Syfte: Syftet med studien var att jämföra två metoder vid undersökning av tårfilmsanalys; tårflödesbeteende-metoden och fluorescein break-up mönster och om deras mätresultat överensstämmer med varandra. Detta för att diskutera om någon av metoderna kunde vara ett bättre alternativ att adaptera ute i praktiken för vårdgivare inom ögonvården. Metod: Initialt fick deltagarna besvara en OSDI-enkät (ocular surface disease index), därefter gjordes resterande mätningar i biomikroskopet. I biomikroskopet utfördes initialt dem icke-invasiva mätningarna som tillhörde tårflödesbeteende-metoden (mätning av TMH (tårmeniskhöjd), debrishastighet efter blinkning och mätning av lipidlagrets interferensmönster) även NiBUT (non-invasive tear break-up time) utfördes. Efter dem icke-invasiva mätningarna tillsattes fluorescein i inferiora temporala fornix och dem invasiva testerna; TBUT (tear break-up time) och FBUP (fluorescein break-up mönster) utfördes tre gånger. Alla mätningar dokumenterades med video. Tre oberoende bedömningar utfördes. Studien undersökte om resultatet av tårflödesbeteendet och FBUP hade ett samband och vilken utav metoderna som fick en högre intravaliditet utefter dem bedömningar som utfördes. Resultat: Totalt deltog 26 deltagare varav 4 exkluderades. Deltagarna var mellan 19–57 år gamla med en genomsnittsålder på 35 år och en standardavvikelse på ±12,3 år. Samtliga presenterade med en OSDI ≥13, alternativt hade en visuellt frekvent blinkfrekvens. Det återfanns ett samband mellan tårflödesbeteende-metoden och FBUP i studien, dock ingen av klinisk signifikans ((bedömare 1, Cramer’s V= 0,46 p=0,06) (bedömare 2, Cramer’s V=0,33 p=0,32) (bedömare 3, Cramer’s V=0,33 p=0,34)). Tårflödesbeteende-metoden (Fleiss kappa =0,29, p=0,40) och FBUP (Fleiss kappa =0,25, p=0,40) uppvisade båda på en mindre god intra-validitet Slutsats: Det finns ett samband mellan tårflödesbeteende-metoden och FBUP men ingen statistisk signifikans. Intravaliditeten är marginellt högre vid tårflödesbeteende-metoden än vid FBUP i studien. / Aim: The aim of this study was to compare two methods used during tear-film analysis; tear-flow behaviour, and fluorescein break-up pattern and if their measured results are in an agreement. This in order to discuss which one of the methods would be more suitable to apply in clinical practice for healthcare workers in the field of optometry.  Method: Initially the participants filled out an OSDI questionnaire (ocular surface disease index), thereafter measurements were done in the biomicroscope. Initially the non-invasive measurements such as the tear-flow behaviour method (measurements of TMH (tear meniscus height), speed of debris movement across the tear film after blinking and measurements of the lipid layer interference) and NiBUT (non-invasive break-up time) were measured. Afterwards fluorescein was applied in the inferior temporal fornix and the invasive measurements; TBUT (tear break-up time) and FBUP (fluorescein break-up pattern) were measured three times. All measurements done in the biomicroscope were documented with video and three independent assessments were done. This study looked at the results from the tear-flow behaviour and FBUP and if they agreed with one another. It also looked into which one of the methods presented a higher intra-validity in regard to the assessments made. Results: The study had a total of 26 participants, of which 4 were excluded. The participants were between the ages of 19-57 years old with an average age of 35 years with a standard deviation of ±12,3 years. The participants presented with an OSDI of ≥13, with exceptions made if a participant visually had a high blink frequency. A correspondence between the tear-flow behaviour method and  FBUP could be observed, however none of statistical significance ((assessor 1, Cramer’s V= 0,46 p=0,06) (assessor 2, Cramer’s V=0,33 p=0,32) (assessor 3, Cramer’s V=0,33 p=0,34)). The intra-validity of the tear-flow behaviour method (Fleiss kappa =0,29, p=0,40) and FBUP (Fleiss kappa =0,25, p=0,40) were both categorized as weak intra-validity.  Conclusion: There is a correspondence between the tear-flow method and FBUP, without statistical significance. The intra-validity was marginally higher within the tear-flow method compared to FBUP in the study.

Das Proteasomsystem ist die Hauptquelle von Peptiden für die MHC Klasse I Antigen-Präsentation. In Vertebraten kann dieses durch die Expression verschiedener Subtypen des 20S Proteasoms moduliert werden. Die häufigsten Subtypen sind konstitutive Proteasomen (c20S) mit den katalytischen Untereinheiten beta1, beta2 und beta5 und Immunoproteasomen (i20S) mit den Immunountereinheiten beta1i, beta2i und beta5i. Die Expression von i20S optimiert in der Regel die MHC Klasse I Antigen-Präsentation, indem die Bildung von Peptiden mit hoher Affinität zu MHC I Molekülen verstärkt wird. Die Bildung von i20S wird momentan durch ein Modell der kooperativen Assemblierung erklärt, das auf der präferentiellen Interaktion zwischen den Immunountereinheiten beruht. In dieser Arbeit wurde die Assemblierung von 20S Proteasomen in beta5i defizienten Mäusen nach Infektion mit Listeria monocytogenes analysiert. In diesem Modell konnte keine präferentielle Interaktion zwischen den Untereinheiten festgestellt werden. Stattdessen zeigen die Ergebnisse, daß die Integration von konstitutiven oder Immunountereinheiten durch direkte Kompetition reguliert wird. Des Weiteren wurde während der Infektion eine beta5i-abhängige Zunahme der zellulären Proteasommenge festgestellt und somit ein neuer Mechanismus zur Regulation des zellulären Proteasomgehaltes entdeckt. Funktionell führt die beta5i-Defizienz zu einer verringerten MHC I Expression auf antigenpräsentierenden Zellen und zu einer verminderten Prozessierung des bakteriellen Antigens LLO296-304. Bei der Analyse der LLO296-304 spezifischen CD8 T Zell Antwort konnte jedoch kein Unterschied zwischen Wildtyp- und beta5i defizienten Mäusen festgestellt werden .Die Kontrolle der Infektion in den beta5i defizienten Mäusen ist jedoch in der Leber verzögert. Dies deutet darauf hin, dass die Erkennung und Elimination infizierter Zellen durch cytotoxische CD8 T Zellen auf Grund der geringeren MHC Klasse I Präsentation bakterieller Antigene behindert wird. / The proteasome-system is the main source of peptides for MHC class I antigen presentation. In vertebrates this system can be modulated by the expression of different subtypes of the 20S proteasome. The most common subtypes are constitutive proteasomes (c20S) with the catalytic subunits beta1, beta2 and beta5 and immunoproteasomes (i20S) with the immunosubunits beta1i, beta2i and beta5i. Expression of i20S generally optimizes MHC class I antigen presentation by increasing the generation of peptides with high affinity to MHC class I molecules. Currently, the formation of i20S is explained by a model of cooperative proteasome assembly, which is based on preferential interactions among the immunosubunits. Here, the assembly of 20S proteasomes was analysed in beta5i deficient mice during an ongoing infection with Listeria monocytogenes. In this model, no preferential interactions among constitutive subunits or immunosubunits could be determined. Instead, the results show that the integration of constitutive subunits or immunosubunits is regulated by direct competition. Further, a beta5i-dependent increase in cellular proteasome quantity was observed following infection. This reveals a novel mechanism for the regulation of cellular proteasome quantity, which is based on the differential expression of beta5i. Functionally, the deficiency in beta5i results in a reduced MHC class I cell surface expression on professional antigen presenting cells and a drastically diminished processing of the bacterial antigen LLO296-304. However, the analyses of LLO296-304 specific CD8 T cells did not reveal differences in the frequencies of these T cells between wild-type and beta5i deficient mice. Still, the control of infection in the liver of beta5i deficient mice was delayed. This phenotype suggests that the recognition and elimination of infected target cells by cytotoxic CD8 T cells is constrained due to the lowered MHC class I presentation of bacterial antigens.

Background Iron deficiency (ID) is the world‟s most prevalent micronutrient deficiency and predominantly affects developing countries, also South Africa. In areas with low fish consumption and high n-6 PUFA vegetable oil intake, there is a risk for having inadequate n-3 PUFA status. Both iron and n-3 PUFA play important roles in the immune response, and supplementation is a strategy to alleviate deficiencies. However, little is known about potential interactive effects between concurrent iron and n-3 PUFA supplementation on the immune system. This is also important in the context that iron supplementation may be unsafe and may increase morbidity and mortality. Aim The overall aim of this thesis was to assess the effects of iron and docosahexaenoic (DHA)/eicosapentaenoic acid (EPA) supplementation, alone and in combination, on the immune system of ID children. More specifically, these effects were investigated on the occurrence and duration of illness and school-absenteeism due to illness, peripheral blood mononuclear cell (PBMC), red blood cell (RBC) and plasma total phospholipid fatty acid composition, iron status, fatty acid-derived immune modulators and targeted PBMC gene expression. Furthermore, association of PBMC, RBC and plasma total phospholipid fatty acid composition with allergic disease, were also examined. Design In a 2-by-2 factorial, randomised, double-blind, placebo-controlled trial, South African children (n = 321, aged 6–11 y) were randomly assigned to receive oral supplements of either 1) iron (50 mg as ferrous sulphate) plus placebo; 2) DHA/EPA (420/80 mg) plus placebo; 3) iron plus DHA/EPA (420/80 mg); or 4) placebo plus placebo for 8.5 mo, four times per week. Absenteeism and illness symptoms were recorded and biochemical parameters for compliance as well as parameters fundamental to immune function were assessed at baseline and endpoint. Furthermore, in a cross-sectional design, associations of allergic disease with baseline fatty acid composition of PBMC, RBC and plasma were examined. Results The combination of iron and DHA/EPA significantly attenuated respiratory illness caused by iron supplementation. DHA/EPA supplementation alone improved respiratory symptoms at school, but increased headache-related absenteeism. DHA/EPA and iron supplementation individually tended to increase and decrease anti-inflammatory DHA and EPA-derived mediators, respectively. Furthermore the anti-inflammatory DHA-derived immune mediator, 17HDHA was higher in the DHA/EPA plus placebo and iron plus DHA/EPA groups than in the iron plus placebo group. Also, the pro-inflammatory arachidonic acid (AA)-derived modulators (5- and 15-hydroxyeicosapentaenoic acid) were significantly lower in the iron plus DHA/EPA group compared to the placebo plus placebo groups. In the study population, 27.2% of the children had allergic disease and AA in PBMC phospholipids was significantly lower in the allergic children than in the non-allergic children. In RBC phospholipids dihomo-gamma-linolenic acid (DGLA) and the ratio of DGLA: linoleic acid (LA) correlated negatively and the n-6:n-3 PUFA ratio positively with total immunoglobulin E (tIgE). Furthermore, trans-C18:1n-9, tended to be higher in the allergic group. Conclusion DHA/EPA prevented respiratory illness caused by iron supplementation and although DHA/EPA on its own reduced respiratory morbidity when the children were present at school, surprisingly it increased the likelihood of being absent with headache and fever. The biochemical findings compliment the clinical results and support previous observations about DHA/EPA supplementation to reduce inflammation, but add to the current knowledge base that a relatively high oral dose of non-haem iron modulates circulating lipid-derived immune modulators and related gene expression. Furthermore, when supplementing with iron and DHA/EPA combined, in this ID population with low fish intake, the anti-inflammatory effect of DHA/EPA is maintained concurrently with attenuation of respiratory morbidity. This finding support the notion that excess iron (probably as non-transferrin bound iron) becomes available for pathogens and is probably why we found that iron increased respiratory infectious morbidity. The improved clinical outcome with combined supplementation seems to be related to increased lipid-mediator synthesis gene expression and the availability of DHA/EPA, leading to a more pro-resolving profile and enhanced immune competence. Overall these results give better insight into immune function and infectious morbidity in relation to n-3 PUFA and iron status and treatment, as well as the possible association of fatty acid status with allergic disease in young South-African school children. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015

Background Iron deficiency (ID) is the world‟s most prevalent micronutrient deficiency and predominantly affects developing countries, also South Africa. In areas with low fish consumption and high n-6 PUFA vegetable oil intake, there is a risk for having inadequate n-3 PUFA status. Both iron and n-3 PUFA play important roles in the immune response, and supplementation is a strategy to alleviate deficiencies. However, little is known about potential interactive effects between concurrent iron and n-3 PUFA supplementation on the immune system. This is also important in the context that iron supplementation may be unsafe and may increase morbidity and mortality. Aim The overall aim of this thesis was to assess the effects of iron and docosahexaenoic (DHA)/eicosapentaenoic acid (EPA) supplementation, alone and in combination, on the immune system of ID children. More specifically, these effects were investigated on the occurrence and duration of illness and school-absenteeism due to illness, peripheral blood mononuclear cell (PBMC), red blood cell (RBC) and plasma total phospholipid fatty acid composition, iron status, fatty acid-derived immune modulators and targeted PBMC gene expression. Furthermore, association of PBMC, RBC and plasma total phospholipid fatty acid composition with allergic disease, were also examined. Design In a 2-by-2 factorial, randomised, double-blind, placebo-controlled trial, South African children (n = 321, aged 6–11 y) were randomly assigned to receive oral supplements of either 1) iron (50 mg as ferrous sulphate) plus placebo; 2) DHA/EPA (420/80 mg) plus placebo; 3) iron plus DHA/EPA (420/80 mg); or 4) placebo plus placebo for 8.5 mo, four times per week. Absenteeism and illness symptoms were recorded and biochemical parameters for compliance as well as parameters fundamental to immune function were assessed at baseline and endpoint. Furthermore, in a cross-sectional design, associations of allergic disease with baseline fatty acid composition of PBMC, RBC and plasma were examined. Results The combination of iron and DHA/EPA significantly attenuated respiratory illness caused by iron supplementation. DHA/EPA supplementation alone improved respiratory symptoms at school, but increased headache-related absenteeism. DHA/EPA and iron supplementation individually tended to increase and decrease anti-inflammatory DHA and EPA-derived mediators, respectively. Furthermore the anti-inflammatory DHA-derived immune mediator, 17HDHA was higher in the DHA/EPA plus placebo and iron plus DHA/EPA groups than in the iron plus placebo group. Also, the pro-inflammatory arachidonic acid (AA)-derived modulators (5- and 15-hydroxyeicosapentaenoic acid) were significantly lower in the iron plus DHA/EPA group compared to the placebo plus placebo groups. In the study population, 27.2% of the children had allergic disease and AA in PBMC phospholipids was significantly lower in the allergic children than in the non-allergic children. In RBC phospholipids dihomo-gamma-linolenic acid (DGLA) and the ratio of DGLA: linoleic acid (LA) correlated negatively and the n-6:n-3 PUFA ratio positively with total immunoglobulin E (tIgE). Furthermore, trans-C18:1n-9, tended to be higher in the allergic group. Conclusion DHA/EPA prevented respiratory illness caused by iron supplementation and although DHA/EPA on its own reduced respiratory morbidity when the children were present at school, surprisingly it increased the likelihood of being absent with headache and fever. The biochemical findings compliment the clinical results and support previous observations about DHA/EPA supplementation to reduce inflammation, but add to the current knowledge base that a relatively high oral dose of non-haem iron modulates circulating lipid-derived immune modulators and related gene expression. Furthermore, when supplementing with iron and DHA/EPA combined, in this ID population with low fish intake, the anti-inflammatory effect of DHA/EPA is maintained concurrently with attenuation of respiratory morbidity. This finding support the notion that excess iron (probably as non-transferrin bound iron) becomes available for pathogens and is probably why we found that iron increased respiratory infectious morbidity. The improved clinical outcome with combined supplementation seems to be related to increased lipid-mediator synthesis gene expression and the availability of DHA/EPA, leading to a more pro-resolving profile and enhanced immune competence. Overall these results give better insight into immune function and infectious morbidity in relation to n-3 PUFA and iron status and treatment, as well as the possible association of fatty acid status with allergic disease in young South-African school children. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015

The MHC class I status of tumour cells during immunotherapy is often underestimated. It represents one of important tumour escape mechanisms and thus can contribute to the failure of most of the cancer clinical trials that are usually based on the induction of cytotoxic T cell responses. Epigenetic changes in the promoters of genes involved in the MHC class I Ag presentation can result in decreased expression of the cell surface MHC molecules on tumour cells. Thus, epigenetic modifiers can restore an expression of the MHC class I molecules and make tumours visible to the CD8+ effector cells. Besides the epigenetic changes on the tumour cells, epigenetic modulators affect cells of the immune system such as dendritic cells (DC). Tumour cells can escape from the immune response not only by changes in the cancer cells, but also by influencing, expanding and/or activating immunoregulatory cell populations, such as regulatory T cells (Treg). This thesis focuses on the potential of the DC-based vaccines against HPV-16-associated tumours with a different MHC class I expression, on the combination of cancer immunotherapy with the treatment using epigenetic modifiers, with special attention paid to their effects on DC, and, finally, on the impacts of the anti-CD25 antibody (used for Treg elimination) on Treg and NKT...