5-Oxo-ETE ist ein chemotaktischer Faktor für Granulozyten, der von der NADP+-abhängigen Dehydrogenase 5h-dh aus dem 5-Lipoxygenaseprodukt 5-HETE gebildet wird. Ziel dieser dreiteiligen Studie war es, die der 5-oxo-ETE-Produktion zugrunde liegenden Regulationsmechanismen aufzuklären. I. Einfluß von myeloider Zelldifferenzierung auf die Expression von 5h-dh in HL-60 und U-937 Zellen. Undifferenzierte HL-60 und U-937 Zellen produzieren vergleichbare Mengen von 5-oxo-ETE wie Monozyten oder Granulozyten. Differenzierung von U-937 Zellen mit PMA verdreifacht die Enzymaktivtät von 5h-dh, während die Behandlung von HL-60 Zellen mit dh-VitD3 diese verdoppelte. Der Einfluß von PMA auf 5h-dh wurde darüber hinaus in Mikrosomen von U-937 Zellen untersucht. Die Behandlung PMA verdreifachte Vmax, liess aber KM unbeeinflußt. II. Regulation der 5-oxo-ETE-Produktion durch oxidativen Stress und Glukose. Da der GSH-Redoxzyklus die Produktion von NADP+ zur Folge hat, stimulierten die Hydroperoxide H2O2 und tBOOH die Synthese von 5-oxo-ETE in U-937 Zellen. Aufgrund seiner Verarbeitung durch den Pentosephosphat Zyklus, der NAD+ in NADPH umwandelt, inhibierte Glucose diesen Effekt von H2O2. Die Synthese von 5-oxo-ETE wurde durch H2O2 auch in humanem Monozyten, Lymphocyten und Thrombozyten, aber nicht in Neutrophilen angeregt. Im Gegensatz zu Monozyten zeigten sich Thrombozyten und Lymphozyten allerdings glukose-resistent. T-BOOH ehöhte auch die Produktion von 5-oxo-ETE nach Zugabe von Ionophore und Arachidonsäure zu mononukleären Blutzellen. III. 5h-dh-Expression in human Strukturzellen. Zunächst rasterten wir mehrere sekundäre Epithelzelllinien und fanden 5h-dh in allen Zellen. Drei Indizien lassen vermuten, daß die epithele 5h-dh der myeloiden entspricht: (i) die enzymatische Aktivtät liegt vor allem in der mikrosomalen Fraktion vor, (ii) bei dem Kofaktor handelt es sich um NADP+ und nicht um NAD+, und (iii) 5S-HETE ist das bevorzugte Substrat. Weitere Studien zeigten, daß auch primäre humane Aorta-Endothelzellen 5h-dh expremieren. Vergleichbar zu Entzündungszellen wird die Produktion von 5-oxo-ETE auch in Endothel- und in Epithelzellen durch oxidativen Stress angeregt. / 5-Oxo-ETE is a highly potent granulocyte chemoattractant that is formed by the NADP+-dependent dehydrogenase 5h-dh by oxidation of the 5-lipoxygenase product 5-HETE. The objective of this study was to investigate underlying regulatory mechanisms of 5-oxo-ETE production in human cells. This matter was addressed from three directions. I. Expression of 5h-dh in HL-60 and U-937 cells and its activity changes during myeloid cell differentiation. Undifferentiated U-937 and HL-60 cells produce similar amounts of 5-oxo-ETE compared to monocytes or neutrophils. Differentiation of U-937 cells with PMA resulted in a 3-fold increase in 5-oxo-ETE production. Similarly, incubation of HL-60 cells with dh-VitD3 induced a 2-fold increase in 5-oxo-ETE production. The impact of PMA on 5h-dh was also investigated in the microsomal fraction of U-937 cells and compared to neutrophil microsomes. PMA treatment leads to a increase of Vmax but does not affect KM. II. Regulation of 5-oxo-ETE by oxidative stress and glucose levels. We found that H2O2 and t-butyl hydroperoxide strongly stimulate 5-oxo-ETE formation by U-937 cells through the GSH redox cycle by providing NADP+. Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, which converts NADP+ back to NADPH. 5-Oxo-ETE synthesis was also strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils. Unlike monocytic cells, lymphocytes and platelets were resistant to the inhibitory effects of glucose. 5-Oxo-ETE synthesis following incubation of peripheral blood mononuclear cells with arachidonic acid and calcium ionophore was also strongly enhanced by t-BOOH. III. Expression of 5h-dh in human structural cells. We screened several secondary epithelial cell lines and detected 5h-dh in all cell lines. Epithelial 5h-dh and the inflammatory cell 5h-dh are identical: (i) the enzymatic activity is localized in microsomes, (ii) the cofactor is NADP+, and (iii) 5S-HETE is the preferred substrate. We also found that primary human aortic endothelial cells express 5h-dh. 5-oxo-ETE production by both endothelial and epithelial cells is regulated by oxidative stress in a manner similar to inflammatory cells.

Nach einer Läsion im Gehirn kommt es trotz der Bildung autoreaktiver T-Zellen zu keiner autoimmunen Neuropathologie. Foxp3+ regulatorische T-Zellen (Tregs) vermitteln möglicherweise Immuntoleranz nach zerebraler Läsion. Deswegen wurde in dieser Studie die Rolle der Tregs 7, 14 und 30 Tage nach einem transienten Verschluss der mittleren Hirnarterie (MCAO), einem Modell für ischämischen Schlaganfall, und nach entorhinaler Kortexläsion (ECL) in der Maus untersucht. Durchflusszytometrisch wurde in beiden Modellen 14 und 30 Tage nach Läsion eine Akkumulation der Tregs in der ipsilateralen Hemisphäre beobachtet. Mikroskopisch wurden an der Läsion Zellkontakte der Tregs mit antigenpräsentierenden Zellen beobachtet. Weitere Experimente wurden ausschließlich nach MCAO durchgeführt. Am Tag 14 und 30 war in der ipsilateralen Hemisphäre eine Akkumulation der Mikroglia zu beobachten. Makrophagen und dendritische Zellen wurden an den Tagen 7, 14 und 30 detektiert. Am Tag 7 und 14 waren ipsilateral im Gehirn ca. 60 % der Tregs positiv für den Proliferationsmarker Ki-67. In zwei Versuchsansätzen wurden naive CD45RBhigh/CD4+ Zellen aus lymphatischen Organen von Foxp3EGFP Mäusen, mit Wildtyp T-Zellrezeptor (TCR), oder 2D2.Foxp3EGFP Mäusen, mit TCR spezifisch gegen Myelin-Oligodendrozyten-Glykoprotein, isoliert. Die Zellen wurden einen Tag vor MCAO in RAG1-/- Mäuse, welche keine adulten T- und B-Zellen besitzen, transferiert. Am Tag 14 nach MCAO war in den RAG1-/- Mäusen keine de novo Induktion Foxp3EGFP+ Tregs zu beobachten. CD25+ Tregs wurden durch die Injektion eines Antikörpers gegen CD25 depletiert, um deren Wirkung nach MCAO zu untersuchen. Nach Depletion konnte bis zu 27 Tage nach MCAO keine Veränderung des Läsionsvolumen und des Gangverhaltens beobachtet werden. In dieser Studie wurde im Gehirn eine späte Präsenz und Proliferation Foxp3+ Tregs nach Läsion nachgewiesen. Mikroglia und periphere Immunzellen sind langfristig an Immunvorgängen im lädierten Gehirn beteiligt. / After brain lesion autoreactive T cells specific against brain antigens are expanded, but no delayed autoimmune neuropathology evolves. Immune suppressive CD4+/Foxp3+ regulatory T cells (Tregs) could have an important role in maintaining immune tolerance in the lesioned brain. Therefore, this study sought to analyse the role of Tregs in mice 7, 14 and 30 days after transient middle cerebral artery occlusion (MCAO), a model for ischemic stroke, and entorhinal cortex lesion (ECL). An accumulation of Tregs was detected in the brain by flow cytometry in both models at days 14 and 30 after lesion. Using immunohistochemistry Tregs were found in close cell-cell contact with antigen presenting cells at the lesion site. Further experiments were performed solely with MCAO. On days 14 and 30 after MCAO a strong accumulation of microglia occurred in the ipsilesional hemisphere. Macrophages and dendritic cells were found ipsilesionally on days 7, 14 and 30. On days 7 and 14 about 60% of Tregs were positive for the proliferation marker Ki-67 in the lesioned hemisphere. In two different setups naïve CD45RBhigh/CD4+ cells were isolated from lymphatic organs of Foxp3EGFP mice, carrying a wild type T cell receptor (TCR), or 2D2.Foxp3EGFP mice, carrying a TCR specific for myelin oligodendrocyte glycoprotein. One day before MCAO naïve CD45RBhigh/CD4+ cells depleted of Foxp3EGFP+ Tregs were transferred into RAG1-/- mice, which lack adult B and T cells. At day 14 after MCAO no de novo generation of Foxp3EGFP+ Tregs was observed. The effects of Tregs on stroke outcome were tested by depleting CD25+/Foxp3EGFP+ Tregs with an antibody against CD25. After depletion no effects on lesion volumes and gait parameters were detected up to 27 days following MCAO. The present study demonstrates for the first time a sustained presence and proliferation of Tregs in the lesioned brain. Local microglia and peripheral immune cells are involved in long-lasting immune processes following brain lesion.

Células mononucleares de sangue de cordão umbilical (SCU) e sangue periférico mobilizado (SPM) com G-CSF, foram cultivadas in vitro com citocinas, na presença ou não de estroma de medula óssea. Os objetivos foram avaliar a capacidade proliferativa de células progenitoras, a ocorrência de apoptose e expressão de integrina. Nas culturas sem estroma, a celularidade aumentou 5 vezes (SCU) e não se alterou nas de SPM. O total de células CD34+ caiu em ambas culturas. Com estroma, o total de células nucleadas aumentou 7 vezes (SCU) e 2,3 vezes (SPM). O total de células CD34+ permaneceu o mesmo. A apoptose foi menor nas culturas de SCU. A expressão de integrina caiu, na população de células CD34+ e de CD45+ / Mononuclear cells from umbilical cord blood (UCB) and G-CSF mobilized peripheral blood (MPB), were cultured in vitro, in the presence of cytokines, with or without bone marrow stroma. The aims were to evaluate the proliferative response of progenitor cells, occurrence of apoptosis and expression of adhesion molecule. In cultures without stroma, cellularity increased 5-fold for UCB, but has not changed for MPB. The number of CD34+ cells has dropped in both culture. With stroma, total nucleated cells had a 7-fold increse (UCB) and a 2,3-fold (MBP), however, CD34+ cells number has not changed. Apoptosis was lower in UCB culture. The expression of integrin decreased, in the CD34+ and CD45+ population

Células mononucleares de sangue de cordão umbilical (SCU) e sangue periférico mobilizado (SPM) com G-CSF, foram cultivadas in vitro com citocinas, na presença ou não de estroma de medula óssea. Os objetivos foram avaliar a capacidade proliferativa de células progenitoras, a ocorrência de apoptose e expressão de integrina. Nas culturas sem estroma, a celularidade aumentou 5 vezes (SCU) e não se alterou nas de SPM. O total de células CD34+ caiu em ambas culturas. Com estroma, o total de células nucleadas aumentou 7 vezes (SCU) e 2,3 vezes (SPM). O total de células CD34+ permaneceu o mesmo. A apoptose foi menor nas culturas de SCU. A expressão de integrina caiu, na população de células CD34+ e de CD45+ / Mononuclear cells from umbilical cord blood (UCB) and G-CSF mobilized peripheral blood (MPB), were cultured in vitro, in the presence of cytokines, with or without bone marrow stroma. The aims were to evaluate the proliferative response of progenitor cells, occurrence of apoptosis and expression of adhesion molecule. In cultures without stroma, cellularity increased 5-fold for UCB, but has not changed for MPB. The number of CD34+ cells has dropped in both culture. With stroma, total nucleated cells had a 7-fold increse (UCB) and a 2,3-fold (MBP), however, CD34+ cells number has not changed. Apoptosis was lower in UCB culture. The expression of integrin decreased, in the CD34+ and CD45+ population

We studied the effects of 30 minutes of exercise on T lymphocyte counts and proliferative responses of peripheral blood mononuclear cells (PBMC) in 25 runners. Exercise resulted in a T lymphocytosis in the immediate post-exercise period in all subjects (p < 0.001), and reduced CD4+/CD8+ ratios in 22/25 subjects (p = 0.001). The change was due primarily to a 2.2-fold increase in CD8+ cells (p < 0.001). Exercise also reduced PBMC mitogenic responses to phytohemagglutinin (PHA) in 13/14 subjects (p = 0.049), and to pokeweed mitogen (PWM) in 11/14 subjects (p = 0.022), but not to concanavalin A. Postrun sera from 5 of 6 subjects inhibited PHA but not PWM responses of resting autologous PBMC with normal CD4+/CD8+ ratios (p < or = 0.05): indomethacin and monocyte depletion blocked the serum inhibition (p = 0.003, p = 0.0006, respectively). We conclude that post-exercise suppression of mitogenic responses to PHA is due to the release of a serum factor(s) capable of inducing prostaglandin synthesis by circulating monocytes, whereas exercise-induced suppression of PWM responses depends primarily on the reversal of CD4+/CD8+ ratios.

This master’s thesis is focused on automating the process of differential leukocyte count in peripherial blood using image processing. It deals with the design of the processing of digital images - from scanning and image preprocessing, segmentation nucleus and cytoplasm, feature selection and classifier, including testing on a set of images that were scanned in the context of this work. This work introduces used segmentation methods and classification procedures which separate nucleus and the cytoplasm of leukocytes. A statistical analysis is performed on the basis of these structures. Following adequate statistical parameters, a set of features has been chosen. This data then go through a classification process realized by three artificial neural networks. Overall were classified 5 types of leukocytes: neutropfiles, lymphocytes, monocytes, eosinophiles and basophiles. The sensitivity and specificity of the classification made for 4 out of 5 leukocyte types (neutropfiles, lymphocytes, monocytes, eosinophiles) is higher than 90 %. Sensitivity of classiffication basophiles was evaluated at 75 % and specificity at 67 %. The total ability of classification has been tested on 111 leukocytes and was approximately 91% successful. All algorithms were created in the MATLAB program.

Vid blodgivning donerar blodgivare blod frivilligt. Blodet kan sedan användas inom sjukvården för exempelvis blodtransfusion, vilket kräver blodprodukter kompatibla med patienten. Förekomst av leukocyter i blodprodukter medför en ökad risk för febrila transfusionsreaktioner hos transfunderade patienter. Därför krävs det att vid framställning leukocytreducera blodprodukter och utföra kvalitetskontroll. Med analysen B-leukocytpartikelkoncentration (LPK) kan totalantalet leukocyter i helblod beräknas. Flödescytometri är en metod som kan analysera optiska och fluorescerande egenskaper hos exempelvis celler i en suspension, vilket kan användas för att kvantifiera cellantal. BD Leucocount™-Kit (BD Biosciences) är avsett för flödescytometrisk analys av antalet kvarvarande leukocyter i leukocytreducerade blodprodukter. Vid framställning av blodprodukter ska helblodspåsen vila vid rumstemperatur i minst 3 timmar efter blodgivning. I Falun används antingen ett dagsprogram där produktion sker samma dag som blodgivningen, eller ett övernattningsprogram där produktion sker dagen därpå. Prover från 505 kontrollerade erytrocytenheter, samlade i Falun, har påvisat en skillnad i leukocytkoncentration beroende på vilket program som använts. Anledningen till att erytrocytenheternas leukocytinnehåll skiljer sig är inte känt. Syftet med denna studie är därav att undersöka om vilotiden har någon effekt på leukocytkoncentrationen i helblodspåsar. LPK varierade mellan helblodspåsarna. Ett ökande leukocytantal observerades över tid i majoriteten av helblodspåsar, inklusive medelvärde. Däremot kunde inte hypotesprövning påvisa statistisk signifikans. Hypotesen om att leukocytantalet ökar över tid går emot grundläggande hematologi. Utifrån resultaten i denna studie kan inte hypotesen bevisas. Vidare studier bör genomföras. / During blood donation, blood donors donate blood voluntarily. The blood can then be used in healthcare for, for example, blood transfusions, which requires blood products compatible with the patient. The presence of leukocytes in blood products increases the risk of febrile transfusion reactions in transfused patients. Therefore, leukocyte-reduction in blood products is necessary during production. Each blood center must perform quality control on produced blood products. With the analysis B-leukocyte particle concentration (LPK), the total number of leukocytes in whole blood can be calculated. Flow cytometry is a method that can analyze the optical and fluorescent properties of, for example, cells in a suspension, which can be used to quantify cell numbers. The BD Leucocount™-Kit (BD Biosciences) is intended for flow cytometric analysis of the number of leukocytes remaining in leukocyte-reduced blood products. When producing blood products, the whole blood bag should rest at room temperature for at least three hours after the donation. In Falun, either a day program is used where production takes place on the same day as the blood was donated, or an overnight program where production takes place the next day. Samples from 505 controlled erythrocyte units, collected in Falun, have shown a difference in leukocyte concentration depending on the program used. The reason why the leukocyte content of erythrocyte units differs is not known. The purpose of this study is therefore to investigate whether the resting period has any effect on the leukocyte concentration in whole blood bags. The LPK varied between the whole blood bags. An increasing leukocyte count was observed over time in most of the whole blood bags. However, hypothesis testing did not show statistical significance. The hypothesis that leukocyte counts increase goes against basic hematology. Based on the results of this study, the hypothesis cannot be proven. Further studies should be conducted. / <p>Vårdförbundet tilldelade Leo Svahn stipendium 2021 för <em>bästa kandidatuppsats inom biomedicinsk laboratorievetenskap</em>.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Vaccinia virus (VV) is the prototypical member of the orthopoxvirus genus of the Poxviridae family, and is currently being evaluated as a vector for vaccine development and cancer cell-targeting therapy. Despite the importance of studying poxvirus effects on the human immune system, reports of the direct interactions between poxviruses and primary human leukocytes (PHLs) are limited. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias towards binding and infecting monocytes among PHLs. VV binding strongly co-localized with lipid rafts on the surface of these cell types, even when lipid rafts were relocated to the cell uropods upon cell polarization. In humans, monocytic and professional antigen-presenting cells (APCs) have so far only been reported to exhibit abortive infections with VV. We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, were permissive to VV replication. The majority of virions produced in MDMs were extracellular enveloped virions (EEV). Visualization of infected MDMs revealed the formation of VV factories, actin tails, virion-associated branching structures and cell linkages, indicating that infected MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. Classical activation of MDMs by LPS plus IFN-γ stimulation caused no effect on VV replication, whereas alternative activation of MDMs by IL-10 or LPS plus IL-1β treatment significantly decreased VV production. The IL-10-mediated suppression of VV replication was largely due to STAT3 activation, as a STAT3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data indicate that PHL subsets express and share VV protein receptors enriched in lipid rafts. We also demonstrate that primary human macrophages are permissive to VV replication. After infection, MDMs produced EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread.