Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-23T21:20:51Z No. of bitstreams: 1 Lorena Pinto. Avaliação ex vivo da proliferação espontanea em PBMC de individuos infectados pelo HTLV-1 por citometria de fluxo.pdf: 1267124 bytes, checksum: 830b004821bb96ca261aed15410bcb82 (MD5) / Made available in DSpace on 2012-07-23T21:20:51Z (GMT). No. of bitstreams: 1 Lorena Pinto. Avaliação ex vivo da proliferação espontanea em PBMC de individuos infectados pelo HTLV-1 por citometria de fluxo.pdf: 1267124 bytes, checksum: 830b004821bb96ca261aed15410bcb82 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / O vírus linfotrópico de células T humanas do tipo 1 (HTLV-1) infecta cerca de 2% da população de Salvador-Bahia. O HTLV-1 é o agente etiológico da ATLL, HAM/TSP e uveíte. Este vírus também está relacionado a outras doenças inflamatórias como artrites, dermatites infectivas, polimiosites, alveolites e Síndrome de Sjögren. Uma das características imunológicas mais marcantes da infecção pelo HTLV-1 é a proliferação espontânea dos linfócitos T induzida por proteínas transativadoras virais, como Tax e HBZ. O papel da proliferação na patogênese da infecção não é conhecido, porém alguns trabalhos indicam que sua intensidade é maior nos pacientes com HAM/TSP, além disso pode ser responsável pela manutenção da carga proviral do HTLV-1, uma vez que o vírus se replica por divisão da célula infectada. A dinâmica da proliferação ex vivo não é muito conhecida, sendo normalmente avaliada por adição de 3[H]-Timidina às culturas, que possui várias desvantagens, entre as quais a toxicidade. Este é um estudo piloto que objetivou padronizar um método para avaliação da dinâmica da proliferação espontânea de linfócitos T utilizando o marcador CarboxyFluorescein diacetate Succinimidyl Ester (CFSE), por citometria de fluxo. Além de quantificar os índices de proliferação celular de linfócitos T totais e das subpopulações linfocitárias T CD4+ e T CD8+, correlacionar proliferação celular com a carga proviral do HTLV-1 e a expressão da proteína viral TAX. Para tal, PBMC de 30 pacientes infectados pelo HTLV-1 (16 assintomáticos e 14 HAM/TSP) foram cultivados por 24, 48, 72, 96 e 120 horas, em presença de CFSE. As células foram adquiridas em citometro de fluxo e os resultados analisados pelo programa FlowJo, através da porcentagem de células divididas e do índice de divisão celular. Observou-se proliferação em 66,7% dos indivíduos infectados pelo HTLV-1. A proliferação espontânea de PBMC foi superior no grupo de pacientes com HAM/TSP (porcentagem de células divididas: 79%, índice de divisão celular: 79%), comparada aos assintomáticos (porcentagem de células divididas: 50%, índice de divisão celular: 56,3%), porém essa diferença não foi significante A proliferação espontânea pode ser detectada nas primeiras 24 horas de cultura. A intensidade de proliferação é semelhante para as subpopulações de linfócitos T CD4 e T CD8, tanto em indivíduos assintomáticos como naqueles com HAM/TSP. Não houve correlação entre a carga proviral e a porcentagem de células divididas (r= -0,009; p= 0,9) e índice de divisão celular (r=0,1; p=0,5). A média de expressão de TAX em linfócitos TCD4+ foi de 2,4±2,4 %. Houve correlação entre a expressão da proteína Tax e a porcentagem de células divididas (r=0,59; p =0,05) e o índice de divisão celular (r=0,60; p=0,05), porém sem significância estatística. No entanto, encontrou-se uma correlação positiva entre a porcentagem de células T CD4+ divididas e a expressão da proteína Tax (r=0,79; p<0,003), bem como entre o índice de divisão das células TCD4+ e a expressão da proteína Tax (r=0,75; p=0,008). Em conclusão, a avaliação da proliferação espontânea por citometria de fluxo, em culturas com CFSE avaliadas pelo programa FlowJo permite identificar a proliferação celular nas 24 horas inicias da cultura e quantificar a proporção de linfócitos T CD4 e TCD8 que proliferam. Este método pode ser útil na avaliação de drogas que modulam a proliferação espontânea em PBMC de indivíduos infectados pelo HTLV-1. / Around 2% of the population of Salvador-Bahia are infected by the Human-T Lymphocyte virus type I (HTLV-1). This virus is the etiologic agent of ATLL, HAM/TSP and uveitis. It is also related to inflammatory illness like arthritis, infective dermatitis, polymyositis, alveolitis and Sjorgen syndrom. The spontaneous proliferation of T-lymphocytes induced by viral transactivating proteins like Tax and HBZ is one of the most outstanding immunological characteristics of the infection. The role of the proliferation in the pathogenesis is still unknown though some studies have shown that it is higher among HAM/TSP patients. Moreover, it could be responsible for the HTLV-1 remaining proviral load since the virus replicate through division of infected cell. The dynamics of the proliferation ex-vivo is not well known and is usually assessed by incorporating 3[H]-Thymidine to the cultures. This method has several disadvantages one of them being the 3[H]-Thymidine toxicity. This pilot study was aimed at the standardization of a method that assesses the dynamics of the spontaneous T-lymphocytes proliferation using flow cytometry and CarboxyFluorescein diacetate Succinimidyl Ester (CFSE) as a marker. Other objectives were to assess the rates of celular proliferation of both total T lymphocytes and CD4+ and T CD8+ subpopulations; to correlate the celular proliferation with the HTLV-1 viral load and the expression of the viral TAX protein. PBMCs of 30 HTLV-1 infected patients (16 assymptomatic and 14 HAM/TSP) were cultivated for 24, 48, 72, 96 and 120 hours in the presence of CFSE. Cells were obtained by flow cytometry and the results were analysed with the software Flowjo through the percentage of divided cells and the rate of celular division. Cell proliferation was observed in 66.7% of the HTLV-1 infected people. The PBMC spontaneous proliferation was higher in the group of HAM/TSP patients (percentage of divided cells:79%, rate of cellular division79%) compared to the assymptomatic individuals (percentage of divided cells: 50%, rate of cellular division: 56,3%) though this difference was not significant. Spontaneous proliferation can be detected in the first 24 hours of cell cultivation. The intensity of proliferation is similar in T CD4 e T CD8 subpopulations of assympomatic individuals and HAM/TSP patients. There was no correlation between proviral load and percentage of divided cells (r= -0,009; p= 0,9) and the rate of cellular division (r=0,1; p=0,5). The average of the TAX expression in T CD4+ was 2,4±2,4 %. There was a correlation between the expression of TAX protein and the percentage of divided cells (r=0,59; p =0,05) and the rate of cellular division (r=0,60; p=0,05), although not statistically significant. Nevertheless, a positive correlation was found between the percentage of divided T CD4+ cells and the expression of TAX protein (r=0,79; p<0,003), as well as between the rate of cellular division of TCD4+ cells and the expression of TAX protein (r=0,75; p=0,008). As a conclusion, the assessment of spontaneous proliferation by flow cytometry in cultures with CFSE analysed with the FlowJo software allows to identify the cellular proliferation in the first 24 hours of cultivation and to quantify the proportion of T CD4 and TCD8 lymphocytes that proliferated. This method could be usefull to assess drugs that modulate the spontaneous proliferation in PBMCs from HTLV-1 infected individuals.

Beeinflussung der Ex-vivo-Chemoresponse von Plattenepithelkarzinomen der Kopf-Hals-Region auf Cisplatin und Docetaxel durch 5-Fluorurazil

Pancreatic ductal adenocarcinoma (PDAC) is a serious oncological disease, which ranks among cancers with the worst prognosis and a three-year life expectancy of 10%. Ex-vivo organoid cultures derived from cancer tissue are popular and reliable research models, which reflect the morphology and histology of the original tissue. Genetic background leading to development PDAC confer typical alterations in genes KRAS, TP53, SMAD4 a CDKN2A. The aim of this thesis was to determine mutations present in organoid cultures derived from human PDAC. We used online genomic databases to estimate specific mutations typical for PDAC. Based on that research we designed protocols for the detection of PDAC genetic alterations and optimized those methods using cultured cells. We applied the approach on primary ex- vivo organoids derived from surgical cancer specimens and detected mutations in KRAS, TP53, SMAD4, or deletion of exons in CDKN2A. Alternatively, we proposed improvements for the analysis of genetic background in PDAC. The data obtained within this thesis will be used for the stratification of metabolomics and biochemical analyses further in the project.

Current epidemiological data showing that the use of the injectable contraceptive progestin Depotmedroxyprogesterone acetate (DMPA) is associated with increased HIV-1 acquisition is controversial. However, animal and ex vivo data reveal plausible biological mechanisms whereby MPA may increase HIV-1 acquisition. Relatively high levels of endogenous progesterone (P4) found in the luteal phase of the menstrual cycle have also been linked to increased HIV-1 acquisition in animal, clinical and ex vivo models. One of the central hypotheses of the present study was that the mechanism of MPA-induced increase in HIV-1 infection occurs via a different mechanism to that of the luteal phase. Furthermore, MPA has been shown to activate both the glucocorticoid receptor (GR) and its target, the progesterone receptor (PR) isoform B (PR-B), which are both transcription factors and regulate genes involved in immune function. Both the GR and PR are expressed in the cervix, the primary site of heterosexual HIV-1 infection. PR is regulated by endogenous estrogen (E2), of which the concentrations fluctuate throughout the menstrual cycle, and GR expression also varies in response to stress hormones, leading to conditions of varied relative levels of GR/PR. The immune-related consequences of changing the relative levels of GR and PR-B are not well understood. Therefore another hypothesis of this study was that changing the relative levels of GR/PR-B modulates HIV-1 infection and immunomodulatory gene expression in response to the GR/PR agonist, MPA. Since GR and PR-B recognize similar DNA target sequences and may regulate the same genes at the same time, the final hypothesis of the present study was that GR and PR-B reciprocally modulate each other’s activity, through possible association. To investigate the effects of exogenous hormones on HIV-1 infection and mechanisms thereof, peripheral blood mononuclear cells (PBMCs) and TZM-bl cervical cells were used as model systems for HIV-1 infection. These cells were stimulated with P4 and E2 at concentrations mimicking the menstrual cycle phases or with levels of MPA at the upper range of peak serum levels detected in DMPA users. Cells were infected with the R-tropic HIV-1 infectious molecular clone, HIV-1Bal_Renilla and luciferase assays were used to measure HIV-1 infection. Levels of HIV-1 CD4 receptor and CCR5 co-receptor protein or mRNA were measured by flow cytometry or qPCR, respectively, while activation of CD4+ T cells using the activation marker CD69 was measured by flow cytometry in PBMCs. To investigate the effects of changing GR/PR-B levels on HIV-1 infection and immune gene regulation, GR/PR levels were altered in End1/E6E7 immortalized endocervical and HeLa/TZM-bl cervical carcinoma cells by GR siRNA knockdown with or without the simultaneous over-expression of PR-B, and cells were stimulated with MPA or the GR agonist Dexamethasone. mRNA expression iii of key immunomodulatory genes in End1/E6E7 and HeLa cells was measured by qPCR. The modulation of GR activity by PR-B was assessed by promoter-reporter assay in COS1 and U2OS cells over-expressing GR and PR and stimulated with GR- and/or PR-specific ligands. Association of GR and PR-B was measured by co-immunoprecipitation in COS1 and MCF-7 cells, while co-localization of GR and PR-B was measured by confocal microscopy and super-resolution structured illumination microscopy in COS1 cells. MPA significantly increased HIV-1 infection in both PBMCs and TZM-bl cells, while luteal phase hormones did so to a lesser extent. However, MPA but not luteal phase hormones increased the ratio of CD4+/CD8+ T cells in PBMCs. MPA but not luteal phase hormones also increased CCR5 protein expression on CD4+ T cells in PBMCs and total CCR5 mRNA expression in TZM-bl cells. In addition, MPA but not luteal phase hormones increased activation of CD4+ T cells in PBMCs. Using a GR antagonist or GR siRNA, it was shown that the GR but not PR-B is required for MPA-, but not luteal phase hormone-induced increased HIV-1 infection in PBMCs and TZM-bls. The presence of PR-B altered the anti-inflammatory, GR-mediated regulation of some key immunomodulatory genes, including GILZ and IL-6, in End1/E6E7 and HeLa cells in response to MPA. In general, basal (unliganded) expression of immunomodulatory genes exhibited a pro-inflammatory profile in the presence of PR-B. Co-immunoprecipitation assays showed that GR and PR-B appeared to associate. Confocal microscopy suggested GR and PR co-localized in the nucleus in response to GR- and/or PRspecific ligands, while super-resolution microscopy showed that co-localization occurred in select regions within the nucleus. Taken together, MPA increases HIV-1 infection in a manner different from that of luteal phase hormones, most likely involving increased CD4+ T cell frequency (CD4+/CD8+ ratio), activation and increased expression of CCR5 on CD4+ T cells, and requiring the GR. Furthermore, PR-B modulates GR-mediated immune function gene regulation, via potential association and region-specific nuclear co-localization. This suggests that the relative levels of GR/PR may play an important role in determining the inflammatory and immune responses and HIV-1 infection in HIV-1 target cells, both in DMPA users and women not using hormonal contraception.

The increase in extracellular dopamine (DA) following cocaine administration plays a major role in cocaine abuse. In vitro, cocaine binds to DA transporters (DAT) and blocks DA uptake. Moreover, cocaine can increase extracellular DA concentration as measured by in vivo neurochemical methods. The present study examined the effects of cocaine and other drugs on DA, NE and 5-HT uptake using an ex vivo assay. Rats were injected i.v. with saline or drug and sacrificed at various time points after injections. Brains were dissected for regional monoamine uptake studies ex vivo. In most brain regions, cocaine given in vivo blocked monoamine uptake as expected. [ H]DA uptake in nucleus accumbens was inhibited with an ED = 22.3 μmol/kg. Cocaine fully inhibited [ H]NE uptake (ED = 4.58 μmol/kg) in the occipital cortex and partially inhibited [ H]5-HT uptake (33% at 30 μmol/kg) in the midbrain. However, under the same conditions [ H]DA uptake in the striatum was not inhibited after injections of cocaine up to 56 μmol/kg. Although the mechanism for this discrepancy is unclear, DA binding and uptake sites may be distinct and/or there may be regional differences in DA transporters.

No description available.

OBJECTIVE: To determine the effects of vitamin C and insulin on osteoblasts harvested from neonatal mouse calvaria. To determine the effects of experimental media (vitamin C and insulin and a combination) on the ex-vivo live bone organ culture model and explore the capacity of addition of osteoblasts to allow for bone formation within a critical defect. To use the chick chorioallantoic membrane (CAM) model to explore bone formation within critical bone defect. METHODS: Osteoblasts were harvested from neonatal mice were tested using four types of experimental media: control DMEM, media prepared with 150 μg/ml vitamin C, 10 nM media, or a combination of both vitamin C, insulin, and a combination of vitamin C and insulin media. Cell were cultured for 18 days at 37°C. Neutral red was done to identify cellular activity and silver nitrate to detect calcium deposits. Two types of scaffolds were inserted into the defect: collagen membrane scaffold and NuOss (xenograft) with collagen scaffold. After 30 days the samples were collected for histological analysis. Neonatal mouse calvaria were harvested and a 2mm critical defect made on each calvaria. Each calvaria received a scaffold of collagen or NuOss with or without osteoblasts with one of three experimental media within the CAM model. After 7 days, the amnion membrane of the egg was dropped and a window was made. The calvaria with the scaffold samples were placed on the amnion membrane. The eggs were incubated for 10 days then the experiment was terminated. Calvaria were collected and processed for histological evaluation. RESULTS: Neutral red and silver nitrate of 2D in-vitro cells revealed calcium deposits in culture well using vitamin C media, cell cultured with insulin media showed calcium deposits and cell morphological change, and cell cultured with a combination of vitamin C and insulin media showed the most calcium deposits and morphological changed. Ex-Vivo samples with collagen scaffold had bone thickening but not enough nutrients for bone regeneration, despite the addition of cells. The collagen scaffold is a more suitable material than xenograft due to particle size. The CAM model showed new bone formation and new vessels were most abundant in areas closest to lining cells in collagen samples. Samples with additional osteoblasts added showed greater results. NuOss scaffold samples did not show the same bone formation or vessel growth. CONCLUSIONS: The results indicate and confirm the basic principles of tissue engineering. In order to have bone regeneration more cells allow for better results. The quality of the scaffold is important and should have stability as well as enough space for cellular migration and recruitment for new blood vessel to support regeneration of bone to its original state.

This dissertation contributed three main areas to flexibility testing and biomechanical characterization of human spinal segments. The first was a literature review of existing testing methodology, the second examined common spine fixation procedures, and the third developed an improved fixation method. Spine biomechanical characterization is the primary technique for assessing the healthy, diseased, and surgically treated response of spinal tissues. However, despite decades of use, no standard testing protocol or reporting methodology for flexibility testing conditions has been established. As part of the present work, a comprehensive, systematic literature review was performed, and the methods sections of 242 relevant journal articles provided key information regarding preparation processes, fixation methods, testing temperatures, loading rates, loading magnitudes, and pre-conditioning procedures. The collated information was utilized to recommend best practices for testing and reporting flexibility testing methodology. Exothermic cementing processes were the dominant spinal fixation method from the reviewed articles. In these cases, fixation occurred when the most superior and inferior vertebrae of a functional spinal unit (FSU) were embedded into a cementing material that bound tightly to the cortical surface of the vertebrae, yielding a strong and geometrically favorable attachment point. The four most common fixation materials are highly exothermic and were tested to quantify the temperature rise in the adjacent intervertebral disc (IVD), as well as any associated thermal iatrogenic damage. Three thermocouples, inserted into three distinct locations of the IVD, measured temperatures throughout the cementing process. Polymethyl methacrylate (PMMA), the most frequently used cementing material, resulted in the greatest temperature increases, with a mean temperature rise up to 57°C above room temperature. Visible macro- and microscopic changes occurred in each of the cemented FSU IVDs. Changes included morphological changes, tissue desiccation, cracks, a breakdown in striations in the annulus fibrosus (AF), and denaturation and cell migration in the nucleus pulposus (NP). Based on these results, alternative vertebral attachment methods were considered and investigated. A structured design process was followed, and a mechanical ex vivo spinal fixation device which comprised a compliant vertebral clamp was designed. The geometry of the vertebral clamp was optimized based on pseudo-rigid body and finite element analyses. The final design was fabricated in titanium alloy (Ti6Al4V) using a 3D laser sintering process. Three clamps were manufactured and functionally validated. The opening/deployment and physiological loads of ± 7.5 N•m were cyclically tested on each clamp. The clamps showed no signs of failure and remained securely attached to the vertebrae. The resultant device/vertebrae interface stiffnesses were 4 to 10x greater than their corresponding FSU stiffnesses. The clamp design eliminated thermal iatrogenic damage, had minimal structural iatrogenic damage, was reusable, adjustable, and resulted in less than 10% of the previous preparation time.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13537号 / 論医博第2277号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 近藤 玄, 教授 齋藤 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Yes / In the study of microbial keratitis, in vivo animal models often require a large number of animals, and in vitro monolayer cell culture does not maintain the three-dimensional structure of the tissues or cell-to-cell communication of in vivo models. Here, we propose reproducible ex vivo models of single- and dual-infection keratitis as an alternative to in vivo and in vitro models. / Wellcome Trust