<p> Mouse spleen leukocytes were stimulated with phytohemagglutinin and infected with Vesicular Stomatitis Virus. The virus titre from stimulated and unstimulated cells was determined and the number of infected cells in stimulated and unstimulated cultures was examined. </p> / Thesis / Master of Science (MSc)

Introduction: Space is an extreme environment exposing astronauts to microgravity and cosmic radiation resulting in immune dysfunction. To overcome the complex challenges of studying astronauts in space, bed rest studies represent an alternative model simulating microgravity exposure on Earth. We sought to characterize the steady state transcriptome changes in leukocytes isolated from two microgravity models: (1) participants to 60 days of bed rest and (2) astronauts to ~6 months of spaceflight. Methods: The bed rest study recruited twenty healthy men receiving a nutritional supplement or not; the spaceflight study had fourteen male and female astronauts participate. For both studies, ten blood samples were collected over three study phases, leukocytes were isolated, and transcriptomes were quantified using high throughput RNA-sequencing. My pipeline of data analysis applied differential expression (DE) methods and functional enrichment to identify gene expression changes and pathways responding to the altered gravity environments of both bed rest and spaceflight models. Results: Temporal differential expression identified transcriptome modulation reflecting multisystem shifts and immune dysregulation in response to the transitions to and from bed rest (2,415 DE genes) and spaceflight (247 DE genes). Interestingly, later bed rest and in-flight timepoints trended towards stable RNA levels with no differential expression. The bed rest study found the nutritional intervention had no mitigating effects on transcriptome changes (0 DE genes), and the spaceflight study revealed down-regulation in response to spaceflight followed by an opposite up-regulation upon return to Earth. Conclusion: The altered gravity environments of bed rest and spaceflight significantly modulated leukocyte transcriptome compositions revealing immune dysfunction at the molecular level. Future analyses utilizing the higher quality bed rest dataset is required to isolate the effect of microgravity from other space stressors and apply validation experiments to develop gene biomarkers indicative of immune deconditioning.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Mouse strains are frequently used to model human disease states, to test the efficiency of drugs and therapeutic principles. However, the direct translation of murine experimental data to human pathological events often fails due to sufficient differences in the organization of the immune system of both species. Here we give a short overview of the principle differences between mice and humans in defense strategies against pathogens and mechanisms involved in response to pathogenic microorganisms and other activators of the immune system. While in human blood mechanisms of immune resistance are highly prevailed, tolerance mechanisms dominate for the defense against pathogenic microorganisms in mouse blood. Further on, species-related differences of immune cells mainly involved in innate immune response as well as differences to maintain oxidative homeostasis are also considered. A number of disease scenarios in mice are critically reflected for their suitability to serve as a model for human pathologies. Due to setbacks in these studies, novel mouse models were created to bridge the immune system of both species: humanized mice. Accordingly, a special section of this review is devoted to new results applying humanized mouse models taking limitations and prospects into account.

Small diameter synthetic vascular grafts have yet to be clinically successful due to luminal narrowing from thrombosis and intimal hyperplasia. Current attempts to address this issue include the development of materials that support endothelialisation and protein modification to the material surfaces that reduce thrombosis. The extracellular matrix protein elastin has been found to be one of the least thrombogenic components of blood vessels, and its purified and recombinant forms have shown reduced thrombogenicity in both in vitro and in vivo models. Biomaterial coatings of elastin-like polypeptides (ELPs) recombinantly produced in the Woodhouse laboratory showed reduced fibrinogen adsorption, platelet adhesion, and platelet activity. However, the reason for their relative non-thrombogenicity is still not fully understood. In this work, the leukocyte response to ELP-coated materials was investigated. In particular, ELP1 and ELP4, which differ in molecular weight and sequence length, were physically adsorbed to a polyethylene terephthalate surface (MylarTM), yielding 0.22 ± 0.13 μg/cm2 and 0.37 ± 0.19 μg/cm2 surface coverage, respectively, as determined by the colorimetric assay, FastinTM Elastin. These surfaces were exposed to flowing citrated whole blood for surface and bulk evaluation of leukocyte activity using scanning electron microscopy and flow cytometry, respectively. Little leukocyte activation was observed on the surface of the controls, low-density polyethylene and uncoated MylarTM. In the bulk, tissue factor (TF) expression (monocytes: ELP1 = 38.6 ± 16.3 %, ELP4 = 33.9 ± 18.1 %) and platelet-leukocyte aggregates determined by CD61 (monocytes: ELP1 = 63.1 ± 17.1 %, ELP4 = 61.8 ± 16.8 %; granulocytes: ELP1 = 62.7 ± 17.0 %, ELP4 = 60.5 ± 20.1 %) were both decreased compared to uncoated MylarTM, while CD11b upregulation (monocytes: ELP1 = 18.7 ± 2.2 %, ELP4 = 19.7 ± 2.7 %; granulocytes: ELP = 21.4 ± 3.7 %, ELP4 = 22.0 ± 3.2 %) was increased. The statistical dependence of TF expression and platelet-monocyte aggregates was tested; however, no correlation was found. Overall, platelet-leukocyte aggregate formation was reduced and there were conflicting results with regards to the reduction of leukocyte activation for the ELP coatings on MylarTM. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-10-10 15:34:51.802

Sirtuins (SIRT) regulate the transcription of various genes involved in the development of diet-induced obesity and chronic disease; however, it is unknown how they change acutely following a high-fat meal. The purpose of this study was to determine the effect of a high-fat meal (65% kcals/d; 85% fat recommendation), on SIRT1-7 mRNA expression in blood leukocytes at 1, 3, and 5-h post-prandial. Men and women (N=24) reported to the lab following an overnight fast (>12H). Total RNA was isolated and reverse transcribed prior to using a Taqman qPCR technique with 18S rRNA as a normalizer to determine SIRT1-7 mRNA expression. An additional aliquot of serum was used to measure triglycerides. Data was analyzed using a RM ANOVA with P<0.05. Triglycerides (P<0.001; 124%) peaked at 3-h. SIRT 1 (P=0.004; 70%), and SIRT 6 (P=0.017; 53%) decreased expression at 3-h. SIRT4 (P=0.024) peaked at 5H relative to baseline (70%) and 3-h (68%). To our knowledge, this is the first study to report that consumption of a high-fat meal transiently alters SIRT mRNA expression consistent in a pattern that mirrors changes in serum triglycerides. Decrease in expression of SIRT1 and SIRT6 combined with an increased SIRT4 would be consistent with an increase in metabolic disease risk if maintained on a chronic basis.

INTRODUCCIÓN. Los leucocitos fecales son utilizados para identificar diarrea invasiva y decidir el uso de antibióticos. Se conoce poco sobre su utilidad en hospitales de países en desarrollo con procesos de laboratorio eficientes. Buscamos evaluar el rendimiento diagnóstico de la prueba en menores de 5 años con diarrea aguda. MATERIAL Y MÉTODOS. Estudio retrospectivo de registros clínicos y de laboratorio en el Hospital de Emergencias Pediátricas, Lima, Perú. Se evaluó los casos a los que se había solicitado sistemática e independientemente leucocitos fecales y coprocultivo. Se calculó sensibilidad, especificidad, valores predictivos, cocientes de probabilidad (CP) y la curva de características operativas del receptor (ROC). RESULTADOS. De 1,804 muestras fecales, 901 (49,9%) fueron positivos para uno o más enteropatógenos bacterianos. La sensibilidad (S), especificidad (E), y el CP positivo variaron para los diferentes umbrales: más de 5 leucocitos por campo (S: 93.2%, E: 21.9%, CP: 1.9), más de 20 (S: 88.4%, E: 34.8%, CP: 1.35), más de 50 (S: 74.9%, E: 56.7%, CP: 1.73), y más de 100 (S: 60.7%, E: 71.9%, CP: 2.17). El área bajo la curva ROC fue 0.69 (IC 95%: 0.67-0.72). CONCLUSIONES. El rendimiento de la prueba es sub-óptimo y continuar su uso rutinario en la práctica clínica no parece justificado, pues promueve el abuso de antibióticos y por otro lado aumenta el riesgo de pasar por alto pacientes con diarrea invasiva. Se necesita estudiar el rendimiento diagnóstico de datos epidemiológicos y clínicos combinados con leucocitos fecales o lactoferrina fecal, para identificar una aproximación más eficiente. / INTRODUCTION. Fecal leukocytes are widely used to identify invasive diarrhea and to make then the decision of prescribing or not antibiotics. This test has been hardly assessed in small hospitals of developing countries with efficient laboratory processes. We aimed to assess the diagnostic performance of different thresholds of fecal leukocytes in children under-five with acute diarrhea. MATERIAL AND METHODS. Retrospective study of clinical and laboratory records in the Pediatric Emergency Hospital, Lima, Peru. All cases with a stool culture and fecal leukocytes independently and systematically performed were studied. Sensitivity, specificity, predictive values, likelihood ratios (LR), and receiver operating characteristics (ROC) curves were calculated. RESULTS. Out of 1,804 stool samples assessed, 901 (49,9%) were positive for one or more bacterial entheropathogens. Sensitivity (Sn), specificity (Sp), and positive LR varied for different thresholds: more than 5 (S: 93.2%, Sp: 21.9%, LR+:), more than 20 (Sn: %, Sp: %, +LR: ), more than 50 (Sn: 74.9%, Sp: 56.7%, +LR: 1.73), and more than 100 fecal leukocytes per high power field (Sn: 60.7%, Sp: 71.9%, LR+: 2.17). The general area under the ROC curve was 0.69 (CI 95%: 0.67-0.72).

The core of the doctoral thesis "Regulation of signal transduction by leukocyte surface proteins" consists of three publications in international peer-reviewed journals dealing with leukocyte signaling both at the level of individual signaling pathways and in the context of a multicellular organism. Most attention is paid to signaling via the T cell receptor (TCR), which plays a central role in the development and function of T cells and represents a key signaling pathway for proper function of the adaptive component of the immune system. Transmembrane protein tyrosine phosphatase CD148 was considered a negative regulator of TCR signaling through dephosphorylation of LAT and PLCγ1 proteins. This study brings evidence that CD148 is able to modulate signaling also at the level of Lck, both positively and negatively. The net effect of CD148 activity on the TCR signaling is determined by the intracellular biochemical context, notably, the presence of another tyrosine phosphatase CD45. The second project dealt with the characterization of a transmembrane adaptor protein PRR7. This adapter inhibits TCR signaling via down-regulation of the intracellular Lck and cell surface TCR levels. The research concerning the signaling in the environment of a multicellular organism is represented by the analysis of...

Este estudo teve por objetivos: i) caracterizar o influxo leucocitário induzido pelo veneno de Bothrops insularis (VBi); ii) avaliar a liberação de PGD2 e PGE2, TXA2 e LTB4 e das quimiocinas MCP-1 e KC induzida pelo VBi; iii) analisar a ação do VBi quanto a expressão protéica das enzimas ciclooxigenases (COX -1 e -2) e iv) avaliar a participação dos metabólitos das COX-1 e -2 e 5-LO no influxo leucocitário. O VBi causou aumento de leucócitos circulantes e do influxo de leucócitos PMN e MN para o local de sua injeção. Ainda, veneno induziu a liberação de PGD2, PGE2, TXA2 e LTB4, da MCP-1, mas não da KC, além da expressão protéica de COX-2. O tratamento dos animais com indometacina, etoricoxibe ou zileuton inibiu o influxo de leucócitos induzido pelo VBi na 12ª h. Em conclusão, o VBi tem capacidade de induzir influxo de leucócitos e liberação de mediadores inflamatórios para o local de sua injeção. O influxo leucocitário relaciona-se aos metabólitos das COX -1 e -2, 5-LO, da MCP-1 e do aumento de leucócitos circulantes / In this study the effects of Bothrops insularis venom (BiV) on the leukocyte influx, on the circulating leukocyte numbers and release of mediators, such as PGD2, PGE2, TXA2, LTB4, MCP-1 and KC into the local of its injection. Moreover, the role of eicosanoids in the BiV- induced leukocyte influx was assessed by selected pharmacological treatments. PMN were accumulated from 6 up to 24 h and MN cells from 3 up to 72 h when injected into the peritoneal cavity of mice. Moreover, BiV increased blood leukocyte at 3 h after injection and incresed the levels of PGD2, PGE2, TXA2, LTB4, MCP-1 at distinct periods of time. In addition, BiV induced the protein expression of COX-2 from 1 up to 12 h and the BiV-induced leukocyte influx was reduced by indomethacin or etoricoxib or zileuton at 12 h after injection. In conclusion, BiV is able to induce leukocyte influx and increase of blood leukocyte numbers. Moreover, the ability of BiV to induce expression of COX-2 and release of inflammatory mediators is relevant for the leukocytes influx into the local of its injection