Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser.

This thesis investigates two new approaches to the use of multiple antibody labels in flow cytometry. On the experimental side, we develop the Multiplex Labelling method, which allows the number of simultaneous antibody labels to exceed the number of fluorochromes, thus overcoming the technical limitations imposed by the number of available fluorochromes and fluorescence measuring channels. On the theoretical side, we construct a dynamic interpretation of immunofluorescent flow cytometry data, which allows information on the kinetics of cell division and differentiation to be extracted. The first part of the thesis discusses multiplex labelling. Chapter 1.1 presents the theory of this method, a reconstruction formula on which the algorithm for multiplex labelling data processing can be based, and a case study illustrating the use of this method in the triple labelling analysis of murine thymocytes. A murine thymocyte subset not previously described by flow cytometry is observed in this study for the first time. Chapter 1.2 describes the Immunofluorescence Tomograph, a microcomputer-controlled device for the preparation of multiplex labelling solutions. This device makes possible the routine use of multiplex triple labelling, by carrying out a complicated and time consuming part of the experimental protocol. The second part of this thesis deals with the dynamic interpretation of the data. Chapter 2.1 describes the theory of skeletal analysis, which is a coarse topological analysis of immunofluorescent flow cytometry data, concerned with the outlines of regions where the distribution is significantly different' from zero. Chapter 2.2 investigates the finer topological details of the distributions resulting from division and/or differentiation. We show that, under certain reasonable conditions, these distributions acquire simple forms, which can be easily analyzed and compared to actual data. Chapter 2.3 presents murine thymocyte triple labelling data obtained by multiplex analysis. These include data on the embryonic thymus (from day 15 to day 20 of embryonic development), as well as the neonate and adult thymus. The methods developed in chapters 2.1 and 2.2 are applied, and two partial thymocyte lineages are defined. One of these lineages has not been previously reported. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

Rheumatoid arthritis (RA) is a chronic, heterogeneous, autoimmune disease that causes inflammation of synovial joints leading to pain, stiffness and swelling. If left untreated, RA results in irreversible joint destruction and long term disability. Initial treatment with glucocorticoids and other immunosuppressive agents suppresses inflammation. However, many of these drugs are not well-tolerated due to extensive side effects or are simply ineffective. The discovery of tumour necrosis factor-α (TNF) as a key mediator of inflammation in RA led to the development of monoclonal anti-TNF antibody therapy. Since then, other biologic drugs targeting immune pathways have been developed for RA, including interleukin-6 (IL-6) blockade, B cell depletion, and T cell co-stimulation blockade. Not all patients will respond to their first biologic drug and currently there is no way to predict which patient will respond to each different class of drug. Generally, 3 – 6 months are required to determine clinical efficacy, during which time joint inflammation proceeds. Therefore, discovering biomarkers to predict treatment response is a research priority. Biologic drugs target immune pathways. As single cell technology advances and has increasing capacity to identify subtle changes in many cell subsets, I hypothesise that studying the blood immune cell landscape will define cellular biomarker profiles relevant to each individual patient’s disease.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

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No description available.

A Research Report submitted to the Faculty of Medicine, Department of Haematology, University of the Witwatersrand, Johannesburg for the Degree of Master of Medicine (in the branch Haematology). / WHSLYP2016

A practical and versatile instrument has been developed both to optimize the chemistry of bio- and chemiluminescence reactions and to measure ultra-trace quantities of associated analytes. The instrument consists of a pneumatic flow injection system linked to a detection cell and a signal processing and readout system. The disk-like cell is defined by a pair of transparent polycarbonate plates and an "0-ring" seal. The lower plate is fitted with a reflective surface. Relative to conventional cells, this design affords a high light collection efficiency due to the large volume element viewed by an end-on photomultiplier detector. Rapid mixing of reagents within the cell is brought about by injection through concentric ports of a commercial burner assembly at a point immediately below the detector. The signal processing and readout system is interfaced to an IBM compatible personal computer and appropriate software was written to automate the instrument and to acquire, store and manipulate luminescence data. With this instrumentation, the chemistry of marine bacteria biolumin-escence was optimized for the determination of cis-11-hexadecenal and, ostensibly, for both the quantification of aldehyde insect pheromones and potential use in the control of insect pests. With the optimized conditions, cis- 11-hexadecenal was determined to 7 fmol. This value is more than an order of magnitude lower than detection limits for aldehyde pheromones reported in the literature. In this research, the less ideal substrates undecanal and heptanal were determined to 570 fmol and 65 pmol, respectively. Marine bacteria bioluminescence was used to quantify several epoxide analytes derivatized to aldehydes. 1,2-epoxyhexadecane and 1,2- epoxytetradecane were determined to 55 and 51 fmol, respectively. 1,2- epoxyoctane and cis-7,8-epoxy-2-methyloctadecane were determined to 100 and 3 pmol, respectively. The latter compound is the sex pheromone of the gypsy moth (Lymantria dispar), a well-known and serious agricultural pest. Epoxides have not been quantified previously with either chemi- or bioluminescence. The instrument was modified for use with corrosive solutions and for possible interfacing with a high performance liquid chromatograph. Lophine chemiluminescence was optimized for the analysis of Cr(VI) samples. With the optimized conditions, aqueous solutions of Cr(VI) were determined to 50 μg /L. A plausible explanation is offered for the dependence of lophine chemiluminescence on the concentration of the chromium species. / Graduation date: 1991

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This Thesis investigates the possibility of an extrathymic pathway of T cell development in the amphibian, Xenopus laevis. Initial studies examined the ontogenetic development of T cell surface antigen expression on both splenocytes and thymocytes in larvae at 7 days through to 6-8 month old adults, using the technique of dual-colour flow cytometry and employing a panel of T and B cell specific monoclonal antibodies. Flow cytometric analysis of splenocytes from thymectomized (Tx) Xenopus was then addressed. This revealed that early (5-7 day) larval thymectomy resulted in the ablation of T cell surface antigen expression, as defined by the monoclonal antibodies 2B1 (anti- CD5), AM22 and F17 (both anti-CD8), XT-1 (anti-XTLA-1) and D4.3 (putative anti-aβ T cell receptor). Lack of these markers was still evident in 8 month old Tx frogs, confirming the effectiveness of the operation. In vitro studies showed that no T cell marker expression could be induced on the surface of splenocytes from Tx animals following stimulation with concanavalin A (ConA), phytohaemagglutinin (PHA) or the potent mitogenic agent, phorbol myristate acetate (PMA). Studies were also carried out to investigate whether in vitro stimulation induced apoptosis. Flow cytometric studies revealed that CD5(^dull) expression could be induced on splenocytes from control Xenopus following stimulation with PMA. The nature of this induced CD5(^dull) expression was investigated further in order to determine why this phenomenon was only seen in control animals. These experiments involving mixing of T and B cell populations, revealed that CD5(^dull) expression was being induced upon the surface of Xenopus B cells, and that this PMA-induced expression required the presence of T cells, and was blocked by a protein kinase C (PKC) inhibitor. Finally, an additional search for extrathymic T cells involved examining the intestine and liver of both control and Tx Xenopus, these tissues being sites of extrathymic T cell development in higher vertebrates. The intestine of control Xenopus was shown to contain T lymphocytes with a surface phenotype distinct to that found in spleen or liver. Studies in Tx Xenopus showed that although expression of some T cell markers was ablated in liver and gut, CD5(^dull) and CD8(^dull) (as determined by the mAb AM22) lymphocytes persisted in these organs. However, proliferative studies showed that these 'T-like' cells were unable to respond to mitogenic stimulation with ConA, suggesting that they are not functional T cells.