not. The presence of macroschizont infected cells in the peritoneal cavity was accompanied by a proliferation of macrophages. Several lines of evidence indicated that natural killer (NK) cells were not effective against T.annulata-infected cells in scid mice, but that macrophages were capable of controlling and eliminating the cells, particularly in the peritoneal cavity. In all the strains of mice examined, subcutaneous T.annulata tumours appeared to be damaged by natural immune mechanisms (macrophages) and neutrophils leading to haemorrhage and necrosis. However, the ability of the mice to completely reject the tumours depended on the presence of T and B-cells. Tumour Necrosis (TNF) was not detected in serum, or in tumour extracts. Attempts to induce tumour rejection using a preparation of rabbit TNF were not successful. Attempts to affect the growth of r.annu/ato-infected cells, injected i/p into scid mice, with human alpha and gamma interferons (HulFN-g, were also unsuccessful. The neutralisation of endogenous murine IFN-g with a monoclonal antibody allowed increased growth of T.annulata and 7".parva-infected cells in the intraperi-toneal site in scid mice. Of the mouse strains examined, the scid mouse was the most favourable host for Theileria-infected cells. Attempts to establish uninfected bovine cells in scid mice were not successful, and these results cast doubt on the use of scid mice as hosts for uninfected cells.

Individuals born with primary immune deficiency diseases (PIDD) have a dysfunctional immune system, and many are treated by lifelong injections of immunoglobulin therapy. Studies have shown that these patients have low health-related quality of life (HRQOL) and well-being (WB) and that these outcomes might be improved by the availability of therapy innovated according to preferences for fewer needle sticks or a shorter infusion time. Regulators at the U.S. Food and Drug Administration (FDA) have approved therapies innovated per these preferences. However, there is limited data demonstrating how these innovations impact HRQOL and WB. Using the biopsychosocial model, the purpose of this cross sectional quantitative study was to evaluate whether patients with PIDD using therapies innovated for fewer needle sticks or a shorter infusion time had a higher mean HRQOL and WB compared to those who were not. The study included 153 patients who completed the Patient Reported Outcomes Measurement Information System (PROMIS)-29 survey. The dependent variables were HRQOL and WB measured by PROMIS-29, and the independent variables were the medical product innovations. Independent samples t tests results showed mean PROMIS-29 scores were not statistically different (p > .05). This suggests patients were optimized according to their treatment preference. A subgroup of patients who had taken the PROMIS-29 survey more than once concurrent with switching to a therapy aligned with patient preferences showed improved HRQOL and WB. These findings have implications for positive social change in that seeking the patient's voice to inform medical product innovation and FDA regulatory decision-making has potential to improve biopsychosocial outcomes.

L'antigene ebna-2, premiere proteine virale exprimee lors de l'infection des lymphocytes b par le virus epstein-barr, concourt a leur immortalisation. Cette proteine est egalement detectee in vivo dans certaines lymphoproliferations associees a l'ebv tels que les lymphomes immunoblastiques dont la frequence est fortement augmentee chez les patients infectes par hiv. Par ailleurs, la proteine ebna-2 presente un polymorphisme qui est a l'origine de la distinction de deux types viraux, ebv-1 et ebv-2 dont les proprietes biologiques in vivo restent largement meconnues. Pour etudier les modalites de l'infection par ebv-1 ou ebv-2 dans une population infectee par hiv, 665 patients hiv-seropositifs ont ete serotypes par le biais de cellules transfectees soit par le gene ebna-2a, soit par le gene ebna-2b. Nous avons ainsi montre que l'infection dominante par ebv-2, dont l'emergence est favorisee par l'immunodepression, est 10 fois plus elevee parmi les patients infectes par hiv que dans la population generale. L'etablissement de lignees spontanees a partir du sang peripherique ou de la salive, a permis la caracterisation de la proteine ebna-2 et le genotypage par southern-blot et pcr du type viral dominant. Par ces methodologies, les resultats du serotypage ont ete confirmes, et le type viral chez des individus ne secretant pas d'anticorps anti-ebna-2 (soit environ 25%) ou n'etant pas serotypables a pu etre identifie. La determination du serotype s'est averee etre le reflet du type viral dominant tant au niveau des lymphocytes b d'un individu, qu'au niveau du reservoir epithelial de l'oropharynx. Enfin, par une etude longitudinale, nous avons observe que le type viral dominant n'est pas toujours stable au cours du temps. Le sequencage d'une region du gene ebna-2 chez 2 patients infectes par hiv-1 a montre l'existence de variants a l'interieur du type ebv-1, specifiques de la population infectee par hiv, puisqu'ils n'ont pas ete observes chez 2 individus sains analyses

Trabectedin (ET-743, Yondelis) is a marine alkaloid isolated from the tunicate Ecteinascidia turbinata, cytotoxic against a variety of tumor cell lines in vitro and human tumor xenografts in vivo. It has been approved by EMEA for the 2nd line therapy of soft tissue sarcomas in 2007 and for the 2nd line therapy of ovarian cancer in 2009. Myxoid liposarcoma (ML) is a specific histological subtype within the family of adults soft tissue sarcomas. Specifically >90% of usual myxoid/round cell liposarcomas (MLS/RCLS) are characterized by the chromosomal translocation t(12;16) (q13; pI I), which produces the FUS-CHOP oncogene. Different chimera subtypes seem to respond differently to trabectedin in clinical setting. To elucidate the mechanisms behind the differential sensitivity to trabectedin, tumor myxoid liposarcomas type II and type III were xenografted in nude mice, treated with trabectedin (0.15 mg/kg was injected i.v.) and the binding of FUS-CHOP to some of its target promoters was monitored by Chromatin immuno precipitation to verify the drug ability to displace the binding. We found that trabectedin was more effective on type II than type In ML xenografts. The response to trabectedin in Type II xenografts was associated with partial regression and pathological response. Type III ML xenografts appeared less sensitive to trabectedin and tumors did not regress unless a more prolonged treatment is performed. Molecular analysis revealed that trabectedin was able to remove FUS-CHOP type II and III from its own gene targets 24 hours after treatment. 72 hours after treatment, FUS-CHOP Type III was attached to its targets whereas FUS-CHOP Type II remained unbound. The results suggest that the difference in sensitivity of type II and type III ML tumors to trabectedin is related to a difference in duration of the drug's ability to detach FUS-CHOP from DNA and that type III ML required more intensive and prolonged treatment to achieve a sufficient respond to trabectedin.

Queratinócitos são células bastante atrativas para a transferência gênica ex vivo e liberação sistêmica, uma vez que as proteínas secretadas por estas células podem atingir a circulação via um mecanismo similar ao processo natural. No presente trabalho, queratinócitos transduzidos mediante um vetor retroviral com o gene do hormônio de crescimento de camundongo (mGH) foram submetidos a um tratamento de aderência ao colágeno e à análise clonal, com o intuito de enriquecer esta população de queratinócitos em células-tronco. O principal resultado foi um aumento da viabilidade celular in vitro dos queratinócitos tratados, que poderá se refletir num aumento da durabilidade da secreção do hormônio in vivo, quando realizado o implante de culturas organotípicas em camundongos anões imunodeficientes (lit/scid). Foi também utilizado um modelo de terapia gênica in vivo, baseado na eletrotransferência de DNA plasmidial (naked DNA), contendo o gene do hormônio de crescimento humano (hGH), no músculo quadríceps de camundongos anões (lit/lit) e anões imunodeficientes (lit/scid). Foram padronizadas as condições de eletroporação em 8 pulsos de 50 V e 20 ms com 0,5 s de intervalo, utilizando um plasmídeo com o promotor da ubiquitina C e a sequência genômica do hGH. A administração de uma dose única de 50 g do plasmídeo pUC-UBI-hGH em camundongos lit/scid, seguida de eletroporação, propiciou pela primeira vez na literatura obtenção de níveis sustentáveis na circulação de 1,5-3,0 ng hGH/ml, durante 60 dias. Esses animais tratados com DNA apresentaram um aumento de peso altamente significativo (P<0,001) de 33,1%, comparado a uma perda de peso, não significativa, no grupo controle (camundongos injetados com salina + eletroporação). Os músculos quadríceps dos animais tratados apresentaram um aumento de 48%, quando comparados aos dos animais do grupo controle (P<0,001). Outro estudo de eletrotransferência foi realizado comparando a utilização da sequência genômica do hGH (gDNA) com a complementar (cDNA), em plasmídeos sob o controle do promotor de citomegalovírus (CMV). Foi observado que o cDNA do hGH foi expresso de maneira mais eficiente na circulação de camundongos lit/scid, por um período de 21 dias. Foram também construídos vetores lentivirais com os genes do hGH (cDNA e gDNA) e do mGH (cDNA), que serão utilizados num próximo estudo, tanto para a transdução de queratinócitos, como para a eletrotransferência in vivo. Vetores lentivirais serão de fato necessários para futuros estudos devido a sua reduzida toxicidade em comparação com os retrovirais. Os resultados deste trabalho, assim como as perspectivas de estudo abertas, têm como meta estabelecer condições para que a terapia gênica seja num futuro próximo uma alternativa viável e segura para o tratamento da deficiência de GH e de outras doenças sistêmicas.
Keratinocytes are very attractive cells for ex vivo gene transfer and systemic delivery, since proteins secreted by these cells may reach the circulation via a mechanism which mimics the natural process. In this study, retrovirally transduced keratinocytes with the mouse growth hormone (mGH) gene underwent a treatment for adhesion to collagen and clonal analysis, in order to enrich in stem cells the keratinocyte population. The main result was an in vitro increase of cell viability for treated keratinocytes, which should result in an increase of the in vivo sustainability of hormone secretion, when implanting organotypic cultures onto immunodeficient dwarf (lit/scid) mice. A model for in vivo gene therapy based on the electrotransfer of human growth hormone (hGH)-coding naked DNA in the exposed quadriceps muscle of dwarf (lit/lit) and immunodeficient dwarf (lit/scid) mice was also utilized. Electroporation conditions were optimized, setting up eight 50-V pulses of 20 ms at a 0.5 s interval, using a plasmid containing the ubiquitin C promoter and the genomic hGH sequence. Administration of a single dose of 50 g of this plasmid led, for the first time in the literature, to sustained levels of circulating hGH of the order of 1.5-3 ng/ml, up to 60 days. The DNA-treated animals presented a highly significant weight gain (P<0.001) of 33.1%, compared to a non-significant weight loss of 4.2% for the control group (saline injected mice + electroporation). The injected quadriceps muscles of the DNA-treated mice showed a 48% weight increase, when compared to the control group (P<0.001). Another electrotransfer study was carried out comparing the use of the genomic hGH sequence (gDNA) with the complementary sequence (cDNA), cloned under the control of the cytomegalovirus (CMV) promoter. It was observed that hGH was more efficiently expressed in the circulation of lit/scid mice, for 21 days, when injecting cDNA. Lentiviral vectors containing the hGH (cDNA and gDNA) and the mGH (cDNA) genes were also constructed and will be used in a next study, both for the transduction of keratinocytes or for in vivo electrotransfer. Lentiviral vectors will in fact be necessary for future studies, considering their reduced toxicity when compared to retroviral vectors. The results of this work, as well as opening perspectives of new studies, will establish in the near future conditions for gene therapy as a feasible and safe option for the treatment of GH deficiency and other systemic diseases.

Doctor of Philosophy
Department of Anatomy and Physiology
Deryl L. Troyer
Many cell types were known to have migratory properties towards tumors and different research groups have shown reliable results regarding cells as delivery vehicles of therapeutics for targeted cancer treatment. Present report discusses proof of concept for 1. Cell mediated delivery of Magnetic nanoparticles (MNPs) and targeted Magnetic hyperthermia (MHT) as a cancer treatment by using in vivo mouse cancer models, 2. Cells surface engineering with chimeric proteins for targeted cancer treatment by using in vitro models. 1. Tumor homing cells can carry MNPs specifically to the tumor site and tumor burden will decrease after alternating magnetic field (AMF) exposure. To test this hypothesis, first we loaded Fe/Fe3O4 bi-magnetic NPs into neural progenitor cells (NPCs), which were previously shown to migrate towards melanoma tumors. We observed that NPCs loaded with MNPs travel to subcutaneous melanoma tumors. After alternating magnetic field (AMF) exposure, the targeted delivery of MNPs by the NPCs resulted in a mild decrease in tumor size (Chapter-2). Monocytes/macrophages (Mo/Ma) are known to infiltrate tumor sites, and also have phagocytic activity which can increase their uptake of MNPs. To test Mo/Ma-mediated MHT we transplanted Mo/Ma loaded with MNPs into a mouse model of pancreatic peritoneal carcinomatosis. We observed that MNP-loaded Mo/Ma infiltrated pancreatic tumors and, after AMF treatment, significantly prolonged the lives of mice bearing disseminated intraperitoneal pancreatic tumors (Chapter-3). 2. Targeted cancer treatment could be achieved by engineering tumor homing cell surfaces with tumor proteases cleavable, cancer cell specific recombinant therapeutic proteins. To test this, Urokinase and Calpain (tumor specific proteases) cleavable; prostate cancer cell (CaP) specific (CaP1 targeting peptide); apoptosis inducible (Caspase3 V266ED3)- rCasp3V266ED3 chimeric protein was designed in silico. Hypothesized membrane anchored chimeric protein (rCasp3V266ED3, rMcherry red) plasmids were constructed. Membrane anchoring and activity of designed proteins were analyzed in RAW264.7 Mo/Ma and HEK293 cells in vitro. Further, Urokinase (uPA) mediated cleavage and release of rCasp3V266ED3 from engineered cells was tested (Chapter-4). Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments. Final chapter of present report shows evidence for immune-deficient line of pigs as a model for human cancers (Chapter-5)

Die Entwicklung innovativer Therapiekonzepte für die Knochenregeneration erfordert validierte segmentale Knochendefekt-Tiermodelle. Dabei ist das Mausmodell für die präklinische Testung von zentraler Bedeutung, jedoch fehlen in der wissenschaftlichen Literatur bislang Angaben zu validierten, extern stabilisierenden kritischen segmentalen Knochendefektmodellen an der immundefizienten Maus. Das Ziel dieser Arbeit war daher die Entwicklung und in vivo Evaluierung eines zuverlässigen und einfach zu handhabenden Modells für extern stabilisierte kritische Knochendefekte an der immundefizienten Maus. Dreißig männliche nu/nu-Mäuse (40,7±2,8 g, 95±2,6 d) wurden mittels Isofluraninhalation narkotisiert und anschließend ein externer Fixateur (MouseExFix, RISystem, AO Research Institute Davos, Schweiz) am rechten Femur angebracht. Femorale Knochendefekte der Länge 1 mm (n=10), 2 mm (n=10) und 3 mm (n=10) wurden erzeugt. Der Wundverschluss erfolgte mit Einzelknopfnähten. Röntgenaufnahmen wurden unmittelbar postoperativ und im Folgenden alle zwei Wochen innerhalb des Beobachtungszeitraums von zwölf Wochen angefertigt und im Hinblick auf Knochenregeneration und –fusion ausgewertet. Weiterhin wurden histomorphologische, histomorphometrische, immunhistochemische und µCT-Analysen zur dreidimensionalen und zellulären Beurteilung der Knochenheilung angefertigt. Alle Tiere überlebten die Operation. Sechs Tiere starben innerhalb des Beobachtungszeitraums als Folge von starkem Blutverlust (n=1), Infektion (n=1), Pinlockerung, welche die Euthanasie erforderlich machte (n=2) und durch Komplikationen bei der Anästhesie (n=2). Die µCT-Analyse nach zwölf Wochen zeigte, dass 3/8 der 1 mm-Defekte, 5/8 der 2 mm-Defekte und 8/8 der 3 mm-Defekte eine Pseudarthrose aufwiesen. Das mittlere Defektvolumen stieg signifikant (p<0,001) mit der Größe des Defektes und betrug 0,36±0,42 mm³ (1 mm-Gruppe), 1,4±0,88 mm³ (2 mm-Gruppe), bzw. 2,88±0,28 mm³ (3 mm-Gruppe). Die mittlere Defektgröße verringerte sich entsprechend um 77,6% (1 mm-Gruppe), 56,8% (2 mm-Gruppe), bzw. 28,6% (3 mm-Gruppe). Die histomorphologischen, histomorphometrischen und immunhistochemischen Analysen zeigten keine statistisch signifikanten Unterschiede zwischen den drei experimentellen Gruppen. Das verwendete MouseExFix-System ist ein zuverlässiges und einfach zu handhabendes Verfahren zur Stabilisierung eines kritischen segmentalen Knochendefekts an der immundefizienten Maus, wenn ein 3 mm-Defekt erzeugt wird. Das im Rahmen der Studie entwickelte und validierte murine extern stabilisierte, segmentale kritische Knochendefektmodell ermöglicht die präklinische Evaluierung von Konzepten zur lokalen Knochenregeneration inklusive der Verwendung allo- und xenogener Zellen
The development of innovative therapies for bone regeneration requires the use of advanced site-specific bone defect small animal models. In this context, murine models are of major importance as they allow for sufficient sample sizes prior to preclinical testing using larger animals. Owing to the small dimensions of the murine femur only a few custom fabricated fixation devices have been described in the literature so far. The aim of this investigation was to develop and validate a new, externally fixated critical size bone defect model for immunodeficient mice. Thirty male nu/nu mice (40.7 ± 2.8 g, 95 ± 2.6 days old) were anesthetized by isoflurane inhalation and an external fixation device (MouseExFix, RISystem, AO Research Institute Davos, Switzerland) was attached to the right femur. Femoral bone defects of 1 mm (n=10), 2 mm (n=10) and 3 mm (n=10) were created. Wounds were closed without any additional treatment. X-ray films obtained immediately after surgery and every 2 weeks postoperatively during the 12 week postoperative observation period were evaluated for bony regeneration and fusion. Furthermore, histomorphology, histomorphometry, immunohistochemistry and µCT analysis were performed. All of the animals survived the operation. Twenty four out of 30 animals reached the twelfth postoperative week. µCT analyses after twelve weeks showed that 3/8 of the 1 mm defects, 5/8 of the 2 mm defects and 8/8 of the 3 mm defects remained as nonunions. The defect volume was 0.36 ± 0.42 mm³ (1 mm group), 1.40 ± 0.88 mm³ (2 mm group), and 2.88 ± 0.28 mm³ (3 mm group) (p<0.001, between all groups). The defect size decreased by 77.6% (1-mm group), 56.8% (2-mm group) and 28.6% (3-mm group) (p=0.152, between all groups). Our method using the MouseExFix device has proven to be a reliable and easy-to-handle external fixation system for the stabilization of critical-size segmental bone defects in immundeficient mice when 3 mm defects are generated. This mouse model allows for high-throughput translational evaluation of concepts for site-specific bone regeneration including strategies using allogenic and xenogenic cell types

A deficiência de hormônio de crescimento (DGH) é a deficiência mais comum entre os hormônios pituitários. A terapia utilizada atualmente consiste de injeções diárias de hormônio de crescimento humano recombinante (r-hGH), entretanto esta terapia apresenta alguns inconvenientes, como a necessidade de frequentes injeções de r-hGH durante um longo período de vida, dependendo da severidade da deficiência, e o alto custo do hormônio, em razão dos dispendiosos processos de purificação. Uma alternativa ao tratamento padrão seria aquele no qual fossem evitados estes tipos de inconvenientes e o processo de liberação da proteína fosse sustentável, por um longo período e promovesse níveis normais e sustentáveis do fator de crescimento semelhante à insulina I (IGF-I), o principal mediador dos efeitos do GH. Uma alternativa é a terapia gênica in vivo, baseada na administração de DNA plasmidial em diversos órgãos/tecidos, seguida de eletroporação. É considerada uma metodologia bastante promissora e que tem sido alvo de vários estudos para diversos tipos de deficiências sistêmicas. Neste trabalho foram realizadas diversas administrações de um plasmídeo contendo o gene do hormônio de crescimento humano, nos músculos quadríceps exposto ou tibial anterior sem exposição, seguidas de eletroporação, em camundongos anões e imunodeficientes (lit/scid) com 40-80 dias de idade, na tentativa de obter uma correção fenotípica do nanismo, mediante a avaliação de parâmetros de crescimento. A administração deste plasmídeo no músculo tibial anterior, em camundongos com a idade inicial de 40 dias, foi capaz de proporcionar uma normalização dos níveis de mIGF-I, quando comparados aos dos camundongos não-deficientes de GH. Além disso, foram obtidos valores de catch-up dos parâmetros de crescimento longitudinal de 36-77%. Visando uma maior eficiência na expressão de GH, foram construídos plasmídeos parentais, e a partir destes, foram produzidos minicírculos de DNA com os promotores do CMV e Ubiquitina C e com os cDNAs de hGH e mGH. Estes minicírculos de DNA foram transfectados em células HEK 293 e foram até 2 vezes mais eficientes em relação aos plasmídeos convencionais com o promotor do CMV. Estes dados são bastantes promissores e abrem caminho para ensaios mais eficientes, utilizando este tipo de protocolo de terapia gênica para a DGH, visando uma normalização de todos os parâmetros de crescimento.
The human growth hormone deficiency (GHD) is the most common deficiency related to pituitary hormones. The current therapy is based on daily injections of recombinant human growth hormone (r-hGH). This therapy, however, presents some disadvantages, as the need for frequent injections of r-hGH during a long life time, depending on the deficiency severity and the high cost of this hormone, due to the expensive purification processes. An alternative to the standard treatment should be to avoid these inconveniences via a sustainable hormone release, acting for a long time and providing normal and sustainable levels of insulin-like growth factor-I (IGF-I). A possible alternative is in vivo gene therapy, based on the administration of plasmid DNA in several organs/tissues, followed by electroporation. This methodology is considered very promising and has been the target of many different studies for several types of systemic deficiencies. In the present work several administrations of a plasmid containing the human growth hormone gene were carried out, in the exposed quadriceps or non-exposed tibialis cranialis muscle, followed by electroporation, using immunodeficient dwarf mice 40-80 days old. The goal was to obtain a phenotypic correction of dwarfism, through the evaluation of different growth parameters. The administration of this plasmid, in the tibialis cranialis muscle of 40 day old mice, was able to provide a normalization of mIGF-I levels, when compared to non GHD mice. Furthermore, catch-up increases of longitudinal growth parameters of 36-77% were obtained. Aiming a high efficiency on GH expression, parental plasmids were constructed and from these DNA minicircles were generated with CMV and Ubiquitin C promoter and hGH or mGH cDNA sequences. These DNA minicircles were transfected into HEK 293 cells and were even 2 times moren efficient than conventional plasmids with CMV promoter. This data are very promising and pave the way for more efficient assays utilizing this type of gene therapy protocol for GHD, aiming at a normalization of all growth parameters.

Thesis (Ph. D.)--University of Georgia, 2002.
Directed by Benjamin G. Brackett. Includes articles submitted to Biology of reproduction, Human reproduction, and Fertility and sterility. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1990.
Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 48-82).

博士
國立陽明大學
生化暨分子生物研究所
105
Enterovirus 71 (EV71) is a major threat to children worldwide. Children infected with EV71 could develop subclinical infection and hand-foot-and -mouth disease (HFMD). In severe cases, patients could develop encephalitis, paralysis, pulmonary edema, and death. A more user-friendly and robust animal model is essential to investigating EV71 pathogenesis. In this study, we established three animal models which support clinical isolates of EV71 in vivo infection. The first animal model was published on the Journal of Virology, titled “Immunodeficient mouse models with different disease profiles by in vivo infection with the same clinical isolate of Enterovirus 71 (Liao and Liou, et al.). ” First, we demonstrated that EV71 infect stat-1 knockout (KO) mice successfully and develop paralysis and death. Levels of infectious EV71, and levels of VP1-specific RNA and protein in muscle, brain, and spinal cord, were compared in stat-1 KO models before, during, and after disease onset. The characteristic pathology of the stat-1 KO model includes (i) a strong tropism of EV71 for the central nervous system, (ii) detection of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brain stem, and spinal cord, (iii) amplification of microglial cells. Since we found that stat-1 KO mice can be infected with clinical isolates of EV71, the rate of disease manifestation was only 30%. A more robust animal model was still required for investigating EV71 pathogenesis. Therefore, we established the second animal model -- a hybrid (hSCARB2 +/+/ stat-1 -/-) mouse strain, which was published on Scientific Reports, titled “A new animal model containing human SCARB2 and lacking stat-1 is highly susceptible to EV71” (Liou, et al., 2016). This hybrid strain was generated from crossbreeding SCARB2 transgenic and stat-1 KO mice, and compared the susceptibilities to EV71 infection and pathogenesis between parental and hybrid mice. In the hybrid strain, VP1 protein can be detected in the streaking nerve fibers in brain and spinal cord. This hybrid mouse strain at 2-week-old age can still be infected with different genotypes of EV71 at 1000-fold lower titer via an i.p. route. Infected hybrid mice developed earlier onset of CNS disease, paralysis, and death at a higher incidence. These advantages of this novel model meet the urgent need from the scientific community in basic and preclinical research in therapeutics and pathogenesis. The third mouse model was an immunocompetent mouse model. The manuscript of this animal model is submitted with title “High therapeutic efficacy with interferon-alpha against clinical isolate of enterovirus 71 in a novel immune competent mouse model”. The title and abstract was attached on page v and vi.

博士
國立陽明大學
臨床醫學研究所
99
Musculoskeletal fibromatosis remains a disease of unknown etiology; surgical excision is the standard of care but recurrence rate remains high. Superficial fibromatosis typically presented as subcutaneous nodules due to rapid myofibroblast proliferation followed by slow involution to dense acellular fibrosis. In this study, we demonstrated that fibromatosis stem cells (FSCs) can be isolated from palmar nodule but not from the cord or normal palm tissues. We demonstrated that FSCs express surface markers such as CD29, CD44, CD73, CD90, CD105 and CD166 but do not express CD34, CD45, and CD133. We also showed that FSCs are capable of expanding into up to 20 passages and possess multipotentiality into the three germ layer cells, including myofibroblast, osteoblast, adipocyte, chondrocyte, hepatocyte and neural cells. When implanted beneath the dorsal skin of nude mice, FSCs recapitulated human fibromatosis nodule. Two weeks after implantation, the cells expressed immunodiagnostic markers for myofibroblasts α-smooth muscle actin (α-SMA) and type III collagen. Two months after implantation, the number of myofibroblasts diminished and type I collagen became evident. Treatment with trichostatin A (TSA), an anti-fibrogenic compound, inhibited proliferation and differentiation of FSCs in vitro. Treatment with TSA before or after implantation further blocked formation of fibromatosis nodules. These results indicated that FSCs act as cellular origin of fibromatosis and may be used as a promising model to develop new therapeutic interventions.

The aim of this thesis was to study the mechanisms of innate imunity involved in the degradation of tumor cells on which the ligands of phagocytic receptors were installed. For this purpose both in vivo experiments using immunodeficient mice, and in vitro experiments based on monitoring the levels of inflammatory cytokines produced in the tumor tissue and on measuring the level of myeloperoxidase released during neutrophil degranulation were performed.

The development of immune response accountable for the ability to control Cryptosporidium muris TS03 infection was studied using immunocompetent and various types of immunodeficient mouse models. Subsequently the immune response was characterized by analysis of leukocyte infiltration and cytokine production in gastric epithelium. Moreover, the potentiality of immunocompetent mice to develop effective immune response to C. andersoni LI03 infection with consequent protection to consequent infection of the same mice with C. muris TS03 was also studied by monitoring oocysts shedding, leukocyte infiltration of the gastric mucosa and cytokine production in ex vivo cultures of splenocytes.

Die Entwicklung innovativer Therapiekonzepte für die Knochenregeneration erfordert validierte segmentale Knochendefekt-Tiermodelle. Dabei ist das Mausmodell für die präklinische Testung von zentraler Bedeutung, jedoch fehlen in der wissenschaftlichen Literatur bislang Angaben zu validierten, extern stabilisierenden kritischen segmentalen Knochendefektmodellen an der immundefizienten Maus. Das Ziel dieser Arbeit war daher die Entwicklung und in vivo Evaluierung eines zuverlässigen und einfach zu handhabenden Modells für extern stabilisierte kritische Knochendefekte an der immundefizienten Maus. Dreißig männliche nu/nu-Mäuse (40,7±2,8 g, 95±2,6 d) wurden mittels Isofluraninhalation narkotisiert und anschließend ein externer Fixateur (MouseExFix, RISystem, AO Research Institute Davos, Schweiz) am rechten Femur angebracht. Femorale Knochendefekte der Länge 1 mm (n=10), 2 mm (n=10) und 3 mm (n=10) wurden erzeugt. Der Wundverschluss erfolgte mit Einzelknopfnähten. Röntgenaufnahmen wurden unmittelbar postoperativ und im Folgenden alle zwei Wochen innerhalb des Beobachtungszeitraums von zwölf Wochen angefertigt und im Hinblick auf Knochenregeneration und –fusion ausgewertet. Weiterhin wurden histomorphologische, histomorphometrische, immunhistochemische und µCT-Analysen zur dreidimensionalen und zellulären Beurteilung der Knochenheilung angefertigt. Alle Tiere überlebten die Operation. Sechs Tiere starben innerhalb des Beobachtungszeitraums als Folge von starkem Blutverlust (n=1), Infektion (n=1), Pinlockerung, welche die Euthanasie erforderlich machte (n=2) und durch Komplikationen bei der Anästhesie (n=2). Die µCT-Analyse nach zwölf Wochen zeigte, dass 3/8 der 1 mm-Defekte, 5/8 der 2 mm-Defekte und 8/8 der 3 mm-Defekte eine Pseudarthrose aufwiesen. Das mittlere Defektvolumen stieg signifikant (p<0,001) mit der Größe des Defektes und betrug 0,36±0,42 mm³ (1 mm-Gruppe), 1,4±0,88 mm³ (2 mm-Gruppe), bzw. 2,88±0,28 mm³ (3 mm-Gruppe). Die mittlere Defektgröße verringerte sich entsprechend um 77,6% (1 mm-Gruppe), 56,8% (2 mm-Gruppe), bzw. 28,6% (3 mm-Gruppe). Die histomorphologischen, histomorphometrischen und immunhistochemischen Analysen zeigten keine statistisch signifikanten Unterschiede zwischen den drei experimentellen Gruppen. Das verwendete MouseExFix-System ist ein zuverlässiges und einfach zu handhabendes Verfahren zur Stabilisierung eines kritischen segmentalen Knochendefekts an der immundefizienten Maus, wenn ein 3 mm-Defekt erzeugt wird. Das im Rahmen der Studie entwickelte und validierte murine extern stabilisierte, segmentale kritische Knochendefektmodell ermöglicht die präklinische Evaluierung von Konzepten zur lokalen Knochenregeneration inklusive der Verwendung allo- und xenogener Zellen.:1 Einleitung 1 1.1 Hintergrund 1 1.2 Das Mausmodell 2 1.3 Übersicht Tierversuche mit Knochendefekten 5 1.4 Frakturheilung 6 1.4.1 Allgemeines 6 1.4.2 Räumliche Gliederung 7 1.4.3 Expression von Proteinen der extrazellulären Matrix 8 1.4.4 Das Vier-Phasen-Modell der Frakturheilung 9 1.4.5 Das anabolisch/ katabolische Modell der Frakturheilung 12 1.4.6 Beeinflussung der Frakturheilung 12 1.4.7 Das Diamantkonzept 14 1.5 Osteosynthesesysteme 14 1.6 Pseudarthrosen 15 1.6.1 Definition 15 1.6.2 Ätiologie 16 1.6.3 Klassifikation 16 1.6.4 Therapie 17 2 Material und Methoden 19 2.1 Versuchstiere 19 2.2 Operationsvorbereitung 19 2.3 Operationsablauf 20 2.4 Postoperatives Vorgehen 27 2.5 Verlaufskontrolle 28 2.6 Entnahme der Präparate 29 2.7 Anfertigung der µCT-Aufnahmen 30 2.8 Anfertigung der histologischen Schnitte 30 2.8.1 Bearbeitung der Femora 30 2.8.2 verwendete Färbungen 31 2.9 Beurteilung der Schnitte 32 2.9.1 Histologische Beurteilung 32 2.9.2 Histomorphometrische Beurteilung 33 2.10 Statistik 33 3 Ergebnisse 34 3.1 Überlebensraten und Gewichtsverlauf 34 3.2 Röntgenauswertung 35 3.3 CT-Auswertung 38 3.4 Histologische Auswertung 41 3.5 Histomorphometrische Auswertung 44 3.5.1 TRAP 44 3.5.2 Osteocalcin 46 3.5.3 Osteopontin 47 3.5.4 Osteonectin 48 4 Diskussion 50 4.1 Diskussion etablierter Modelle für kritische Knochendefekte 50 4.1.1 Diskussion der Konzeption der Modelle 50 4.1.2 Diskussion der durchgeführten Anästhesieverfahren 54 4.1.3 Diskussion der Ergebnisse 55 4.1.4 Diskussion der Defektlängen 56 4.1.5 Diskussion der verschiedenen Osteosynthesesysteme 57 4.1.6 Diskussion der Ausfaller 59 4.2 Anwendung und Nutzen immundefizienter Tiermodelle 60 4.3 Vergleich des tibialen und des femoralen murinen Frakturmodells 63 4.4 Diskussion der Beurteilung der Knochenheilung mittels bildgebender und histologischer Verfahren 63 4.5 Anwendungsmöglichkeiten 65 4.6 Schlussfolgerungen 66 5 Zusammenfassung 67 5.1 In deutscher Sprache 67 5.2 In englischer Sprache 68 6 Literaturverzeichnis 70 7 Anhang 92 7.1 Danksagung 92 7.2 Lebenslauf 93 7.3 Veröffentlichungen 95 7.4 Wertetabellen 96 7.5 Erklärungen zur Eröffnung des Promotionsverfahrens 109
The development of innovative therapies for bone regeneration requires the use of advanced site-specific bone defect small animal models. In this context, murine models are of major importance as they allow for sufficient sample sizes prior to preclinical testing using larger animals. Owing to the small dimensions of the murine femur only a few custom fabricated fixation devices have been described in the literature so far. The aim of this investigation was to develop and validate a new, externally fixated critical size bone defect model for immunodeficient mice. Thirty male nu/nu mice (40.7 ± 2.8 g, 95 ± 2.6 days old) were anesthetized by isoflurane inhalation and an external fixation device (MouseExFix, RISystem, AO Research Institute Davos, Switzerland) was attached to the right femur. Femoral bone defects of 1 mm (n=10), 2 mm (n=10) and 3 mm (n=10) were created. Wounds were closed without any additional treatment. X-ray films obtained immediately after surgery and every 2 weeks postoperatively during the 12 week postoperative observation period were evaluated for bony regeneration and fusion. Furthermore, histomorphology, histomorphometry, immunohistochemistry and µCT analysis were performed. All of the animals survived the operation. Twenty four out of 30 animals reached the twelfth postoperative week. µCT analyses after twelve weeks showed that 3/8 of the 1 mm defects, 5/8 of the 2 mm defects and 8/8 of the 3 mm defects remained as nonunions. The defect volume was 0.36 ± 0.42 mm³ (1 mm group), 1.40 ± 0.88 mm³ (2 mm group), and 2.88 ± 0.28 mm³ (3 mm group) (p<0.001, between all groups). The defect size decreased by 77.6% (1-mm group), 56.8% (2-mm group) and 28.6% (3-mm group) (p=0.152, between all groups). Our method using the MouseExFix device has proven to be a reliable and easy-to-handle external fixation system for the stabilization of critical-size segmental bone defects in immundeficient mice when 3 mm defects are generated. This mouse model allows for high-throughput translational evaluation of concepts for site-specific bone regeneration including strategies using allogenic and xenogenic cell types.:1 Einleitung 1 1.1 Hintergrund 1 1.2 Das Mausmodell 2 1.3 Übersicht Tierversuche mit Knochendefekten 5 1.4 Frakturheilung 6 1.4.1 Allgemeines 6 1.4.2 Räumliche Gliederung 7 1.4.3 Expression von Proteinen der extrazellulären Matrix 8 1.4.4 Das Vier-Phasen-Modell der Frakturheilung 9 1.4.5 Das anabolisch/ katabolische Modell der Frakturheilung 12 1.4.6 Beeinflussung der Frakturheilung 12 1.4.7 Das Diamantkonzept 14 1.5 Osteosynthesesysteme 14 1.6 Pseudarthrosen 15 1.6.1 Definition 15 1.6.2 Ätiologie 16 1.6.3 Klassifikation 16 1.6.4 Therapie 17 2 Material und Methoden 19 2.1 Versuchstiere 19 2.2 Operationsvorbereitung 19 2.3 Operationsablauf 20 2.4 Postoperatives Vorgehen 27 2.5 Verlaufskontrolle 28 2.6 Entnahme der Präparate 29 2.7 Anfertigung der µCT-Aufnahmen 30 2.8 Anfertigung der histologischen Schnitte 30 2.8.1 Bearbeitung der Femora 30 2.8.2 verwendete Färbungen 31 2.9 Beurteilung der Schnitte 32 2.9.1 Histologische Beurteilung 32 2.9.2 Histomorphometrische Beurteilung 33 2.10 Statistik 33 3 Ergebnisse 34 3.1 Überlebensraten und Gewichtsverlauf 34 3.2 Röntgenauswertung 35 3.3 CT-Auswertung 38 3.4 Histologische Auswertung 41 3.5 Histomorphometrische Auswertung 44 3.5.1 TRAP 44 3.5.2 Osteocalcin 46 3.5.3 Osteopontin 47 3.5.4 Osteonectin 48 4 Diskussion 50 4.1 Diskussion etablierter Modelle für kritische Knochendefekte 50 4.1.1 Diskussion der Konzeption der Modelle 50 4.1.2 Diskussion der durchgeführten Anästhesieverfahren 54 4.1.3 Diskussion der Ergebnisse 55 4.1.4 Diskussion der Defektlängen 56 4.1.5 Diskussion der verschiedenen Osteosynthesesysteme 57 4.1.6 Diskussion der Ausfaller 59 4.2 Anwendung und Nutzen immundefizienter Tiermodelle 60 4.3 Vergleich des tibialen und des femoralen murinen Frakturmodells 63 4.4 Diskussion der Beurteilung der Knochenheilung mittels bildgebender und histologischer Verfahren 63 4.5 Anwendungsmöglichkeiten 65 4.6 Schlussfolgerungen 66 5 Zusammenfassung 67 5.1 In deutscher Sprache 67 5.2 In englischer Sprache 68 6 Literaturverzeichnis 70 7 Anhang 92 7.1 Danksagung 92 7.2 Lebenslauf 93 7.3 Veröffentlichungen 95 7.4 Wertetabellen 96 7.5 Erklärungen zur Eröffnung des Promotionsverfahrens 109

Aujourd'hui, l'un des modèles de souris humanisées le plus robuste est obtenu en injectant des cellules souches hématopoïétiques humaines (HSC) issues de foie fœtal humain et en implantant du thymus fœtal autologue. Ce modèle, appelé BLT (Bone marrow/Liver/Thymus), s'est révélé capable de supporter une reconstitution, une maturation et une sélection optimales des cellules T. Les souris BLT sont utilisées pour de nombreuses études telles que la compréhension de la biologie du VIH ou plus récemment en médecine régénérative. Grâce à ce modèle, nous avons pu d’une part étudier le rôle des cellules dendritiques plasmacytoïdes (pDC) lors de l’infection par le VIH mais aussi mieux comprendre la formation in vivo de tératomes lors de l’utilisation d’iPSC. Cependant, l'une des principales limites de cette technique réside dans l'obtention du tissu fœtal. Ici, nous avons décrit un nouveau protocole de souris humanisées greffées avec du thymus humain en utilisant des matériaux plus accessibles: du thymus humain retiré lors d’une chirurgie cardiaque chez des nouveaux-nés ou des enfants, et des HSC de sang de cordon. Des morceaux de ces thymus ont été implantés dans les quadriceps de souris immunodéficientes, après avoir été mis en culture. Ces souris CCST (Cord blood and Cardiac Surgery Thymus) ont permis une prise de greffe importante et un meilleur développement des lymphocytes T humains que les souris humanisées sans thymus. Les lymphocytes T des souris CCST et BLT ont montré une fonction similaire, évaluée par des tests de prolifération ex vivo et par rejet de lignées de cellules leucémiques allogéniques in vivo. Nous avons testé l’intérêt de cette nouvelle stratégie dans le modèle de l’infection au VIH-1, qui représente le modèle type de l’utilité des BLT. Nous avons montré que les souris CCST sont sensibles à l'infection par le VIH-1 par voie muqueuse ou intrapéritonéale, comme l'indique la détection de l'ADN du VIH et des cellules p24 +, similairement aux souris BLT. Les souris CCST ont présenté des réponses de lymphocytes T spécifiques du VIH-1 ex vivo plus efficaces que les BLT. Lors du traitement antirétroviral, les souris CCST, comme les BLT, ont vu leur charge virale diminuer. Ces résultats démontrent que les souris CCST représentent une alternative au modèle de souris BLT classique. Ces thymus, éthiquement plus facile à obtenir, peuvent être utilisés pour générer un grand nombre de souris par rapport aux thymus fœtaux.
Immunodeficient mice engrafted with human immune system provide an exciting in vivo model for a better understanding of its functioning and for development of new therapies. Today, one of the most robust humanized mouse model is achieved by injecting human hematopoietic stem cells (HSC) from fetal liver along with an implantation of autologous fetal thymic tissue. This model, called BLT, was shown to be able to support an optimal T cell reconstitution, maturation and selection. BLT mice are extensively used for many studies such as understanding HIV biology or in regenerative medicine. Indeed, our work used BLT mice on one hand to study the role of plasmacytoid dendritic cells (pDC) during the HIV infection and on the other hand to better understand the formation of teratomes from iPSCs in vivo. However, one of the biggest limitations of this technique is the procurement of the fetal tissue. Here we describe a new protocol to do humanized mice engrafted with human thymus pieces by using more accessible materials: human thymus obtained during cardiac surgery and cord blood HSC. Indeed, thymus is spontaneously removed during cardiac surgery in neonates and young children, thus it is an easy and ethical way to obtain this tissue. Those thymuses pieces were implanted in the quadriceps of a immunodeficient mice, after being put in culture. CCST mice (Cord blood and Cardiac Surgery Thymus) exhibited a significant engraftment of T-cells, compared to humanized mice without thymus. T-cells from both CCST and BLT mice showed a similar function as evaluated by proliferation assays upon PHA stimulation ex vivo and rejection of allogeneic leukemic cells lines in vivo. CCST mice were susceptible to HIV-1 infection via mucosal or intraperitoneal route, as shown by detectable viral load, HIV DNA and p24+ cells, at similar levels to those of BLT mice. Importantly, CCST mice displayed more effective ex vivo HIV-1-specific T-cell responses compared to BLT. Upon antiretroviral treatment, CCST mice, like BLT, were able to diminish the viral load. Our data suggest that CCST mice represent an alternative to the regular BLT mouse model. Those easy-to-access thymuses can be used to generate a large number of mice compared to fetal thymuses.