Dissertations / Theses on the topic 'Peripheral blood mononuclear cells methylation'
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Siu, Vincent. "MGMT promoter methylation and expression in glial tumours and peripheral blood mononuclear cells." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86563.
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O6-Methylguanine DNA methyltransferase (MGMT) is an inducible DNA repair protein that acts to repair damage by DNA alkylating agents currently used in chemotherapy, such as temozolomide. MGMT removes the alkyl group placed at the O6-position of guanine by these alkylating agents, decreasing their efficacy. It has been shown that epigenetic methylation of the O6-MGMT DNA promoter region in tumour tissue from glioblastoma multiforme (GBM) is associated with improved survival from patients treated with temozolomide and concomitant radiotherapy. We wanted to assess the levels of MGMT promoter methylation, RNA and protein expression of cell lines to determine if there is a correlation between these three variables. We also hypothesized that MGMT promoter methylation mosaicism exists in glial tumours and would affect response to temozolomide. To assess this mosaicism we sampled multiple regions of each tumour intra-operatively and analyzed them using methylation specific PCR. Blood was drawn from these patients and the aforementioned MGMT assays were assessed in the peripheral blood mononuclear cells (PBMCs) to determine its usefulness as a prognostic tool. Our results show that MGMT promoter methylation is not a binary event, as currently calculated, but that an intermediate levels of promoter methylation percentage can be assessed. Promoter methylation also does not correlate with RNA or protein expression, but they do trend together. MGMT promoter methylation and RNA expression also vary intratumourally. MGMT promoter methylation can also be assayed in the PBMC fraction of blood in certain patients with high grade gliomas. These methylation levels appear to be associated with recurrence of the tumour and were altered after resection. Our study shows that promoter methylation may need to be looked at as a percentage-based variable and not as a binary system. Furthermore, due to intratumoural heterogeneity more areas of a tumour may need to be assessed for promoter meO6-Methylguanine DNA methyltransferase (MGMT) est une protéine qui répare l'ADN à la suite de dommages génétiques causées par des traitements de chimiothérapiques tel que Temozolomide (TMZ). MGMT corrige l'addition alkyle à la position O6 de guanine et par conséquence, diminue l'efficacité des agents alkylateurs. La méthylation épigénétique de la région promoteure de MGMT dans les tissues de glioblastomes corrèle avec l'augmentation de la survie des patients traités avec le TMZ et la radiothérapie. Le but de notre recherche était d'évaluer le niveau de méthylation du promoteur de MGMT, de l'ARN, et de l'expression de la protéine dans des lignées cellulaires afain de déterminer si une corrélation existe entre ces trois facteurs. De plus, nous avons prédit que le niveau de méthylation du promoteur de MGMT varierait entre chaque échantillon de tumeur cérébrale. Nous avons testé plusieurs régions d'une tumeur par
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Horsburgh, Steven. "An investigation into exercise-induced modifications to DNA methylation-regulatory enzymes in human peripheral blood mononuclear cells." Thesis, Northumbria University, 2016. http://nrl.northumbria.ac.uk/32545/.
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DNA methylation, an epigenetic modification which can regulate gene transcription independently from alterations to the nucleotide sequence, can be manipulated by lifestyle factors such as diet and exercise, hypothetically reversing aberrant DNA methylation associated with disease pathogenesis. The underlying mechanisms by which these changes occur are currently poorly characterised, however, in vitro data suggest that inflammatory mediators are involved. Furthermore, regular exercise appears to reduce inactivity-associated systemic inflammation, possibly by alterations to the methylome, thereby suggesting a cyclic relationship between exercise, inflammation, and epigenetic modification. The aims of this research programme, therefore, were to: characterise the acute changes that occur to the de novo DNA methyltransferases following exercise in peripheral blood mononuclear cells (PBMCs), and the role of exercise-induced systemic inflammation in this process; investigate how these changes then translate into functional modifications to the methylome; and to determine whether a training programme utilising sedentary individuals manipulates DNA methylation of genes involved in chronic systemic inflammation associated with physical inactivity. Pilot investigations corroborated previous in vitro data that recombinant IL-6 is able to regulate nuclear concentrations of DNMT3A and DNMT3B in PBMCs. In order to isolate the influence of circulating proteins independently from genetic polymorphisms that may influence susceptibility to epigenetic change, cells were stimulated with exercise-conditioned plasma following intense endurance exercise which elicited significant alterations in nuclear concentrations of DNMT3A and DNMT3B. Eccentric exercise, which is typically not associated with elevations in circulating cytokines, did not cause any significant changes in nuclear or cytoplasmic DNMT concentration, or global DNA methylation; this supports the hypothesis that transient systemic elevations in inflammatory cytokines are important regulators of epigenetic modifications associated with exercise. Lack of transcriptional changes in DNMT3A following both exercise training and an acute maximal bout suggests that, in line with in vitro data, that the observed elevations in nuclear DNMT concentration are largely due to cellular relocalisation and not gene expression of this enzyme. It remains to be elucidated whether the training regime, and the subsequent response to an acute maximal bout, is able to elicit differential methylation of IL6, NFκB2, and ASC, however, in vitro stimulation of PBMCs with the cytokines IL-6 and IL-1β did cause significant changes to IL6 promoter methylation, further supporting the role of these proteins in epigenetic regulation. The data presented in this thesis support the postulation that exercise-induced changes to DNA methylation in PBMCs likely occur due to systemic elevations of inflammatory proteins, in particular IL-6, which causes manipulation of de novo DNMT nuclear concentrations due to cellular translocation of the enzymes themselves. While it was not possible to determine whether exercise directly modified gene-specific methylation, in vitro experiments suggest that inflammatory cytokines are able to regulate IL6 promoter methylation in human PBMCs.
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LOTTO, VALENTINA. "Nutrient-gene interactions within one-carbon metabolism and effects on epigenetic regulation through dna methylation in peripheral blood mononuclear cells." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2010. http://hdl.handle.net/10281/18016.
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Epigenetics is a field of molecular biology that copes with the study of gene function regulation without variations in DNA structure or nucleotide sequences.
Among the main epigenetic phenomema in eukaryotic cells there are DNA methylation and post-traslational mechanisms among which the major are histone methylation and acetylation.
Epigenetic changes are potentially reversible phenomena that are controlled also by nutritional factors as the methyl-donors involved in the folate cycle.
Plasma levels of B vitamins, among which “in primis” plasma folate concentrations, are implicated in epigenetic modulation so that it can be hypothesized that they may affect the modulation of gene expression through epigenetic mechanisms.
Epigenetic modifications represent one of the earliest events in the genesis of some complex pathologies, therefore the study of the interaction between epigenetics and nutritional status is of great interest either to define the physiopathological mechanisms of development of some illnesses, and for possible personalized strategies of prevention.
The present work has been articulated, at first, on the analysis of gene-nutritional interaction mechanisms within the folate cycle through the study of polymorphisms of enzymes involved in the metabolism of methyl-group donors; the aim was to study their possible role on the modulation of genomic DNA methylation in relationship to different plasma levels of idrosoluble B vitamins. In this regard, the most important functional polymorfisms known on the genes of one-carbon metabolism and their relationship with methylation status of polymorphonuclear cells DNA have been analyzed from a cohort of around 800 subjects within a clinical study, underlining the role of the key folate-related enzymes in the modulation of DNA methylation.
Besides the function of genomic DNA methylation, the methylation status at specific sites has been also approached with the specific intent of considering a possible interrelationship between the role of promoter methylation and the co-presence of functional polymorphisms in the same genic site for a gene for which a precise functional effect is well-known. To address this issue the promoter region of coagulation factor VII gene was evaluated for both genetic and epigenetic modifications as a possible model of genetic-epigenetic interaction in the modulation of gene product regulation. The results showed the key importance of genetic-epigenetic interactions, so far unknowm, in modulating gene-expression at promoter gene sites.
The role of other vitamins involved in one-carbon metabolism in major chronic diseases, and specifically the emerging role of B6 vitamin, have been also studied.
Furthermore, a clinical study is now in progress to evaluate the function of gene-specific methylation in liver tissue where most of the folate cycle functions take place. The aim of this project is the evaluation of both genome-wide and gene-specific methylation status in the liver in comparison to that observed in peripheral blood mononuclear cells DNA to define whether methylation status of peripheral blood DNA may be regarded as a good systemic biomarker for this epigenetic feature of DNA in relation to B vitamins nutritional status in cancer disease. Results from this study may help to define possible functional markers of gene-nutrients interactions with effects on epigenetic modulation for future preventive or therapeutic strategies. With that purpose, a novel high-throughput array-based technique for the detection of gene-specific methylation at promoter sites has been optimized in our laboratory.
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Silva, Carolina Sousa. "Protemic characterization of peripheral blood mononuclear cells." Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/15872.
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Mestrado em Biotecnologia - Biotecnologia MolecularPeripheral blood mononuclear cells (PBMCs) play quite diverse and important roles in monitoring immune homeostasis. Thus, these subset of blood cells may provide access to potential physiological relevant biomolecules, namely proteins. For this reason, PBMCs represent a promising biological sample in scientific research, particularly as a source of potential biological markers discovery of the most diverse diseases. Prior studies of proteomic characterization of PBMCs from healthy individuals lack either the identification of a large number of proteins or its quantification in a way that is compatible with the search of potential biomarker candidates. Therefore, this study aimed to provide a comprehensive PBMCs proteome characterisation as well as to create a SWATH library. It was also evaluated if by using the BD Vacutainer® CPT™ tubes for PBMCs isolation, it would be possible to identify a larger number of immunologically relevant proteins in comparison to plasma samples. The enrichment test assay revealed that it is possible to identify more immune-related proteins from isolated PBMCs than from plasma. Moreover, the majority of the quantified proteins with an “immune system” GO term assigned is present in higher amounts in PBMCs samples. 2D LC-MS/MS proved to be the best approach to use in qualitative analysis of PBMCs and in the construction of a SWATH library, since it resulted in an increase of both identified and quantified proteins (66.3% and 16.9%, respectively) in comparison to 1D LC-MS/MS. A total of 2071 proteins were identified and it was possible to quantify 922 different proteins among six distinct samples. From these proteins, 445 were commom between all individuals. In conclusion, this work provides a comprehensive PBMCs proteome dataset that will be useful in further studies that focus on the search for potential biological markers of various pathologies in these cells. Additionally, SWATH-MS proved to be a reproducible and effective acquisition method to quantify PBMCs proteins.
As células mononucleares do sangue (CMS) desempenham diversos e importantes papéis na monitorização da homeostasia do sistema imunitário. Assim sendo, esta subpopulação de células sanguíneas pode providenciar acesso a potenciais biomoléculas relevantes a nível fisiológico, nomeadamente proteínas. Por esta razão, as CMS representam uma amostra biológica promissora na investigação científica, particularmente na descoberta de potenciais marcadores biológicos de diversas doenças. Estudos anteriores de caracterização proteómica das CMS de indivíduos saudáveis falharam quer na identificação de um grande número de proteínas, quer na sua quantificação, de forma compatível com a pesquisa de potenciais biomarcadores. Portanto, este estudo teve como objectivo providenciar uma caracterização proteómica abrangente, bem como a criação de uma biblioteca SWATH. Foi igualmente avaliado se usando tubos CPT™ disponíveis na BD Vacutainer® para o isolamento das CMS, seria possível identificar um maior número de proteínas imunologicamente relevantes comparativamente a amostras de plasma. O teste de enriquecimento revelou que é possível identificar mais proteínas associadas ao sistema imunitário em CMS isoladas do que em amostras de plasma. Também se verificou que a maioria das proteínas quantificadas com ontologia genética “sistema imunitário” estão presentes em maior quantidade nas amostras de CMS. 2D LC-MS/MS mostrou ser a melhor abordagem na análise qualitativa das CMS e na elaboração da biblioteca SWATH, uma vez que o número de proteínas identificadas e quantificadas apresentou um aumento de 66,3% e 16,9%, respectivamente, comparativamente à 1D LC-MS/MS. No total foram identificadas 2071 proteínas e foi possível quantificar 922 proteínas diferentes em seis amostras distintas. Destas, 445 proteínas eram comuns a todos os indivíduos. Em conclusão, este trabalho disponibiliza um amplo conjunto de dados do proteoma das CMS que será útil a estudos futuros que pretendam centrar-se na pesquisa de potenciais marcadores biológicos, nas CMS, das mais diversas patologias. Além disso, comprovou-se que o método de aquisição SWATH-MS é reprodutível e eficaz na quantificação das proteínas das CMS.
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Moser, Stephanie. "In vitro effects of psychopharmaceuticals on peripheral mononuclear blood cells." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-144429.
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Drake, Mary. "Characterisation of mononuclear cells in peripheral blood stem cell harvests." Thesis, Queen's University Belfast, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.287206.
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Parkinson, Nicholas J. "Endotoxin-induced microRNA expression in equine peripheral blood mononuclear cells." Thesis, Virginia Tech, 2016. http://hdl.handle.net/10919/81766.
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The innate immune response to lipopolysaccharide (LPS) mediated by toll-like receptor 4 (TLR4) contributes substantially to the morbidity of equine gastrointestinal disease, neonatal sepsis and other diseases. MicroRNAs (miRNAs), small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in TLR4 signaling regulation in other species. The central hypothesis of this study was that LPS induces differential expression of miRNAs in equine peripheral blood mononuclear cells (PBMCs).
PBMCs were isolated from healthy adult horses and cultured with LPS or medium only for 2, 4 and 8 hours. Concentrations of inflammatory cytokines were measured in supernatants by immunoassay. Illumina Next-Generation Sequencing of the miRNA transcriptome was performed in PBMCs at 0, 2 and 4 hours. Selected expression changes were verified by qRT-PCR.
327 mature miRNAs were detected in equine PBMCs. Only miR-155 was significantly upregulated by LPS. 9 miRNAs showed statistically significant expression changes with time. Tumor necrosis factor-α concentration was significantly higher in supernatants from LPS-treated cells than controls from 2 hours, while interleukin-10 and interferon-γ were increased at 8 hours. miR-155 expression was correlated to all three cytokines.
These data provide a foundation for future research into miRNA involvement in equine inflammatory responses. miR-155 is the principal LPS-induced miRNA in horses. Bioinformatic target predictions support roles in regulation of innate and adaptive immune responses including TLR4 signaling, as in humans. It is thus likely to influence the acute inflammatory response to LPS. Further research will be necessary to establish its role in naturally occurring disease.Master of Science
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Sibbons, Charlene. "Characterisation of polyunsaturated fatty acid synthesis in peripheral blood mononuclear cells." Thesis, University of Surrey, 2018. http://epubs.surrey.ac.uk/845807/.
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Conversion of the essential n-3 (18:3n-3) and n-6 (18:2n-6) fatty acids to longer chain polyunsaturated fatty acids (PUFA) involves sequential desaturation and elongation reactions. Previous studies have reported gender differences in n-3 PUFA synthesis, whereas the effect of age is less clear. n-3 PUFAs are reported to have important effects on immune cell function. A previous study reported long chain PUFA synthesis in mitogen stimulated but not quiescent peripheral blood mononuclear cells (PBMCs). However, the underlying mechanism is not known. PUFA synthesis was investigated in PBMCs incubated with [1-13C]18:3n-3 for 48 h. Activation with the T-lymphocyte mitogen concanavalin A (Con A) increased PUFA synthesis. 22:6n-3 synthesis was not detected. [1-13C] incorporation was greatest for 20:3n-3 suggesting initial chain elongation is an important fate for 18:3n-3. Con A increased expression of three key genes (FADS2, FADS1 and ELOVL5) involved in PUFA synthesis, suggesting upregulation of the pathway is controlled at the transcriptional level. ELOVL2 expression was negligible, possibly explaining the lack of 22:6n-3 synthesis. Con A increased methylation of 12 CpGs in the FADS2 promoter contradicting the general view that DNA methylation represses transcription. Subsequent 5’RACE analysis verified that activated PBMCs were not using an alternative promoter for FADS2 transcription. Contrary to expectation, 18:3n-3 conversion in activated PBMCs was not affected by gender or menopausal status and there was no clear age effect. PUFA synthesis was constitutive in the Jurkat T-lymphocyte leukaemic cell-line and was higher than in PBMCs. FADS2, FADS1 and ELOVL5 mRNA expression was also higher in Jurkat cells and was associated with 50% lower methylation of 17 CpGs in the FADS2 promoter, suggesting transcriptional dysregulation of PUFA synthesis in Jurkat cells involves altered DNA methylation. These findings have provided novel insights into the regulation of PUFA biosynthesis in PBMCs and upregulation of the pathway in activated PBMCs suggests that newly synthesised PUFAs may be important for cell function.
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Monteiro, Flavia Regina Goncalves. "Peripheral Blood Mononuclear Cells Cytokine Expression in Horses Treated with Dexamethasone." Thesis, Virginia Tech, 2005. http://hdl.handle.net/10919/34753.
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Glucocorticoids are widely used in horses for a variety of autoimmune and inflammatory conditions. Its potent antiinflammatory properties have been associated with the suppression of a number of different inflammatory cytokines. The purpose of the study was to evaluate the effect of dexamethasone treatment in horses on mRNA cytokine expression, including interleukin-1Î , interferon-gamma, interleukin-4 and interleukin-6, during a five day treatment period and a five day post treatment period.
A randomized complete block design was performed on 16 healthy horses. Group I (8 horses) received 0.1 mg/kg of dexamethasone sodium phosphate by intravenous injection once daily for 5 days. Group II (8 horses) received an equivalent volume of sterile saline by intravenous injection daily for 5 days. A sample of 5x10 mililiters of blood in acid citrate dextrose was obtained prior to initial treatment. Thirty minutes after each treatment injection (placebo or dexamethasone) a sample of blood was obtained during the 5 day treatment period and 24, 48, 72, 96 and 120 hours after the last treatment injection was administered. Peripheral-blood mononuclear cells were isolated from the blood samples and stimulated with concavalin A. RNA was isolated using the QIAGEN RNeasy kit. cDNA first strand synthesis was achieved using QIAGEN's OMMISCRIPT RT KIT. cDNA was also constructed for the house keeping gene Î actin. Primer pairs specific for each cytokine were designed using equine cytokine sequences available on Genbank. cDNA for each cytokine and Î -actin was amplified using Real Time PCR technique.
Interleukin-4, interleukin-6 and interferon-gamma mRNA expression was statistically significant suppressed in horses treated with dexamethasone when compared to control horses. Interleukin-1Î was only significantly suppressed on day 5. Interleukin-4, interleukin-6 and interferon-gamma mRNA expression suppression was initially observed on day 2 and lasted 24 hours after the last dose of dexamethasone was administered. Interleukin-6 mRNA expression was significantly higher when compared to control group on day 10.
Our results suggest that dexamethasone treatment of healthy horses suppresses mRNA expression of several cytokines, including interleukin-4, interleukin-6 and interferon-gamma. This effect could explain part of corticosteroid's mechanism of action for controlling inflammation in a variety of disease conditions. The time-course effect of dexamethasone showed that the effect on mRNA cytokine expression suppression is only observed on day 2 of treatment and mRNA suppression is maintained for 24 hours after discontinuation of treatment.
Master of Science
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Pattanaik, Malisha. "Separation of cancer cells from peripheral blood mononuclear cells using pH control and dielectrophoresis." Diss., [La Jolla] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p1469581.
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Thesis (M.S.)--University of California, San Diego, 2009.Title from first page of PDF file (viewed October 22, 2009). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 56-59).
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Kopechek, Mary E. "Expression of CD163 on bovine alveolar macrophages and peripheral blood mononuclear cells." Connect to resource, 2007. http://hdl.handle.net/1811/25220.
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Thesis (Honors)--Ohio State University, 2007.Title from first page of PDF file. Document formatted into pages: contains 17 p.; also includes graphics. Includes bibliographical references (p. 16-17). Available online via Ohio State University's Knowledge Bank.
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Clark, Megan Frances. "Effects of endotoxin on cationic amino acid transport in peripheral blood mononuclear cells." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298251.
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Papageorgiou, Konstantina. "Gene delivery to peripheral blood mononuclear cells using Herpes Simplex Virus Type I." Thesis, University College London (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406514.
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Egawa, Haruto. "Peripheral blood mononuclear cells in early pregnancy promote invasion of human choriocarcinoma cell line, BeWo cells." Kyoto University, 2004. http://hdl.handle.net/2433/147458.
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Bazzard, H. L. "Exploring peripheral blood mononuclear cells as the source of interleukin-6 in polymyalgia rheumatica." Thesis, University of the West of England, Bristol, 2014. http://eprints.uwe.ac.uk/25257/.
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Polymyalgia rheumatica (PMR) is a chronic inflammatory condition which affects the elderly, causing aching and stiffness of the neck, shoulders and pelvis, as well as more systemic manifestations such as fever, malaise and fatigue. PMR shares symptoms with rheumatoid arthritis (RA) and both are associated with significantly elevated circulating concentrations of interleukin-6 (IL-6), which is thought to play a key role in the pathogenesis of these diseases. In RA, IL-6 is derived from synovial cells in the joints. In PMR, however, the source of IL-6 is unknown. PMR patients do not exhibit the same joint involvement as in RA but they do have elevated circulating IL-6 concentrations, thus, it was hypothesised that the source of IL-6 in PMR may be one of the circulating peripheral blood mononuclear cell (PBMC) types, which are all capable of producing IL-6. Blood samples were taken from untreated PMR patients, RA patients with active disease and healthy controls (HC) of similar age and gender. To account for known circadian variations in circulating IL-6, samples were taken at a standard time. IL-6 was quantified in plasma and serum using cytometric bead array (CBA) and enzyme-linked immunosorbent assay. The biological activity of the IL-6 was tested for the first time in PMR using a B cell proliferation assay. Using immunostaining and flow cytometry, constitutive intracellular IL-6 was measured in CD3+, CD14+, CD19+, CD123+ and CD11c+ PBMCs. Intracellular IL-6 was also determined following PBMC stimulation in vitro, to determine potential differences in cell responsiveness. Concentrations of secreted IL-6 in the culture supernatants of resting and stimulated PBMC were determined by CBA in parallel cultures. Other cytokines were also quantified in order to examine PMR and RA pathologies more broadly. Finally, the results of the cytokine assays were compared with patient reported severity of fatigue and the four different components of fatigue (emotional, living, physical and cognitive). Circulating IL-6 was significantly elevated above HC in both serum and plasma of PMR and RA patients, and this IL-6 was found to be biologically active. PBMC in all subjects constitutively produced low levels of intracellular IL-6 and very low concentrations of secreted IL-6 in parallel cultures. Overall, responses to in vitro stimulation were variable but no significant differences were observed between PMR, RA and HC samples. Secreted IL-6, in contrast, increased dramatically following stimulation of all cultures, suggesting intracellular staining may not reflect the secretory capability of these cells, but also confirming that there were no differences between PBMC responses of PMR, RA and HC groups. A significant correlation was observed between circulating IL-6 concentrations in PMR and RA patients, and physical fatigue, living fatigue and total fatigue. Broader cytokine analysis demonstrated that IL-6 alone was significantly elevated in PMR patients. Taken together, circulatory IL-6 in active PMR is found to be elevated in the absence of other inflammatory cytokines. It is biologically active and correlates strongly with physical and living aspects of fatigue. Circulating PBMCs are not the source of this elevated IL-6 in PMR patients, suggesting that neutrophils, vascular endothelium or muscle tissue may instead be involved.
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Frellstedt, Linda. "Induction and characterization of endotoxin tolerance in equine peripheral blood mononuclear cells in vitro." Thesis, Virginia Tech, 2010. http://hdl.handle.net/10919/44307.
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Endotoxemia is responsible for severe illness in horses. Individuals can become unresponsive to the endotoxin molecule after an initial exposure; this phenomenon has been called developing a state of â endotoxin toleranceâ (ET). ET has been induced in horses in vivo; however, cytokine expression associated with ET has not been investigated. The purpose of this study was to develop and validate a method for inducing ET in equine peripheral blood mononuclear cells (PBMCs) in vitro, and to describe the cytokine profile which is associated with the ET. Blood was collected from 6 healthy horses and PBMCs were isolated. ET was induced by culturing cells with three concentrations of endotoxin given to induce ET, and evaluated after a second dose of endotoxin given to challenge the cells. The relative mRNA expression of IL-10 and IL-12 was measured by use of quantitative PCR.
ET was induced in all cells (n=6) exposed to the 2-step endotoxin challenge. In PBMCs treated with 1.0 ng/ml of endotoxin followed by challenge with 10 ng/ml of endotoxin, the relative mRNA expression of IL-10 in tolerized cells was not different from positive control cells. In contrast, the relative mRNA expression of IL-12 in tolerized cells was decreased by 15-fold after the second endotoxin challenge compared with positive control cells.
This experiment demonstrated a reliable method for the ex vivo induction of ET in equine PBMCs. A marked suppression of IL-12 production is associated with ET. The production of IL-10 was not altered in ET in our model.
Master of Science
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Van, Heerden Johannes Hendrik. "Peripheral blood mononuclear cells as non-invasive diagnostic indicators of stress-associated neural states." Master's thesis, University of Cape Town, 2008. http://hdl.handle.net/11427/4347.
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Includes abstract.Includes bibliographical references (leaves 155-181).
Researchers have demonstrated the ability to predict psychopathological states from human peripheral immune tissure transcriptional profiles, using microarrays. Although evidence in support of such an approach as a viable diagnostic avenue within psychiatric settings is accumulating, it remains to be demonstrated, in an animal model, that transcriptional changes in peripheral tissue targets are paralleled by specific gene expression changes in neural tissues.
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Moser, Stephanie [Verfasser], and Markus [Akademischer Betreuer] Schwarz. "In vitro effects of psychopharmaceuticals on peripheral mononuclear blood cells / Stephanie Moser. Betreuer: Markus Schwarz." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1024243303/34.
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Rony, Sharmin Aqter [Verfasser]. "Helminth antigen-induced innate immune response in porcine peripheral blood mononuclear cells / Sharmin Aqter Rony." Bonn : Universitäts- und Landesbibliothek Bonn, 2017. http://d-nb.info/1150183071/34.
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Li, Ying. "Analyses of peripheral blood mononuclear cells in operational tolerance after pediatric living donor liver transplantation." Kyoto University, 2006. http://hdl.handle.net/2433/143875.
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Fiocco, Daniela. "α-DEFENSINS EXPRESSION IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH HEPATITIS C VIRUS INFECTIONS." Doctoral thesis, La Sapienza, 2005. http://hdl.handle.net/11573/916796.
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Onodera, Rie. "Bone marrow mononuclear cells versus G-CSF-mobilized peripheral blood mononuclear cells for treatment of lower limb ASO: pooled analysis for long-term prognosis." Kyoto University, 2010. http://hdl.handle.net/2433/131879.
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James, Francine O. "The rhythmic expression of circadian clock genes in human peripheral blood mononuclear cells : investigating the functional clock in circulating white blood cells." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103024.
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The rhythmic expression of an autoregulatory loop of circadian clock genes underlies the intrinsic circadian rhythmicity in the central circadian pacemaker of the suprachiasmatic nucleus (SCN). Clock genes are also known to be rhythmically expressed outside the SCN and outside the brain. While these peripheral circadian oscillators are functional, their expression is chiefly coordinated by the central circadian pacemaker of the SCN. This thesis presents the first evidence of a functional circadian oscillator in human peripheral blood mononuclear cells (PBMCs). Measuring levels of RNA transcripts in PBMCs sampled throughout specific behavioural protocols permitted the characterization of these peripheral circadian oscillators in humans. The expression of clock genes HPER1, HPER2, and HPER3 peaks early after the time of typical awakening and demonstrates a significant circadian rhythmicity in PBMCs sampled from healthy young men that persists in time isolation and under the constant behavioural conditions of a constant routine (CR). The functional circadian oscillator in human PBMCs is also observable in the presence of the sleep/wake cycle. Using frequent sampling over 72 hours, it was determined that the patterns of HPER1 and HPER2 expression are comparable when sampled in the presence of a habitual sleep/wake cycle or during a CR. The expression of HBMAL1 in PBMCs was more variable under different behavioural conditions. The pattern of light and darkness exposure including bright light during night shifts, shielding from morning light and sleep in darkened quarters can induce adaptive phase delays in the endogenous circadian rhythms of cortisol secretion in night shift workers. In the presence of such a light intervention, the cortisol rhythm in night shift workers re-assumes an appropriate temporal alignment with the shifted sleep/wake schedule and cortisol levels peak near the shifted time of awakening. Following nine days of simulated night shift work, HPER1 and HPER2 expression in PBMCs is aligned to the shifted sleep/wake schedule of a typical night shift worker in the presence of a comparable light/darkness intervention. This thesis will consider the implications of a functional clock in human white blood cells in light of the demonstration of a functional and shiftable circadian oscillator in human PBMCs.
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Lacelle, Chantale. "Blood sample processing for the study of aging, and characterization of caspase mRNA expression in peripheral blood mononuclear cells." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82906.
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Centenarian population studies are one of several approaches currently used to study the aging process and characterize successful aging. I have described a methodology permitting the simultaneous generation of RNA, DNA, protein, and plasma samples, as well as fixed peripheral blood mononuclear cells (PBMC) and frozen blood aliquots, from a single 10- to 30-ml sample of peripheral blood obtained from donors of any age, and showed that although extremely old individuals are somewhat anemic, it is possible to obtain enough biological material from their blood to conduct aging studies.I investigated the possibility of immortalizing B-lymphocytes from extremely old individuals, using the Epstein-Barr virus (EBV), and found that although extremely old individuals (90+ years) possess low levels of circulating B-lymphocytes, it is possible to immortalize B cells present in less than one milliliter of their blood using EBV.
Using biological material obtained from blood samples of individuals of all ages by the method for blood sample processing I have described, I studied the mRNA expression of cell death (specifically caspase) genes in nonagenarians and centenarians, successful models of aging who have survived or avoided age-associated diseases, as well as in their younger counterparts, to determine whether apoptotic genes may be part of the genetic determinants of longevity. I found that a population of extremely old individuals (90+) shows a unique pattern of caspase mRNA expression, characterized by high levels of caspase-1 and -3, and low levels of caspase-8, mRNA, while slightly less aged individuals (70--89) are characterized by high levels of caspase-8 mRNA expression. Furthermore, I showed that these changes in caspase mRNA do not appear to result from age-related changes in PBMC composition, such as decreases in CD24. Therefore, I suggest that unique patterns of caspase mRNA result from the regulation of message abundance on a per cell basis, via a putative regulation of caspase genes at the transcription or RNA processing level, rather than age-associated changes in immune profiles.
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Walker, Mollie. "Characterisation of α-1 adrenergic receptors in peripheral blood mononuclear cells of complex regional pain syndrome." Thesis, Walker, Mollie (2017) Characterisation of α-1 adrenergic receptors in peripheral blood mononuclear cells of complex regional pain syndrome. Honours thesis, Murdoch University, 2017. https://researchrepository.murdoch.edu.au/id/eprint/38078/.
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Complex regional pain syndrome (CRPS) is a chronic pain condition that may occur after injury or trauma to a limb. The underlying pathophysiology of CRPS is largely undetermined, although CRPS patients commonly present with a persisting inflammatory response in the early stage of the condition and overexpress the α-1 adrenergic receptor (α-1AR) on nociceptors and keratinocytes in the affected limb. In other chronic inflammatory conditions, increased α-1AR mRNA expression in peripheral blood mononuclear cells (PBMCs) has been shown, and is thought to contribute to persisting inflammation. The α-1AR are expressed on a range of cells, including nerve, smooth muscle, skin and immune cells, and bind to adrenaline and noradrenaline released after activation of the sympathetic nervous system. The aims of this project were to examine the mRNA expression of α-1AR subtypes (α-1A, α-1B and α-1D) by qPCR in PBMCs isolated from fractionated blood of CRPS patients, and to quantify the percentages of various PBMC subpopulations compared to healthy controls. Subpopulations of PBMCs were determined by flow cytometry using a panel of fluorescent antibodies that identified CD4+ T cells, CD8+ T cells, CD4+ CD8+ T cells, CD4+ CD25+ T cells, CD8+ CD25+ T cells, B cells, natural killer (NK) cells, NKT cells, and subsets of monocytes. No differences in expression of α- 1AR subtypes in PBMCs of CRPS patients were found when compared to healthy controls. However, a significant increase in the concentration of total PBMCs isolated per mL of blood in CRPS patients and a shift from CD16- to CD16+ monocyte subpopulations was identified when compared to healthy controls. These results show there is an inflammatory component to CRPS and provide preliminary evidence that the persisting inflammation in CRPS patients may originate from over-proliferation of PBMC progenitors in the bone marrow, which is sympathetically modulated by α-1AR, and/or an expansion of other PBMC cell types that were not examined in this project.
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Miller, Danielle. "Intestinal absorption of colostral leukocytes, peripheral blood mononuclear cells, and porcine umbilical cord matrix stem cells by neonatal pigs." Thesis, Manhattan, Kan. : Kansas State University, 2008. http://hdl.handle.net/2097/916.
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Zarkesh-Esfahani, Sayyed Hamid. "Effects of growth hormone and leptin on cytokine production and proliferation of human peripheral blood mononuclear cells." Thesis, University of Sheffield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392501.
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Martinez, Francisca. "The effect of selected drugs on pokeweed mitogen-stimulated IgG synthesis by human peripheral blood mononuclear cells." Thesis, University of Liverpool, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.279718.
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Olivier, Brenda Jean. "Increased osteoclastogenesis and bone resorption by peripheral blood mononuclear cells in chronic liver disease patients with osteopenia." Diss., University of Pretoria, 2007. http://hdl.handle.net/2263/27189.
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BARZAGO, CLAUDIA. "Identification of a new molecular signature in peripheral blood mononuclear cells from patients affected by myasthenia gravis." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2016. http://hdl.handle.net/10281/105298.
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La miastenia grave è una malattia autoimmune T-dipendente mediata da autoanticorpi diretti contro proteine della giunzione neuromuscolare. È caratterizzata da un danno alla trasmissione neuromuscolare che causa affaticamento e debolezza muscolare. In circa l’80% dei pazienti, la malattia è associata alla produzione di autoanticorpi diretti contro il recettore dell’acetilcolina (AChR) localizzato a livello della membrana post-sinaptica della giunzione neuromuscolare. Numerose evidenze sperimentali suggeriscono che il processo autoimmune si sviluppi nel timo; tuttavia, i meccanismi molecolari sottostanti la perpetuazione di tali processi in periferia non sono del tutto noti. Abbiamo studiato, mediante sequenziamento dell’intero trascrittoma, il profilo trascrizionale delle cellule mononucleate da sangue periferico derivate da pazienti miastenici ad esordio precoce di malattia (minore di 50 anni di età) e positivi per gli anticorpi anti-AChR (AChR-EOMG), che rappresentano il sottogruppo clinico meglio studiato. Come controllo, sono state incluse nello studio cellule derivate da individui sani di pari età e sesso. I risultati di trascrittomica combinati con l’analisi dei pathway, tramite il software Ingenuity, hanno mostrato che 128 trascritti codificanti e 9 precursori di microRNAs (miRNAs) erano differenzialmente espressi tra pazienti AChR-EOMG e controlli. In particolare, 22 su 128 (17%) trascritti codificanti appartenevano alla categoria ‘malattie infettive’ e 59 su 128 (46%) alle categorie ‘malattie infiammatorie’ e ‘risposta infiammatoria’. La validazione dei livelli di espressione dei trascritti tramite qPCR ha mostrato che, tra i trascritti associati a ‘malattie infettive’, i livelli di espressione di ETF1, NFKB2, PLK3 e PPP1R15A erano aumentati, mentre quelli di CLC ed IL4 erano diminuiti nei pazienti AChR-EOMG rispetto ai controlli; nella categoria ‘infiammazione’, i livelli di espressione di ABCA1, FUS e RELB erano aumentati, suggerendo una perdita delle funzioni immunomodulatorie di queste molecole. Partendo dai dati di trascrittomica abbiamo inoltre applicato una selezione sulla
base delle interazioni putative tra miRNAs e trascritti target. La successiva validazione tramite qPCR ha mostrato che i livelli di espressione dei miRNA miR-612, miR-3654 e miR-3651 erano aumentati, mentre i target predetti di miR-612, AKAp12 e HRH4, come anche il target putativo di miR-3651, CRISP3, erano diminuiti nei pazienti AChR-EOMG, ad ulteriore sostegno dell'ipotesi di un’alterazione dei processi di immunoregolazione in periferia.
In conclusione, abbiamo identificato un nuovo profilo molecolare associato ad AChR-EOMG, dimostrando un ruolo cruciale di molecole associate ad ‘infezione’ ed ‘infiammazione’ nella patogenesi della miastenia grave. Ulteriori indagini sulle molecole scoperte in questo studio potranno contribuire ad una migliore conoscenza delle basi molecolari della patogenesi della malattia con un possibile impatto su nuove strategie terapeutiche.Myasthenia gravis (MG) is a T-cell dependent humoral-mediated autoimmune disease characterized by neuromuscular transmission impairment, resulting in fatigability and muscular weakness. In approximately 80% of MG patients, the disorder is associated with the production of autoantibodies against acetylcholine receptor (AChR) localized at the post-synaptic membrane of the neuromuscular junction. Growing body of evidences suggests that the autoimmune reaction develops in the thymus; nevertheless, the molecular mechanisms underlying the perpetuation of the autoimmune processes in the periphery are not fully characterized. We studied the transcriptional profile of peripheral blood mononuclear cells from AChR-positive early onset (< 50 years old) (AChR-EOMG) patients, the best studied clinical subgroup, and age- and sex-matched healthy controls, by using whole-transcriptome sequencing. Transcriptome data together with Ingenuity Pathway Analysis showed that 128 coding transcripts and 9 microRNA (miRNAs) precursors were differentially expressed between AChR-EOMG patients and healthy controls. In particular, 17% (22 out of 128) of the coding transcripts were related to ‘infectious disease’ category and 46% (59 out of 128) to ‘inflammatory disease’ and ‘inflammatory response’ categories. Selection of the genes of interest and further qPCR validation of the transcript levels revealed that among the ‘infectious disease-associated’ transcripts, ETF1, NFKB2, PLK3, and PPP1R15A were increased, whereas CLC and IL4 were decreased in AChR-EOMG patients versus healthy controls; in the ‘inflammation’ categories, ABCA1, FUS, and RELB were upregulated, suggesting of a possible loss of immunomodulatory function. Additional transcriptome data analysis and validation were also centered on miRNA-mRNA putative interactions. We observed that miR-612, miR-3651, and miR-3654 were upregulated, whereas miR-612-putative AKAp12 and HRH4 target transcripts and also miR-3651-predicted CRISP3 target were decreased in AChR-EOMG samples, further suggesting a loss of immunoregulatory processes. Taken together, our findings disclose a novel peripheral molecular signature associated with AChR-EOMG, and suggest a key role of ‘infectious’ and ‘inflammation-related’ molecules in disease pathogenesis. Future studies on the molecules discovered here will allow a better understanding of the molecular basis of AChR-EOMG pathogenesis that could be helpful for the development of new therapeutic interventions.
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MacDonald, Elizabeth Steward. "The influence of equine bone marrow derived stem cells on the response of cultured peripheral blood mononuclear cells to endotoxin." Thesis, Virginia Tech, 2015. http://hdl.handle.net/10919/76722.
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Endotoxemia is a major cause of morbidity and mortality in horses. The presence of large amounts of circulating endotoxin inititates a number of cell signaling pathways leading to a systemic inflammatory response. Activation of these pathways causes the release of a number of pro- and anti-inflammatory mediators. An overwhelming release of these mediators leads to the development of clinical signs associated with endotoxemia. Treatment options are limited mostly to supportive care at this time. Mesenchymal stem cells (MSCs) have been shown to have anti-inflamamtory and immune modulatory effects that may have some benefit for the treatment of horses with endotoxemia.
To evaluate the effect of equine MSCs on the response to endotoxin challenge, the study was performed on two different stem cell lines with peripheral blood mononuclear cells (PBMCs) used as controls. After stimulation with endotoxin, secretion of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and interferon gamma (IFN-γ) were determined by ELISA. The immunogenic properties of MSCs were assessed with a one-way mixed lymphocyte reaction. In addition, the ability of MSCs to alter production of cytokines from stimulated PBMCs was assessed.
TNF-α was not produced by MSCs when compared to PBMCs (p = < 0.001). There was no significant difference between MSCs and PBMCs in the production of IL-6. IL-10 production was significantly different (p = <0.001) at 6 and 12 hours with MSCs producing more than PBMCs in one stem cell line only. MSCs did not stimulate proliferation of PBMCs. Co-incubation of MSCs with PBMCs decreased the production of TNF-α in both stem cell lines although it was not statistically significant (p = 0.4 and 0.9) at either time point. IL-6 secretion was suppressed at twelve hours with co-incubation. IL-10 production was increased with co-incubation in one stem cell line. MSCs secrete soluble factors that can alter PBMC cytokine production and they do not appear to be immunostimulatory. These findings have potential implication for treatment of equine inflammatory conditions.Master of Science
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Wooding, Anita. "Antiviral efficacy of nine nucleoside reverse transcriptase inhibitors against feline immunodeficiency virus in feline peripheral blood mononuclear cells." Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-182515.
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The purpose of the study reported here was to compare the antiviral efficacy against feline immunodeficiency virus (FIV) and cytotoxicity in feline peripheral blood mononuclear (PBM) cells of 9 nucleoside reverse transcriptase inhibitors (NRTIs), three of which had not been evaluated against FIV in feline cells before. PBM cells were isolated from the blood of three specific pathogen-free (SPF) cats.
The cytotoxic effects of the test compounds were determined by colorimetric quantification of a formazan product resulting from bioreduction of a tetrazolium reagent by viable PBM cells. Each compound was tested in 12 concentrations ranging from 0.001 to 500 M. Uninfected cells from one SPF cat were used in these assays. PBM cells (from all three SPF cats) were infected with the molecular clone FIV pPPR and the antiviral efficacy of the test compounds was assessed using a FIV p24 antigen capture enzyme-linked immunosorbent assay. Each compound was tested in 5 concentrations ranging from 0.1 to 10 M.
Cytotoxic effects in feline PBM cells were observed only at concentrations over 10 M for all 9 NRTIs. Comparison of the cytotoxic effect at the highest concentration investigated (500 M) revealed that didanosine and amdoxovir were significantly less toxic than abacavir. As no cytotoxicity was noted up to a concentration of 10 M, this was set as the highest concentration for the second part of this study investigating the anti-FIV efficacy of the test compounds. All drugs induced a dose-dependent reduction of FIV replication. When compared at the highest concentration investigated, there was no significant difference in the antiviral efficacy among the test compounds. The EC50 could not be determined as none of the test compounds achieved 50% viral inhibition.
The evaluated NRTIs had low cytotoxicity against feline PBM cells and appear to be safe options for further in vivo evaluation for the treatment of FIV-infected cats. There was no evidence suggesting that the newly evaluated compounds would be superior to the existing NRTIs for reducing the FIV burden of infected cats.
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Palombo, Philipp [Verfasser]. "Investigation of stress biomarkers in human peripheral blood mononuclear cells in response to chronic isoproterenol treatment / Philipp Palombo." Konstanz : KOPS Universität Konstanz, 2018. http://d-nb.info/1234912260/34.
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Varelias, Antiopi. "Studies of CD44 variant isoform expression and function on activated human peripheral blood mononuclear cells and in renal transplantation." Title page, summary and contents only, 2001. http://web4.library.adelaide.edu.au/theses/09PH/09phv293.pdf.
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Singla, Sunit, Tong Zhou, Kamran Javaid, Taimur Abbasi, Nancy Casanova, Wei Zhang, Shwu-Fan Ma, et al. "Expression Profiling Elucidates a Molecular Gene Signature for Pulmonary Hypertension in Sarcoidosis." UNIV CHICAGO PRESS, 2016. http://hdl.handle.net/10150/622494.
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Pulmonary hypertension (PH), when it complicates sarcoidosis, carries a poor prognosis, in part because it is difficult to detect early in patients with worsening respiratory symptoms. Pathogenesis of sarcoidosis occurs via incompletely characterized mechanisms that are distinct from the mechanisms of pulmonary vascular remodeling well known to occur in conjunction with other chronic lung diseases. To address the need for a biomarker to aid in early detection as well as the gap in knowledge regarding the mechanisms of PH in sarcoidosis, we used genome-wide peripheral blood gene expression analysis and identified an 18-gene signature capable of distinguishing sarcoidosis patients with PH (n = 8), sarcoidosis patients without PH (n = 17), and healthy controls (n = 45). The discriminative accuracy of this 18-gene signature was 100% in separating sarcoidosis patients with PH from those without it. If validated in a large replicate cohort, this signature could potentially be used as a diagnostic molecular biomarker for sarcoidosis-associated PH.
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Selinger, Christina Imanta. "Identification of RANKL-Regulated Genes Involved in Osteoclast Differentiation and Resorption." Thesis, Griffith University, 2008. http://hdl.handle.net/10072/367396.
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Peripheral blood mononuclear cells (PBMCs) are pluripotent for osteoclast and macrophage cell lineages. The differentiation of macrophages and osteoclasts from a common monocyte precursor is induced following exposure to macrophage-colony stimulating factor (M-CSF), or both M-CSF and receptor activator of nuclear factor B ligand (RANKL) respectively. Differential gene expression resulting from cytokine treatment of PBMCs was examined over time using differential display PCR (DD-PCR) and quantitative real-time PCR (Q-PCR). Q-PCR analysis verified the expression of a new chemokine, FAM19A1, in addition to Special AT-rich binding sequence 1 (SATB1), solute carrier family 16 member 6 (SLC16A6) and LIM kinase 1 (LIMK1) in primary human osteoclasts, however, only LIMK1 was significantly up-regulated by RANKL.
Highly efficient delivery of small interfering RNA (siRNA) transfection to primary human osteoclasts was developed, and represents a technical milestone due to the inherent phagocytic tendencies of the PBMC lineage. The development of RNA interference for use in primary human osteoclasts was conducted using siRNA synthesised by Dicer enzyme, to verify the role of candidate genes in osteoclast differentiation and osteoclast bone resorption. Cathepsin K (CTSK) is the key proteinase expressed by osteoclasts, and was used as a benchmark for the optimisation of siRNA inhibition in primary human osteoclasts. Transfection of primary human osteoclasts with siRNA to CTSK significantly diminished bone resorption, with a 60% reduction in area resorbed (P=1.3x10-2), and a 50% reduction in pit number (P=1.8x10-2). Normal bone remodelling is dependent on both the rate of osteoclast formation and resorption. A number of genes were examined for their contribution to osteoclast formation and resorption using siRNA. Nuclear factor of activated T cells, calcineurin dependant 1 (NFATc1) inhibition was found to significantly deplete osteoclast formation (P=4.0x10-3), confirming other NFATc1 inhibition studies, and the necessity of NFATc1 in osteoclast differentiation. In pre-differentiated osteoclasts, siRNA targeting NFATc1 did not reduce osteoclast bone resorption, rather it significantly increased area resorbed (P=1.0x10-3), with no significant difference in cell number. This result suggests that NFATc1 may act in accordance with its regulator calcineurin, which has been found to enhance osteoclast differentiation, but inhibit osteoclast resorption in mature cells.
The inhibition of LIMK1 by targeted small interferring RNA (siRNA) was found to significantly diminish osteoclast formation (P=1.0x10-3), pits resorbed (P=4.2x10-2), as well as area resorbed (P=4.0x10-3). LIMK1 is a signalling kinase, identified as RANKL-regulated in murine osteoclasts, notwithstanding, this is the first study that confirms LIMK1 involvement in osteoclast formation and activity. LIMK1/cofilin-mediated actin reorganisation is critical to progenitor cell migration to stromal cells, and also regulates the stability of F-actin formation. F-actin rings were analysed in LIMK1 depleted pre-differentiated osteoclasts, which seemed to have formed properly and did not appear dissimilar from controls.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Marques, Graça Susete Costa de Carvalho. "Establishing a cell biology platform: isolation and preservation of human blood products." Master's thesis, Faculdade de Ciências e Tecnologia, 2014. http://hdl.handle.net/10362/11009.
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Dissertação para obtenção do Grau de Mestre em
Genética Molecular e BiomedicinaThe use of human primary cells provide researchers in different areas with irrefutable more biologically relevant data than using cell lines or animal blood cells. The work was performed in the scope of the Cell Biology Services @ CEDOC, aiming to provide viable and trustful human primary cells and products. We had three main objectives: protocol optimizations for blood cell isolation, culture and cryopreservation; cost estimation and divulgation of the services. We have reviewed standard protocols and compared different strategies for blood cell isolation. The impact of those methodologies was evaluated regarding cell yield and purity, cell functional characteristics and cost. We also developed a method for serum isolation from human plasma in blood buffy coats. The resultant sera were sterile and suitable to be used in leukocyte cultures. Different protocols for T cells isolation were compared: positive versus negative immunomagnetic selection and isolation using nylon wool fiber columns. Positive selection provided the highest isolation yield (32.35%), while negatively selected cells had the highest purity (92.81%). Although nylon wool fiber column was the fastest and cheapest method, unlike the immunomagnetic methods, it did not allow complete separation of T from B lymphocytes. Positive selection of monocytes was compared using two widely used commercial kits. Miltenyi’s kit provided the highest isolation yield (25.92%), recovery rate (86.70%) and purity (95.01%). Monocytes isolated with StemCell kit presented a higher cell complexity, and when differentiated into dendritic cells (DCs), showed a more mature phenotype. Differences between both kits are probably caused by the nature of the magnetic beads, suggesting caution when choosing one or other kit, as it may have an impact on DCs’ function. Overall, although dealing with apparently straight forward methodologies, our results show that testing commercial products and optimizing protocols is very important and contribute for a better quality of products and services.
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Elhussiny, Mohammed lyad Ezat Roba. "Analysis of cytokine induced phosphorylation of STAT3 in peripheral blood mononuclear cells by flow cytometric and western blot assays." Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/40836.
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Signal transducer and activator of transcription (STAT) is a family of intracellular proteins that are responsible for carrying the signal from the cell surface to the nucleus in response to specific ligands. Once in the nucleus, STATs activate the transcription of specific genes. To date, seven human STATs have been identified. Among these STATs, STAT3 is considered as oncogenic. It activates genes that block apoptosis and inhibits antitumor immune responses (1). STAT3 is also essential in early embryogenesis and plays a role in cell growth and survival, differentiation and apoptosis depending on the target tissue.
Analysing STAT3 signalling provides insights into pathology and can be used as a tool for diagnosis, prognosis and therapy development. Traditionally, western blot has been used to analyse cell signalling but it is impractical in analysing rare cell populations or providing information at the single cell level. Moreover, it is a demanding and time consuming technique that offers qualitative and less sensitive analysis. The rapid evolution in the multi-parametric flow cytometry and the availability of both epitope specific antibodies and sophisticated software facilitate the wide application of this technology in cell signalling studies. Flow cytometry has the ability to resolve different subcellular sets in a heterogeneous population, collects data at a single cell level and correlates multiple markers simultaneously. However, it requires highly standardized protocols for maximal sensitivity.
The aim of this study was to assess the dose and the time response of both total STAT3 and pSTAT3 to in vitro stimulation with either IL-6 or IL-10 in peripheral blood mononuclear cells (PBMC). This assessment was done using both the flow cytometry and the western blot techniques.
The results of this study showed that lower doses of IL-6 (1 & 10 ng/ml) were not sufficient to induce phosphorylation of STAT3. However, following stimulation with 100 ng/ml of IL-6, no significant change in the level of total STAT3 could be detected in either lymphocytes or monocytes from 3 different donors using either the FC500 or the Accuri cytometer. Using the FC500 cytometer, a small but insignificant increase in the pSTAT3 was seen in the lymphocytes and monocytes. A significant increase in STAT3 phosphorylation was only observed for monocytes after 15 minutes stimulation with 100 ng/ml of IL-6 using the Accuri flow cytometer. xii
When the fluorescent labelled antibodies used in the flow cytometric assays were used for western blot probing, western blot analysis of stimulated cell lysates with 100 ng/ml IL-6 detects proteins of a low molecular weight than STAT3 or pSTAT3 which may explain the flow cytometric results of IL-6 stimulation.
In IL-10 stimulation experiments, lower doses (1 and 10 ng/ml) tested by flow cytometric and western blot techniques demonstrated insignificant STAT3 phosphorylation induction. Following stimulation with either 50 or 100 ng/ml IL-10, no significant change in the total STAT3 was seen in either lymphocytes or monocytes when using the Accuri flow cytometer. However, stimulation with 100 ng/ml IL-10 induces STAT3 phosphorylation from 10 minutes through 30 minutes in both lymphocytes and monocytes. Longer times were required and high inter-individual variability was noticed for the activation of STAT3 after stimulation with 50 ng/ml IL-10.
By using different antibodies from those used in the flow cytometric assay; the western blot results were comparable with the flow cytometric findings following stimulation with 100 ng/ml IL-10.
The addition of phosphatase inhibitors during the flow cytometric protocol didn’t show any increase in the STAT3 phosphorylation. However, using paraformaldehyde for fixation and methanol for permeabilisation significantly decreased the mean fluorescence intensity of the PE conjugated antibodies comparing to the BD commercial fixation and permeabilisation buffers. The onset and the signal intensity of “in house” chemiluminescence mixture for western blot detection of STAT3 were comparable to the commercial ECL reagent used. However, the background of the “in house” mixture increased with time and was higher than with the commercial product. Upon longer exposure, the background increased enough to cause signal loss.
In spite of the number of advantages of the flow cytometric assay compared to the western blot assay, these results are highly dependent on the specificity and the selectivity of the used antibodies. Furthermore, flow cytometry requires a highly standardized protocol to be able to assess the normal level of signalling proteins which could be later applied to detect abnormalities. It is suggested that the antibodies used in the flow cytometric assay be tested by western blot to confirm their selective detection of the target protein before their use in the flow cytometric analysis.Dissertation (MSc)--University of Pretoria, 2013.
gm2014
Pharmacology
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AGOSTINI, L. P. "AMENDMENT Of Gene Expression In Mononuclear Cells Of Human Peripheral Blood Submitted To Exposure With Herbicide Based On Glyphosate." Universidade Federal do Espírito Santo, 2018. http://repositorio.ufes.br/handle/10/10333.
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Previous issue date: 2018-06-27O Glifosato [N-(fosfonometil)glicina] é um herbicida pós-emergente, não seletivo e sistêmico. No processo de criação das formulações comerciais de herbicidas a base de glifosato (GBHs, do inglês glyphosate-based herbicides), como o Roundup®, são adicionados surfactantes com o intuito de aumentar a eficiência do composto base. A rota prioritária de degradação do glifosato por micro-organismos no solo resulta na formação do ácido aminometilfosfônico (AMPA). As respostas moleculares ao glifosato têm sido extensivamente estudadas em espécies de plantas e em alguns vertebrados. Em humanos, apesar dos estudos até agora realizados, não se conhece exatamente quais os riscos e mecanismos de atuação que explicariam a toxicidade ao glifosato relatada em alguns experimentos. Sendo assim, a hipótese dessa tese é de que a exposição rápida ao Roundup® e ao AMPA leva à alterações de expressão gênica em importantes processos celulares. Dessa forma, o objetivo desse trabalho é identificar genes diferencialmente expressos (DEGs, do inglês differentially expressed genes) em células mononucleares do sangue periférico (PBMCs, do inglês, peripheral blood mononuclear cells) humano submetidas à exposição rápida com herbicida à base de glifosato (Roundup®) e AMPA. O teste de MTT [3(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio brometo], realizado em triplicatas, foi utilizado para avaliar a viabilidade celular e para a escolha das condições de tratamento utilizadas na técnica de microarray (GeneChip® Human Transcriptome Array 2.0, Affymetrix). As condições analisadas foram controle (3 chips), AMPA (10 mM; 3 chips) e Roundup® (0,05%; 2 chips), expostos durante 3 horas. Utilizando um valor de p<0,05 e fold-change de 1,5 foram identificados 5 DEGs no tratamento com o AMPA e 26 no tratamento com Roundup®. As análises de enriquecimento mostraram que os genes com expressão alterada após exposição ao Roundup® estavam associados a 33 processos celulares, principalmente relacionados à regulação destes processos. A plataforma digital Pathview foi utilizada para identificar a atuação dos DEGs após exposição ao Roundup® em diferentes vias. Os genes TNF, LTA, TAB2 e ATM foram relacionados à via de sinalização NF-kappa β; BCL2L11 e ATM à via de sinalização FoxO; SESN3 e ATM à via de sinalização p53; e TNF, BCL2L11 e ATM à apoptose. Dessa forma, os resultados sugerem que o Roundup® altera o padrão de expressão gênica de diversos genes associados com o controle do ciclo celular, regulação de processos celulares e apoptose.
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Strydom, Aliki Veruschka. "Extraction and biomedical application of peripheral blood stem cells in sheep and horses." Thesis, Stellenbosch : University of Stellenbosch, 2007. http://hdl.handle.net/10019.1/1146.
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Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2007.SUPERFICIAL digital flexor tendon injury has a serious negative impact on the competitive horse industry. Injured horses require up to a year of rest for recovery and likelihood of re-injury upon return to normal activity is as high as 80 %. Tendon healing requires (a) production of collagen by fibroblasts, to provide tensile strength and elasticity to the tendon, (b) minimisation of restrictive fibrosis, which compromises tendon gliding function and (c) minimisation of peritendinous adhesions. We review conventional treatments for tendon healing before exploring stem cell application as a therapeutic alternative. We promote the use of hematopoietic and mesenchymal stem cells derived from adult peripheral blood - as opposed to bone marrow-derived stem cells or embryonic stem cell sources - and review published research output in this regard. In conclusion, we outline our research objectives and present and discuss our results in the chapters that follow. Mononuclear cells - consisting of hematopoietic stem cells, mesenchymal stem cells and leucocytes – were isolated from the peripheral blood of sheep and horses through red blood cell lysis and blood plasma extraction. Cell counts and propidium iodide dye exclusion viability tests were conducted on the cell pellets. Sheep sub samples were tested for CD45 expression and horse sub samples for CD4 and CD11a/18 cell surface markers by flow cytometry for characterisation purposes. In both cases, separate sub samples were incubated with matched immunoglobulin (IgG) isotypes, conjugated to fluorescein isothiocyanate (FITC), to serve as controls. For the culture of mononuclear cells, 4.5 x 106 cells were selected for autologous sheep injections, 3 x 106 CD45- cells for allogeneic sheep injections (the latter excluding leucocytes that may induce an immune response) and 72 x 106 cells for horse injections. These cells were incubated with bromo-deoxyuridine (BrdU), cultured and subsets were extracted for a second round of cell counts and viability tests before being resuspended in blood plasma. For the horse samples an additional 1 x 106 mononuclear cells were incubated until reaching 60 % confluence and tested for myogenic differentiation. Low cell mortality and lack of fluorescence from IgG-FITC controls reflected effective protocols and a lack of false positive results. The fact that the equine cell population differentiated into myotubes verified the presence of mesenchymal stem cells in injections. We tested whether surgical incisions or collagenase injections best mimicked naturally occurring tendon injuries and compiled macroscopic and microscopic descriptions of tendon injury sites at seven weeks post-injury. The superficial digital flexor tendons of 27 sheep received an incision, a collagenase injection or a saline control injection. After one week a number of sheep were sacrificed while the remainder received further saline treatment and were sacrificed after another seven weeks. Tendons were examined through clinical observations, image analysis of maximum tendon diameter, mechanical testing and histological sectioning of affected tissues. Collagenase-induced injury resembled tendonitis more closely than surgically-induced injury. Collagenase-injured tendons (a) induced lengthier lameness in affected limbs, (b) were more swollen and difficult to palpate, (c) assumed the bow appearance characteristic of natural injury, (d) experienced extensive haemorrhage due to collagen lysis, (e) had decreased elasticity and capacity to carry loads and stress, (f) displayed decreased stiffness due to collagen fibre disruption and (g) developed severe inflammation. After seven weeks injured tendons displayed increased vascularisation in the areas of haemorrhage and in the adjacent collagen matrix. High inflammation rates and low collagen levels however still persisted. Collagenase injections were used to induce tendonitis in the superficial digital flexor tendons of 27 sheep. After one week these tendons received treatment with a control saline solution, autologous peripheral blood mononuclear cells (MNCs) or allogeneic peripheral blood CD45- MNCs. Healing rates were compared after a further seven week period by conducting ultrasonographic evaluations, clinical observations, image analyses of maximum tendon diameter, mechanical tests and histological investigations. Tendons treated with MNCs displayed an improvement in echogenicity and fibre linearity, higher and more organised collagen levels, stronger mechanical properties and less swelling. Although these improvements were not always significant, they provided strong evidence to suggest marked healing benefits over a longer time period. Collagenase injections were used to induce tendonitis in the superficial digital flexor tendons of four horses. After one week these tendons received treatment with either a control saline solution or autologous peripheral blood mononuclear cells (MNCs). Healing rates were compared after a further seven week period by conducting ultrasonographic evaluations, clinical observations, image analysis of maximum tendon diameter and histological investigations. Tendons treated with MNCs displayed significant improvements in fibre linearity in the direct vicinity of the lesion, as well as recovery rate thereof, and experienced less swelling when compared with their untreated counterparts. Healing trends suggested that, given a longer period of observation post-injury, more significant improvements may become apparent. Human adipose tissue is known be an easily accessible and high yielding source of multipotent mesenchymal stem cells. These stem cells could potentially be used for therapeutic advancement of tendon regeneration. Our first goal was to examine the in vitro myogenic differentiation potential of adipose-derived, adherent mononuclear cells (MNCs) from six adult sheep. The second goal was to characterise the population of cells isolated through various available ovine specific, non-mesenchymal stem cell surface markers, namely, CD1, CD31, CD34 and CD45. After incubation, only four of the six MNC cultures started to proliferate. These four cultures all exhibited high myogenic differentiation ability. The isolated cell populations did not express any of the non-mesenchymal stem cell specific cell surface markers. In conclusion, our data suggests that peripheral blood stem cells and adipose-derived stem cells are important candidate cell types for therapeutic application to improve tendon repair in horses and sheep. Sufficient time must be allowed following injury and prior to stem cell treatment (at least one month) and a controlled exercise program should be followed posttreatment. A larger sample size is required and at least six months of recovery before macroscopic and histological repair can be analysed more accurately and conclusively. Ultrasonography should be carried out on a continuous basis, as it is a non-invasive method of monitoring change over time.
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Hellgren, Sofie. "Impact of cryopreservation and characterization of peripheral blood mononuclear cells and subsets in healthy donors by multicolor flow cytometry analysis." Thesis, Uppsala universitet, Institutionen för kvinnors och barns hälsa, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-413035.
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Introduction: Immune therapy plays a larger role in cancer treatment these days, but in order to find new therapies and improve already existing ones, more knowledge about the immune system is needed. The peripheral blood contains many different cell types, some extensively studied, some less well-known. By using multicolor flow cytometry, the immune status of an individual can be displayed in relatively short time. Aim: The aim of this study was to develop a multicolor flow cytometry method in order to examine the distribution of 31 cell types, with a focus on immune regulatory cells, in peripheral blood in healthy donors, as well as examine the impact of cryopreservation on the different subsets. Methods: After isolating the mononuclear cells from peripheral blood using Ficoll separation, each sample (n = 19) were analyzed with three flow cytometry panels. The remaining cells were cryopreserved in -190°C and later thawed and analyzed the same way as the fresh samples were. Results: The cell distribution in fresh samples were mostly consistent with other studies. While the percentage of many cell types remained unchanged after thawing, the total percentage of T helper cells and some subsets were decreased in frozen samples, leading to a decrease in total T cells. Furthermore, the percentage of total monocytes were increased and the distribution of monocyte subsets were altered in frozen samples, among others. Conclusion: This study confirms the results of other studies of the human immune system and provides valuable knowledge about the impact of cryopreservation.
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Sofrenovic, Tanja. "Evaluation of an Enhanced (Sialyl Lewis-X) Collagen Matrix for Neovascularization and Myogenesis in a Mouse Model of Myocardial Infarction." Thesis, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/22747.
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In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis.
Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.
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Lin, Ming-Cheng, and 林明政. "Relationship between C-myc proto-oncogene expression and DNA methylation in peripheral blood mononuclear cells from patients with systemic lupus erythematosus." Thesis, 1993. http://ndltd.ncl.edu.tw/handle/86791823012568583410.
Full textAbstract:
碩士高雄醫學院
醫學研究所
81
Systemic Lupus Erythematosus (SLE) is a multisystem in fam- matory disorder characterized by the production of antibodies that react with many different self-antigens. However,little is known about their molecular pathogenesis. In this study, the peripheral blood mononuclear cells (PBMC) expression of c-myc proto-oncogene and 5-methylcytosine content C-myc proto- oncogene in samples of normal individuals and of patients who fulfilled the criteria of the American Rheumatism Association for the classification of SLE were investigated. The SLE patients ex- pressed an average of 1.6-fold increase c-myc mRNA levels. The methylation-pattern of the human c-myc proto- oncogene in patient with SLE does not differ from that of normal controls. These results indicated that methylation status of the gene are not responsible for the enhanced c-myc expression in PBMC of SLE patients.
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Bispo, Cláudia Simões Martins 1988. "The role of peripheral blood mononuclear cells in atherogenesis." Master's thesis, 2011. http://hdl.handle.net/10451/4929.
Full textAbstract:
Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2011Atherosclerosis (ATH) is recognized as a chronic inflammatory condition and it is the leading cause of cardiovascular disease. Atherogenesis is characterized by the infiltration of low density lipoprotein (LDL) through the endothelial layer, and migration of activated peripheral blood lymphocytes (PBL) and monocytes (PBMN) that contribute to a pro-inflammatory state in specific locations. However, the functional interaction between immunity and LDL metabolism is still not fully understood. One hypothesis for the etiopathogeny of ATH may be associated with an ongoing inflammatory process caused by a pro-oxidant/anti-oxidant imbalance induced by metals such as iron (Fe) or copper (Cu). Interestingly, ceruloplasmin (Cp) is a multicopper oxidase with a relevant role in Fe metabolism and oxidation of LDL, but also an acute-phase protein involved in the inflammatory process. Herein, we intended to study by flow cytometry the effect of putative pro-atherogenic immune stimuli on the expression of Cp at the surface of human peripheral blood mononuclear cells (PBMC), and search for a putative association with LDL oxidation. Additionally, a population of Familial Hypercholesterolemia (FH) patients was used as a clinical model of ATH to clarify the physiologic interplay between inflammation, Fe/Cu and lipid metabolism at systemic level in this disease. The obtained results showed that higher cell surface expression of Cp was consistently observed in PBMN compared to PBL, in activated vs non-activated cells and in non-T cells vs T cells. Also, PBMC surface expression of Cp was differently modulated by several tested treatments. In particular, various modulators caused opposite effects on Cp expression of PBMN compared to PBL, suggesting a specific cell-type regulation for this protein. Specifically, it was observed that different sources of Fe might activate specific regulation mechanisms of Cp. Until now, due to technical limitations it was not possible to demonstrate an association between PBMC surface Cp expression and oxidation of LDL, but this issue should be addressed in future experiments. Of notice, IL-1β which was found to significantly increase PBMN surface Cp expression in vitro, was increased in serum from FH patients and positively correlated with total cholesterol and ApoB. However, the fact that no correlation was found between IL-1β and serum Cp (sCp)/oxidated LDL suggest that at systemic level, IL-1β may not be a contributing factor for oxidation of LDL by sCp. However, the role of PBMC surface Cp expression on LDL oxidation, namely in specific inflammatory states, remains to be established. In summary, the results of this study demonstrate that specific pro-atherogenic conditions are involved in the modulation of Cp expression at surface of PBMC and thus, suggest a possible a role of Cp in ATH.
A aterosclerose (AT) é a principal causa de doenças cardiovasculares (DCV). Muito embora esta doença tenha sido numa primeira fase considerada como uma simples deposição de lípidos, avanços substanciais nas áreas da ciência experimental permitiram revelar o importante papel da inflamação nesta patologia. Actualmente, a AT já não é reconhecida como uma simples consequência de envelhecimento considerando os factores de risco tradicionalmente aceites no seu envolvimento, mas antes como uma doença inflamatória crónica que pode ser convertida num evento clínico agudo por ruptura de placas ateroscleróticas e consequente trombose. A formação da placa aterosclerótica (aterogénese) caracteriza-se por uma infiltração e activação precoce de monócitos e linfócitos (células mononucleares) que contribuem para um estado pró- inflamatório localizado em regiões de endotélio disfuncional. Adicionalmente, a passagem de lipoproteínas de baixa densidade (LDL) através da camada endotelial e a sua oxidação constituem eventos precoces da aterogénese. Contudo, os mecanismos fisiológicos envolvidos na interacção funcional entre as células imunitárias e a oxidação das LDL não estão completamente esclarecidos. Uma hipótese para a etiopatogenia da AT poderá estar associada à existência de um processo inflamatório contínuo relacionado com stress oxidativo induzido por metais, tais como ferro (Fe) ou Cobre (Cu). A ceruloplasmina (Cp) é uma proteína de fase aguda da família das multicobre oxidases com funções importantes no metabolismo do Fe, principalmente devido à sua actividade ferroxidásica. Para além disso, esta proteína apresenta potencial pró-e anti-oxidante, actividades relevantes no contexto do stress oxidativo característico deste processo. O objectivo geral deste estudo foi investigar e caracterizar in vitro alguns dos mecanismos subjacentes à relação funcional entre inflamação, metabolismo do Fe/Cu e a homeostase lipídica, de forma a adquirir novos conhecimentos sobre a fisiopatologia da AT. Em particular, estudaram- os efeitos de status de Fe e Cu alterado, citocinas pró-inflamatórias e outros imunomoduladores na expressão de Cp à superfície de células mononucleares de sangue periférico (PBMC). Este estudo foi realizado através da utilização de citometria de fluxo, que possibilitou caracterizar a expressão da Cp membranar em subpopulações leucocitárias específicas de monócitos (PBMN) e linfócitos (PBL) de sangue periférico. Posteriormente, avaliou-se a possível associação entre a modulação da expressão de Cp membranar nas condições testadas e a oxidação de LDL. Adicionalmente, realizou-se a caracterização laboratorial de uma população de doentes diagnosticados com Hipercolesterolemia Familiar (HF), como um modelo clínico de AT. Neste contexto, pretendeu-se complementar os estudos in vitro procurando a existência de associações entre os parâmetros do metabolismo do Fe/Cu, lípidos e inflamação medidos a nível sistémico. Os resultados obtidos através de citometria de fluxo mostraram consistentemente que a expressão de Cp à superfície de PBMN é maior comparativamente com PBL e que em ambas as populações as células activadas apresentam maiores níveis de expressão desta proteína que as suas homólogas não activadas. Além disso, mostrou-se que dentro das subpopulações de PBL, são as células não-T que possuem maior expressão de Cp à sua superfície em comparação com as células T. Por outro lado, os resultados obtidos através das experiências de modulação mostraram que a Cp membranar das duas populações celulares em estudo é diferentemente regulada na presença dos diversos estímulos. Em condições de status de Fe alterado foi observado que a Cp à superfície de PBMC é diferentemente regulada nos dois tipos celulares testados, e parecem existir mecanismos de regulação específicos consoante o dador de Fe utilizado. Adicionalmente, a alteração do status de Cu não produziu modificações significativas na expressão de Cp à superfície de PBMC. Estas observações não corroboraram resultados obtidos anteriormente por outros autores e devem-se provavelmente ao curto espaço de tempo de incubação que não possibilitou visualizar o efeito de variação da concentração deste metal na estabilidade da Cp. Em relação aos tratamentos com as citocinas e outros imunomodulatores observou-se de uma forma geral que a interleucina (IL)-1ß, lipopolissacarídeo, e forbol 12-miristato 13-acetato induziram o aumento da expressão de Cp à superfície dos PBMN enquanto que a diminuíram nos PBL, sugerindo que a Cp membranar destas populações celulares poderá ser regulada especificamente devido às funções particulares e diferenças de cada tipo celular durante a resposta inflamatória. Os tratamentos com interferão-. e IL-2 diminuíram a expressão de Cp à superfície de PBMN e PBL em incubações de longa duração, enquanto os tratamentos efectuados com os mediadores IL-6, IL-8, factor estimulador de colónia de macrófagos e o factor de necrose tumoral- a não resultaram variações na expressão membranar de Cp em ambos os tipos celulares. Provavelmente, a expressão de Cp à superfície de PBMC poderá ser nestes casos modulada em curtos espaços de tempo, pelo que as condições experimentais utilizadas não permitiram confirmar esta hipótese. A influência de espécies reactivas de azoto na expressão de Cp à superfície de PBMC foi igualmente testada realizando o tratamento com S-nitroso-N-acetilpenicilamina(dador de óxido nítrico), verificando-se que na presença deste tipo de condições de stress oxidativo são especificamente os PBMN que aumentam a expressão de Cp à sua superfície. Estes resultados sugerem que em estado pró-inflamatórios e de stress oxidativo a Cp expressa à superfície dos PBMN parece estar mais associado ao papel de proteína de fase aguda descrito para a forma circulante da Cp enquanto nos PBL esta proteína parece estar mais associada à regulação de status de Fe intracelular de modo a permitir a proliferação deste tipo de células após estimulação. Posteriormente, avaliou-se uma possível associação entre a indução de Cp à superfície de PBMC e oxidação das LDL no meio. Contudo, limitações técnicas impediram a confirmação desta hipótese neste trabalho. No entanto, a detecção de níveis de LDL oxidada (oxLDL) no sobrenadante de culturas em que Cp membranar de PBMN fora induzida por IL-1ß, sugere a existência de potencial pró-oxidante na forma membranar de Cp. Esta observação carece contudo de confirmação após optimização de procedimento experimental utilizado. Os resultados decorrentes da caracterização clínica do grupo de indivíduos diagnosticados com HF revelaram que estes doentes apresentam diferenças nos parâmetros relacionados com o metabolismo dos lípidos, consistentes com o seu diagnóstico molecular que mostra a existência de mutações genéticas em componentes do metabolismo lipídico. Nestes doentes foram ainda encontrados valores mais elevados de ferritina, oxLDL e IL-1ß comparativamente com os indivíduos saudáveis. Além disso, nos doentes HF observou-se a existência de correlações positivas entre Cp circulante (sCp) e oxLDL, concordante com potencial pró-oxidante desta proteína, e entre sCp e proteína C reactiva -duas proteínas de fase aguda presentes em processos inflamatórios envolvidos na progressão desta doença. Foi também encontrada a correlação positiva de IL-1ß com níveis de colesterol total e ApoB, parâmetros associados com risco aumentado de DCV. Contudo, o facto de não ter sido observada nenhuma correlação entre os níveis de IL-1ß e sCp / oxLDL nestes indivíduos sugere que a nível sistémico a IL-1ß parece não contribuir para o aumento de acção pró- oxidante da sCp. No entanto não é de excluir a possibilidade de um possível papel fisiológico de Cp expressa à superfície das PBMC a nível local nas placas ateroscleróticas. Em conclusão, os resultados deste estudo demonstram que modulatores específicos de Fe/Cu e mediadores inflamatórios putativamente associados a estados pró-aterogénicos podem estar, pelo menos em parte, envolvidos na modulação da forma membranar da Cp em PBMC, sugerindo um possível papel da Cp à superfície dessas células durante a aterogénese.
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Tsai, Pei-Wen, and 蔡佩雯. "Regulation of Telomerase Activity in Human Peripheral Blood Mononuclear Cells." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/20485747326756971054.
Full textAbstract:
碩士長庚大學
基礎醫學研究所
88
Abstract Telomerase is a specialized ribonucleoprotein reverse transcriptase. Its main function is to synthesize the tandem repeat sequence (TTAGGG) of telomere on the human chromosome end. The telomerase activity is low or undetectable in normal somatic cells, but is reactivated in most cancers and immortal cells. Therefore, the expression of telomerase activity appears to play a crucial role in the process of cell immortalization and carcinogenesis. As yet, little is known concerning the mechanism of telomerase regulation in human cells. In this study, human peripheral blood mononuclear cells (PBMC) were used to investigate the regulation of telomerase activity. PBMC express low or undetectable telomerase activity. Treatment of PBMC with phytohemagglutinin (PHA) activates T cells, and the telomerase activity was increased 14 folds. Here, I have demonstrated that the increased telomerase activity during T cell activation was attributed to the induction of hTERT, the reverse transcriptase subunit of telomerase. Preceding the induction of hTERT, there is a rapid induction of c-myc, suggesting that c-myc may be one of the factors that upregulates hTERT expression. To explore the signal transduction pathways involved in the up-regulation of telomerase activity during T cell activation, I have employed inhibitors for protein tyrosine kinase (PTK), phospholipase C (PLC), protein kinase C (PKC), MAP kinase kinase (MEK) and nuclear factor κB (NF-κB) to study their effect on the telomerase activity. In the presence of PTK or PKC inhibitor, the induced expression of c-myc, hTERT and telomerase activity was completely inhibited, while the other inhibitors produce either partial inhibition or no inhibition. Therefore, PTK- and PKC- mediated signaling pathways are involved in the up-regulation of telomerase expression during T cell activation. Finally, we have employed PMA and ionomycin to further explore the potential role of c-myc in the up-regulation of hTERT expression. PBMC treated with PMA resulted in an ordered induction of c-myc, hTERT and telomerase activity. On the other hand, PBMC treated with ionomycin resulted in an early transient increase of c-myc that was followed by an increased expression of hTERT. However, c-myc expression was not detected following longer treatment of ionomycin, yet the expression of hTERT continued to increase. These results indicate that while c-myc may be one of the factors that are involved in the up-regulation of hTERT expression, there is a c-myc-independent mechanism for the up-regulation of telomerase expression.
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Tu, Jian-Ching, and 涂建勍. "Chromosomal Rearrangements Detected in Peripheral Blood Mononuclear Cells of Normal Adults." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/17606316073746722415.
Full textAbstract:
碩士國立臺灣大學
醫學檢驗暨生物技術學研究所
98
Variations in the human genome consists of single nucleotide polymorphisms (SNPs) sites, and structural variation (SVs); which contains deletion, insertion, inversion, translocation and duplication. Up to date, more than 10 million SNPs have been used to study human genetic and phenotypic changes. In addition, data on human genome variation database (Database of Genomic Variants, DGV) to larger fragments of the structural variation information data has collected 90,000 document information, among which 99% is copy number variation. The current technology array-CGH can detect changes of copy number variation, but not the balanced structural variation, including inversion and translocation. Our laboratory has previously established the technique Restriction Enhanced Capturing Of Rearranged DNA (RECORD) to determine the target genes structure variation and was shown to detect three kinds of gene translocation, the neonatal and infant leukemia common translocation gene MLL, child care type of bone cancer common translocation gene EWSR1, and child-type lymphoblastic Leukemia translocation gene TCF3 in human sperm cells. In this study, we set to determine whether chromosomal rearrangement also occur in human Peripheral Blood Mononuclear Cells (PBMCs) using MLL as target gene. The RS4; 11 cell lines with t(4; 11)(q21; q23) balanced translocation of chromosomes was first used to determine the experimental conditions and sensitivity of the RECORD. Among the PBMCs from 25 healthy individuals, some random translocation chromosome structure was observed. The object of its translocation chromosomes was scattered in different locations, and the orientation suggested by the sequence can be grouped into three types of chromosomes, the two-center (dicentric), no center (acentric), and single-center (derivative) chromosomes. Flow cytometry sorting was next used to determine the cell types, B, T, and non-B/non-T groups, in which translocations occur. Our preliminary results suggest that germ cell chromosomal rearrangement mechanisms can be retained to differential cells. Our study results provide some information about the mechanism of evolution within individual, yet whether it can be related to some sporadic gene-related diseases and the potential mechanisms require further exploration.
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You, Chiou-Mien, and 游秋綿. "Responses of Human Peripheral Blood Mononuclear Cells to Glucosyltransferases of Streptococcus mutans." Thesis, 1999. http://ndltd.ncl.edu.tw/handle/61623403828504586490.
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碩士國立臺灣大學
微生物學研究所
88
The investigation of peptide vaccine against dental caries in oral cavity is an interesting subject in mucosal immune response. Streptococcus mutans had been identified as the principal pathogen of the human dental caries. Protein antigen I/II (AgI/II) and glucosyltransferases (GTFs) are important virulence factors responsible for the colonization of S. mutans and these proteins have been shown to be good vaccine candidates in animal model. Immunization with either AgI/II or GTFs could induce protective immune response against experimental dental caries. In humans, major B cell and T cell epitopes of AgI/II have been mapped. But the cellular response to the GTFs in human is still not clear. Our laboratory has found that significant difference existed in the human natural antibody responses against three S. mutans GTFs (GtfB/C/D). The purpose of this study is to find out if GtfB/C/D could induce T-cell proliferation to the different degree and to search for possible T cell epitope candidates for the design of peptide vaccine. The recombinant GtfC, GtfD, and fragments of GtfD fusion proteins were expressed in E. coli and purified with either His-tag or glutathione S-transferase (GST) affinity chromatography. The identity and homogeneity of the purified fusion proteins were confirmed by SDS-PAGE and Western blot analysis. The enzymatic activities of purified GtfC and GtfD were confirmed by activity gel staining and sucrose hydrolysis assay. Crude extracellular cell-free (CF) and cell wall- associated (CA) protein extracts were prepared from S. mutans MT8148. Control antigens for lymphocyte proliferation assays were purified tetanus toxoid (TT), staphylococcal enterotoxin subunit B (SEB, a superantigen) and phytohemagglutinin (PHA, a mitogen). In addition, we have also tested the T cell responses to three synthetic peptide fragments; Gtf-P1, Gtf-P2, and AgI/II-P of 19, 21 and 22 amino acid residues, respectively. Gtf-P1 and -P2 were putative protective B-cell epitopes and AgI/II-P was a major T-cell epitope of S. mutans AgI/II found in European people. All these antigens were tested initially by lymphocyte proliferation assay using peripheral blood mononuclear cell (PBMC) for titration of the proper antigen concentration. We have previously found that anti-GtfD antibody level was significantly higher than that of anti-GtfB/C in both serum and saliva. We hypothesized that differential response of GtfD could result from difference in T-cell responses to GtfB/C. A total of 6 cord and 22 adult blood samples were taken and PBMC were isolated for [3H]thymidine lymphocyte proliferation assay. MHC class II DRB1 molecular typing confirmed typical Chinese DRB1 distribution and random distribution in tested blood samples. The stimulatory index (SI) value (mean standard deviation) in cord blood PBMC were 173.181.27 by PHA, 13.17.4 by GtfC, 11.87.1 by GtfD, 7.15.5 by CA, CF by 2.451.03, 0.740.46 by TT, and 35.5 by SEB, respectively. Therfore, GTFs could stimulate cord blood PBMC with a capacity far below the superantigen SEB. SI values (m sd) for PBMC from adult samples were 180.5117.3 by PHA, 8.610.4 by GtfC, 18.615.78 by GtfD, 3.73.0 by CA, 2.681.71 by CF, and 8.85.88 by TT, respectively. Therefore, GtfD exhibited greater stimulation effect on PBMC than GtfC and TT, and presumably GtfD is a better candidate for T-cell epitopes. Gtf-P1, -P2 and AgI/II-P did not stimulate PBMC proliferation even in higher concentrations. To confirm the stimulatory effect of GtfC/D on PBMC of cord blood samples, blocking experiments by monoclonal antibody WR18 that recognized anti-human HLA DP, DQ, DR was conducted and results indicated that GtfC/D preparation may not exhibit mitogenetic effect. Antibody levels to GtfD in both plasma and saliva of tested samples were stronger than GtfC. These results confirmed our previously findings and suggested that differential responses to GTFs identified in antibody responses might be attributed by a stronger T cell response against GtfD.
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Weng, Wen-Ting, and 翁文婷. "Infection and stimulation of human peripheral blood mononuclear cells by enterovirus 71." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/28870550285426454647.
Full textAbstract:
碩士國立陽明大學
微生物及免疫學研究所
101
Enterovirus 71 (EV71) is an RNA virus which belongs to Picornaviridae family. EV71 mainly infects children and often causes hand, foot, and mouth disease (HFMD) or even causes neurological or systemic complications which may lead to sequelae or death. Until now, it remains unknown about how EV71 progresses to the central nervous system (CNS) after infection via the gastrointestinal tract. EV71-infected patients with pulmonary edema had significantly lower circulating lymphocytes and reduced cytokine production and cell proliferation in peripheral blood mononuclear cells (PBMCs), suggesting the association between EV71 infection and host cellular immune responses. To further confirm whether EV71 could infect human PBMCs directly, we used EV71-GFP, a reporter virus that can express green fluorescent protein (GFP) upon viral replication. We found that CD14+ monocytes in PBMCs could be infected by EV71-GFP. The viral titer in PBMCs reached a peak and was about 10-fold higher at 8 hrs after EV71 infection. Our results show that EV71 infects and replicates in PBMCs. To further study the impact on monocytes, we used qPCR and ELISA to measure the cytokine levels after PBMCs co-cultured with UV-inactivated virus or live virus. We found cytokine levels, such as TNF-α, IL-1β, and IL-6, in virus-infected PBMCs are higher than that in UV-inactivated virus-treated PBMCs. The levels of CD69 and MHC classII on monocytes were induced after EV71 infection, however, the level of CD86 was reduced in the presence of EV71. In conclusion, we demonstrated that EV71 may infect monocytes directly and stimulate the monocyte-related cytokine secretion. Moreover, EV71 infection may also affect monocytes activation and regulate their ability as an antigen presenting cells.
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Yi-Chen, Lai, and 賴怡辰. "Effects of Toll-like receptor agonists on porcine peripheral blood mononuclear cells." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/42735994699424982539.
Full textAbstract:
碩士國立屏東科技大學
動物疫苗科技研究所
101
Toll-like receptor (TLR), a type of pattern recognition receptor (PRR), identifies different pathogen associated molecular patterns (PAMPs). Their binding with PAMPs induces immune responses and thus plays an important role in immune system. CpG oligodeoxynucleotides (CpG-ODN), FliC, and PolyI:C are different TLR agonists. CpG contains unmethylated CG pairs of nucleotides module (CpG motif). Action of mode of CpG motif is mainly mediated through TLR9. CpG motif can increase antibody production in B cells, cytokines secretion in non-specific immune cell and thereby increase activities of T and NK cells. FliC, a flagellin protein recognition by TLR5, can promotion cell-mediate immunity. PolyI:C, a synthetic double-stranded RNA recognited by TLR3, induces type I interferon production. However, research regarding the activation of porcine antigen-presenting cells stimulated with TLR agonists are still few. In this study, peripheral mononuclear cells (PBMC) were isolated from two different breeds of pigs at six to eight weeks of age, and then stimulated with CpG and FliC prepared in our lab, or commercially available polyI:C. The results showed that CpG and polyI:C significantly stimulated the secretions of IL-18 and IFN-r but did not affect the IL-10 secretion while FliC significantly enhanced IL-10 secretion. CpG significantly increased gene expression of TLR9. FliC significantly promoted TLR5 and TLR8 gene expressions. PolyI:C significantly increased gene expression of TLR3. In summary, TLR agonists exhibit the function of modulating activities of immune cells and can potentially act as vaccine adjuvants or immune-modulatory agents.
50
Tsern, Jy-Mei, and 岑枝梅. "Regulation of Areca Nut Extracts on Functional Activation of Peripheral Blood Mononuclear Cells." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/87843347867264811491.
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碩士國立陽明大學
口腔生物研究所
88
Abstract The habit of betel quid chewing, very common in Taiwan, has long been associated with an increased risk of oral submucous fibrosis and oral cancer. In animal models and some tissue culture studies, it was shown that areca nut or its chemical components can promote tumor formation and cause cell death. However, the pathogenesis of betel quid related oral diseases is not fully understood yet. In this study, we aimed to investigate the regulation of areca nut extract (ANE) on cytokine elaboration of peripheral blood mononuclear cells (PBMC), and its effect on gingival fibroblast proliferation. First, freshly isolated PBMC were stimulated with various concentrations of ripeANE (10-100 μg/ml), tender ANE (20-200 μg/ml) or arecoline (10-100 μg/ml) for 8, 24, and 48 hours. The mRNA and protein of proinflammatory cytokines were then detected by RT-PCR and ELISA separately. We found that both ripe and tender ANE (but not arecoline) could stimulate the production of TNF-αand IL-1β. Ripe ANE caused steady and persistent upregulation of cytokines, whereas the regulatory effect of tender ANE was abrupt and short-lived. Moreover, the PBMC response to ripe or tender ANE stimulation varied among different donors with regard to their sensitivity and released cytokine level. Secondly, gingival fibroblasts were cultured with the condition medium from PBMC incubated with 50 μg/ml ripe ANE or 200 μg/ml tender ANE for 8 or 24 hours. With BrdU labeling, the modulation of fibroblast proliferation was monitored. The result showed that the culture supernatant from unstimulated PBMC could increase fibroblast proliferation, but the effect of conditional medium of ANE-stimulated PBMC was variable. In summary, areca nut extracts can induce PBMC elaborating TNF-αand IL-1β, which may play important modulatory roles in the process of disease development. However, its mechanisms and biological significance remain to be further investigated.