A doença de Behçet tem sua fisiopatologia caracterizada por hiperatividade neutrofílica, particularmente em relação à quimiotaxia, e períodos de atividade da doença podem ser desencadeados por exposição a estreptococos. Uma vez que células do sistema imune inato são ativadas pelo ácido lipoteicoico (LTA) de bactérias gram-positivas via receptor toll-like (TLR) 2 e CD14, cujas expressões são reguladas pelos fatores estimulantes de colônias de granulócitos (G-CSF) e granulócitos-macrófagos (GM-CSF), o objetivo principal deste estudo foi determinar se há hiperexpressão de TLR2 em neutrófilos de DB ativa e se a quimiotaxia de polimorfonucleares (PMN) neutrófilos na DB poderia ser hiperestimulada pelo LTA. Além do TLR2, foram medidas as expressões de TLR4, CD14, CD114 (receptor de G-CSF) e CD116 (receptor de GM-CSF) nos neutrófilos e nos monócitos de pacientes com doença de Behçet (DB), as concentrações séricas de CD14 solúvel (CD14s) e as respostas quimiotáxicas dos PMNs de DB sob diferentes estímulos. A expressão dos receptores foi medida pela citometria de fluxo, as concentrações séricas por ELISA e as respostas quimiotáxicas foram avaliadas em câmara de Boyden. Nos PMNs, os receptores foram igualmente expressos nos dois grupos e, estimulados com LTA, suas respostas quimiotáxicas também foram similares. Somente à incubação com plasma os PMNs de DB desenvolveram hiperquimiotaxia em relação aos PMNs controles. A expressão do TLR2 foi maior em monócitos de DB em relação aos controles, e a concentração de CD14s sérica, de origem monocitária, foi maior nos pacientes com DB ativa. Em conjunto, os resultados demonstram que PMNs de DB, isoladamente, não reagem exacerbadamente ao LTA, e suas respostas migratórias são estritamente dependentes de fatores estimulantes solúveis. / Expressions of toll-like receptor (TLR) 2, TLR4, CD14, CD114 and CD116 were assessed on polymorphonuclear (PMN) neutrophils and monocytes of patients with Behçets disease (BD). PMN chemotactic responses under different stimulations were also measured. The objective was to determine if BD PMN chemotaxis may be overstimulated by lipoteichoic acid (LTA) from gram-positive bacteria. Receptor expressions were measured by flow cytometry and PMN chemotaxis was assessed in a Boyden chamber. Only TLR2 expression was higher on monocytes of the BD group than in control group. On PMNs, however, TLR2 expression was similar in both groups and, when stimulated with LTA, BD PMN cells showed chemotactic responses similar to the controls. These cells only exhibited increased chemotaxis when incubated with plasma. In conclusion, isolated BD PMN did not overreact to LTA, and its hyperchemotaxis is strictly dependent on soluble stimulating factors

No description available.

Seit 20 Jahren unterteilt man Monozyten in eine klassische CD14++/CD16-Population und eine proinflammatorische CD16+ Population. Letztere macht 10-20 % der peripheren Blutmonozyten aus und ist unter verschiedenen pathologischen Zuständen drastisch erhöht. Seit Kurzem wird die CD16+ Fraktion in zwei weitere Untergruppen aufgeteilt, die CD14++/CD16+ und die CD14+/CD16+ Monozyten. In der hier vorliegenden Arbeit wurde versucht, die relativ neue und wenig charakterisierte Subpopulation der CD14++/CD16+ Monozyten näher zu beschreiben. Es sollte die Frage beantwortet werden, ob diese sich von den übrigen Subpopulationen abzugrenzen lässt und damit als eigenständige Zellgruppe anzusehen ist. Die von gesunden Spendern gewonnen peripheren mononukleären Blutzellen (PBMCs) wurden mithilfe einer Magnetsäulenvorseparation von einem Großteil der T-, B- und NK-Zellen befreit. Die restlichen Zellen wurden mit Anti-CD14-FITC und Anti-CD16-PE markiert. In einem Hochgeschwindigkeitssortierer wurden diese, anhand ihres Fluoreszenzmusters, in die drei Subpopulationen CD14++/CD16- (Sub1), CD14++/CD16+ (Sub2) und CD14+/CD16+ (Sub3) aufgeteilt. Durch dieses Verfahren konnte ein hoher Reinheitsgrad erreicht werden. Die erhaltenen Subpopulationen wurden mit heterologen CD4+ T-Lymphozyten zusammen gebracht. Die Reaktion dieser T-Zellen auf die verschiedenen Subpopulationen wurde im Proliferations- und Sekretionsassay (IFN-γ, IL-4) studiert. Des Weiteren wurde die Zytokinsekretion der Monozytenarten nach definierter Stimulierung (LPS, Zymosan, aktivierte T-Zellen) analysiert. In einem zweiten Versuchskomplex wurden aus den jeweiligen Subpopulationen unreife dendritische Zellen (Sub-DCs) differenziert und diese auf bestimmte Oberflächenmarker bzw. deren Verhalten mit heterologen CD4+ T-Zellen im Proliferations-/Sekretionsassay (IFN-γ, IL-4) untersucht. Nach 7 Tagen Inkubation fanden sich im Sub2- und Sub3-Ansatz ca. 10 % mehr proliferierende T-Zellen als im Sub1-Ansatz. Dabei lag der Anteil der CD25+ T-Zellen in diesen beiden Versuchsansätzen ebenfalls um 10 % höher. Der Proliferationsassay über 14 Tage zeigte für die Sub3 ca. 10-15 % mehr proliferierende T-Zellen als für die anderen Populationen. Das Niveau der CD25-Expression der T-Zellen war hierbei in allen drei Ansätzen gleich. Im Sekretionsassay induzierten alle drei Subpopulationen eine Th1-Antwort, jedoch mit einem leicht höheren Anteil IFN-γ-positiver T-Zellen für die CD16+ Monozyten. Durch LPS-Gabe produzierten die CD14+/CD16+ Monozyten am meisten TNF-α, gefolgt von den CD14++/CD16+. Bei den CD14++/CD16- fanden sich die geringsten Mengen an TNF-α. Zusammen mit aktivierten T-Zellen demonstrierten die CD14++/CD16+ Monozyten die stärkste TNF-α-Sekretion unter allen anderen Subpopulationen. Für die beiden CD16+ Populationen wurden nach LPS-Stimulation höhere Werte für IL-1β bestimmt als für die klassischen Monozyten. Sowohl im LPS- als auch im Zymosanansatz lag die IL-6-Sekretion der CD14++/CD16- Subpopulation über der der anderen zwei Fraktionen. Unter allen drei Stimuli wurden die höchsten Werte für IL-8 bei den CD14++/CD16- Monozyten detektiert, gefolgt von den CD14++/CD16+. Am niedrigsten lag die IL-8-Produktion bei den CD14+/CD16+. Die IL-10-Sekretion im LPS- und im Zymosanansatz der CD14++/CD16- und CD14++/CD16+ Monozyten war gegenüber der CD14+/CD16+ Subpopulation erhöht. Nach Entwicklung der unreifen dendritischen Zellen aus den entsprechenden Monozytenarten weisen diese eine differenzierte Morphologie auf. Die DCs der CD14+/CD16+ Monozyten hatten eine stärkere HLA-DR-Expression in der mittleren Fluoreszenzintensität als die anderen beiden Sub-DC-Populationen, wobei sich keine Unterschiede in der HLA-DRExpressionsintensität zwischen Sub1- und Sub2-DCs feststellen ließen. Der Anteil der CD11c positiven Zellen war bei den Sub1-DCs deutlich größer als bei den Sub2-DCs. Dagegen exprimierten die Sub3-DCs praktisch kein CD11c. Im Proliferationsassay über 7 Tage ergaben sich keine signifikanten Differenzen zwischen den Subpopulationen. Allerdings zeigte sich eine Tendenz zu geringeren Werten im Anteil proliferierender bzw. CD25-positiver T-Lymphozyten für den Sub2-DC-Ansatz. Nach 14 Tagen im Assay war der Anteil der proliferierenden T-Zellen bei den Sub1-DCs um 10 % höher als bei den Sub2-DCs. Es fanden sich ca. 10 % mehr CD25+ T-Zellen im Sub1-DC-Ansatz als in den anderen beiden Ansätzen. Der Sekretionsassay erbrachte für alle Sub-DCs eine Th1-Induktion der T-Zellen. Dabei ließen sich keine Abweichungen im Anteil IFN-γ-positiver T-Zellen zwischen den einzelnen Sub-DC-Kulturen nachweisen. Die gewonnen Ergebnisse dieser Arbeit verdeutlichen auf der einen Seite das proinflammatorische Potential (z.B. TNF-α-Sekretion, erhöhte Proliferation), auf der anderen Seite die antiinflammatorische Komponente (IL-10-Sekretion) der CD14++/CD16+ Monozyten. Eine Rolle in der Regulation von entzündlichen und infektiologischen Erkrankungen erscheint für diese Subpopulation denkbar. Die Subpopulation 2 kann dabei als eigenständige Monozytenfraktion betrachtet werden. Die funktionellen Unterschiede zwischen den analysierten Monozytensubpopulationen zeigten sich auch nach deren Differenzierung zu unreifen dendritischen Zellen. In Anbetracht des erhöhten Anteils der CD16+ Monozyten im Rahmen diverser autoimmuner Krankheiten und der immer klarer werdenden Unterteilung in eine CD14++/CD16+ und eine CD14+/CD16+ Subpopulation, sollten weitere Untersuchungen zur klinischen Relevanz dieser Monozytengruppen durchgeführt werden. Die in der vorliegenden Arbeit erzielten Ergebnisse könnten bei der Zuordnung und Interpretation in diese zukünftigen klinischen Befunde behilflich sein. / For more than 20 years monocytes were subdivided in the classical CD14++/CD16- population and the proinflammatory CD16+ populations. The latter one includes about 10-20 % of the peripheral blood monocytes and is dramatically expanded under different pathological circumstances. Recently, the CD16+ fraction was further subdivided into two subpopulations: The CD14++/CD16+ and CD14+/CD16+ monocytes. In the present paper, the subpopulation of CD14++/CD16+ monocytes was characterized in order to answer the question if this subset can be distinguished as an independent cell population. T cells, B cells and NK cells were depleted from peripheral mononuclear blood cells (PBMCs) from healthy donors using immunomagnetic cell separation. Subsequently, the pre-separated cells were stained with anti-CD14-FITC and anti-CD16-PE and separated by fluorescence activated cell sorting (FACS) in three subpopulations: The CD14++/CD16- (Sub1), the CD14++/CD16+ (Sub2) and the CD14+/CD16+ (Sub3). The achieved purity was sufficient for the subsequent experiments. The obtained subpopulations were co-cultured together with heterologous CD4+ T lymphocytes. The reaction of these T cells to the different subpopulations was studied in the proliferation and secretion assay (IFN-γ, IL-4). Furthermore, the cytokine secretion of the monocyte subsets after defined stimulation (LPS, Zymosan, activated T cells) was analysed. In a second set of experiments, immature dendritic cells were differentiated from the monocyte subpopulations (Sub-DCs) and phenotypically and functionally characterized according to the expression of cell surface markers and the response to heterologous CD4+ T cells in the proliferation/secretion assay (IFN-γ, IL-4). After 7 days of incubation, the Sub2 and Sub3 of the monocytes induced approximately 10 % higher proliferation rates of T cells than the Sub1. The resulting frequency of CD25+ T cells in these two co-culture settings was also 10 % higher. The proliferation analysis after 14 days again showed for the Sub3 ca. 10-15 % more proliferating T cells than for the other populations. At this, the frequency of CD25 expression was equal in all co-cultures. In the secretion assay all three subpopulations induced a Th1 response, but the range of IFN-γ-positive T cells was somewhat higher for the CD16+ monocytes. Under LPS stimulation the CD14+/CD16+ monocytes produced the highest amounts of TNF-α followed by the CD14++/CD16+. The CD14++/CD16- showed the lowest amounts of TNF-α. In co-culture with activated T cells the CD14++/CD16+ monocytes demonstrated the strongest TNF-α secretion from all subpopulations. After LPS stimulation, a higher level of IL-1β was measured for both CD16+ populations than for the classical monocytes. In the LPS and the Zymosan stimulated cultures, the IL-6 secretion of the CD14++/CD16- subpopulation was higher than in the two other fractions. Under all three stimuli the highest levels of IL-8 were detected for the CD14++/CD16- monocytes followed by the CD14++/CD16+. The lowest IL-8 production was found by the CD14+/CD16+. The IL-10 secretion of the CD14++/CD16- and CD14++/CD16+ monocytes was increased compared to the CD14+/CD16+ subpopulation after LPS and Zymosan stimulation. The in vitro generated immature dendritic cells from the different monocyte subsets showed a differentiated morphology. The DCs of the CD14+/CD16+ monocytes had the strongest HLA-DR expression compared to the other two Sub-DC populations. No differences in the HLA-DR intensity were found between the Sub1- and Sub2-DCs. The rate of CD11c-positive cells was significantly higher in the Sub1-DCs than in the Sub2-DCs. However, the Sub3-DCs expressed no CD11c altogether. The proliferation assay over 7 days showed no significant differences between the subpopulations. Nevertheless, a tendency for lower levels of proliferating or CD25-positive T lymphocytes was seen in T cells co-cultured with the Sub2-DC. After 14 days, the ratio of proliferating T cells was 10 % higher with the Sub1-DCs than with the Sub2-DCs. The Sub1-DC co-culture yielded ca. 10 % more CD25+ T cells than the other two. The secretion assay revealed for all Sub-DCs a Th1 response of the T cells, with no differences in the amount of IFN-γ-positive T cells between the Sub-DC cultures. The results illustrate on one hand the proinflammatory potential (e.g. TNF-α secretion, higher proliferation), on the other hand the antiinflammatory effect (IL-10 secretion) of CD14++/CD16+ monocytes. A role in the regulation of inflammatory and infectious diseases seems to be possible for this subpopulation. The subpopulation 2 can be regarded as an independent fraction of monocytes. The functional differences between the analyzed monocyte subpopulations are further underscored following differentiation into immature dendritic cells. Considering the increased proportion of CD16+ monocytes in various autoimmune diseases and their clear subdivision in a CD14++/CD16+ and a CD14+/CD16+ subpopulation, new investigations about the clinical relevance are warranted. The findings obtained in the work presented could be in the basis for these future clinical studies.

Seit 20 Jahren unterteilt man Monozyten in eine klassische CD14++/CD16-Population und eine proinflammatorische CD16+ Population. Letztere macht 10-20 % der peripheren Blutmonozyten aus und ist unter verschiedenen pathologischen Zuständen drastisch erhöht. Seit Kurzem wird die CD16+ Fraktion in zwei weitere Untergruppen aufgeteilt, die CD14++/CD16+ und die CD14+/CD16+ Monozyten. In der hier vorliegenden Arbeit wurde versucht, die relativ neue und wenig charakterisierte Subpopulation der CD14++/CD16+ Monozyten näher zu beschreiben. Es sollte die Frage beantwortet werden, ob diese sich von den übrigen Subpopulationen abzugrenzen lässt und damit als eigenständige Zellgruppe anzusehen ist. Die von gesunden Spendern gewonnen peripheren mononukleären Blutzellen (PBMCs) wurden mithilfe einer Magnetsäulenvorseparation von einem Großteil der T-, B- und NK-Zellen befreit. Die restlichen Zellen wurden mit Anti-CD14-FITC und Anti-CD16-PE markiert. In einem Hochgeschwindigkeitssortierer wurden diese, anhand ihres Fluoreszenzmusters, in die drei Subpopulationen CD14++/CD16- (Sub1), CD14++/CD16+ (Sub2) und CD14+/CD16+ (Sub3) aufgeteilt. Durch dieses Verfahren konnte ein hoher Reinheitsgrad erreicht werden. Die erhaltenen Subpopulationen wurden mit heterologen CD4+ T-Lymphozyten zusammen gebracht. Die Reaktion dieser T-Zellen auf die verschiedenen Subpopulationen wurde im Proliferations- und Sekretionsassay (IFN-γ, IL-4) studiert. Des Weiteren wurde die Zytokinsekretion der Monozytenarten nach definierter Stimulierung (LPS, Zymosan, aktivierte T-Zellen) analysiert. In einem zweiten Versuchskomplex wurden aus den jeweiligen Subpopulationen unreife dendritische Zellen (Sub-DCs) differenziert und diese auf bestimmte Oberflächenmarker bzw. deren Verhalten mit heterologen CD4+ T-Zellen im Proliferations-/Sekretionsassay (IFN-γ, IL-4) untersucht. Nach 7 Tagen Inkubation fanden sich im Sub2- und Sub3-Ansatz ca. 10 % mehr proliferierende T-Zellen als im Sub1-Ansatz. Dabei lag der Anteil der CD25+ T-Zellen in diesen beiden Versuchsansätzen ebenfalls um 10 % höher. Der Proliferationsassay über 14 Tage zeigte für die Sub3 ca. 10-15 % mehr proliferierende T-Zellen als für die anderen Populationen. Das Niveau der CD25-Expression der T-Zellen war hierbei in allen drei Ansätzen gleich. Im Sekretionsassay induzierten alle drei Subpopulationen eine Th1-Antwort, jedoch mit einem leicht höheren Anteil IFN-γ-positiver T-Zellen für die CD16+ Monozyten. Durch LPS-Gabe produzierten die CD14+/CD16+ Monozyten am meisten TNF-α, gefolgt von den CD14++/CD16+. Bei den CD14++/CD16- fanden sich die geringsten Mengen an TNF-α. Zusammen mit aktivierten T-Zellen demonstrierten die CD14++/CD16+ Monozyten die stärkste TNF-α-Sekretion unter allen anderen Subpopulationen. Für die beiden CD16+ Populationen wurden nach LPS-Stimulation höhere Werte für IL-1β bestimmt als für die klassischen Monozyten. Sowohl im LPS- als auch im Zymosanansatz lag die IL-6-Sekretion der CD14++/CD16- Subpopulation über der der anderen zwei Fraktionen. Unter allen drei Stimuli wurden die höchsten Werte für IL-8 bei den CD14++/CD16- Monozyten detektiert, gefolgt von den CD14++/CD16+. Am niedrigsten lag die IL-8-Produktion bei den CD14+/CD16+. Die IL-10-Sekretion im LPS- und im Zymosanansatz der CD14++/CD16- und CD14++/CD16+ Monozyten war gegenüber der CD14+/CD16+ Subpopulation erhöht. Nach Entwicklung der unreifen dendritischen Zellen aus den entsprechenden Monozytenarten weisen diese eine differenzierte Morphologie auf. Die DCs der CD14+/CD16+ Monozyten hatten eine stärkere HLA-DR-Expression in der mittleren Fluoreszenzintensität als die anderen beiden Sub-DC-Populationen, wobei sich keine Unterschiede in der HLA-DRExpressionsintensität zwischen Sub1- und Sub2-DCs feststellen ließen. Der Anteil der CD11c positiven Zellen war bei den Sub1-DCs deutlich größer als bei den Sub2-DCs. Dagegen exprimierten die Sub3-DCs praktisch kein CD11c. Im Proliferationsassay über 7 Tage ergaben sich keine signifikanten Differenzen zwischen den Subpopulationen. Allerdings zeigte sich eine Tendenz zu geringeren Werten im Anteil proliferierender bzw. CD25-positiver T-Lymphozyten für den Sub2-DC-Ansatz. Nach 14 Tagen im Assay war der Anteil der proliferierenden T-Zellen bei den Sub1-DCs um 10 % höher als bei den Sub2-DCs. Es fanden sich ca. 10 % mehr CD25+ T-Zellen im Sub1-DC-Ansatz als in den anderen beiden Ansätzen. Der Sekretionsassay erbrachte für alle Sub-DCs eine Th1-Induktion der T-Zellen. Dabei ließen sich keine Abweichungen im Anteil IFN-γ-positiver T-Zellen zwischen den einzelnen Sub-DC-Kulturen nachweisen. Die gewonnen Ergebnisse dieser Arbeit verdeutlichen auf der einen Seite das proinflammatorische Potential (z.B. TNF-α-Sekretion, erhöhte Proliferation), auf der anderen Seite die antiinflammatorische Komponente (IL-10-Sekretion) der CD14++/CD16+ Monozyten. Eine Rolle in der Regulation von entzündlichen und infektiologischen Erkrankungen erscheint für diese Subpopulation denkbar. Die Subpopulation 2 kann dabei als eigenständige Monozytenfraktion betrachtet werden. Die funktionellen Unterschiede zwischen den analysierten Monozytensubpopulationen zeigten sich auch nach deren Differenzierung zu unreifen dendritischen Zellen. In Anbetracht des erhöhten Anteils der CD16+ Monozyten im Rahmen diverser autoimmuner Krankheiten und der immer klarer werdenden Unterteilung in eine CD14++/CD16+ und eine CD14+/CD16+ Subpopulation, sollten weitere Untersuchungen zur klinischen Relevanz dieser Monozytengruppen durchgeführt werden. Die in der vorliegenden Arbeit erzielten Ergebnisse könnten bei der Zuordnung und Interpretation in diese zukünftigen klinischen Befunde behilflich sein. / For more than 20 years monocytes were subdivided in the classical CD14++/CD16- population and the proinflammatory CD16+ populations. The latter one includes about 10-20 % of the peripheral blood monocytes and is dramatically expanded under different pathological circumstances. Recently, the CD16+ fraction was further subdivided into two subpopulations: The CD14++/CD16+ and CD14+/CD16+ monocytes. In the present paper, the subpopulation of CD14++/CD16+ monocytes was characterized in order to answer the question if this subset can be distinguished as an independent cell population. T cells, B cells and NK cells were depleted from peripheral mononuclear blood cells (PBMCs) from healthy donors using immunomagnetic cell separation. Subsequently, the pre-separated cells were stained with anti-CD14-FITC and anti-CD16-PE and separated by fluorescence activated cell sorting (FACS) in three subpopulations: The CD14++/CD16- (Sub1), the CD14++/CD16+ (Sub2) and the CD14+/CD16+ (Sub3). The achieved purity was sufficient for the subsequent experiments. The obtained subpopulations were co-cultured together with heterologous CD4+ T lymphocytes. The reaction of these T cells to the different subpopulations was studied in the proliferation and secretion assay (IFN-γ, IL-4). Furthermore, the cytokine secretion of the monocyte subsets after defined stimulation (LPS, Zymosan, activated T cells) was analysed. In a second set of experiments, immature dendritic cells were differentiated from the monocyte subpopulations (Sub-DCs) and phenotypically and functionally characterized according to the expression of cell surface markers and the response to heterologous CD4+ T cells in the proliferation/secretion assay (IFN-γ, IL-4). After 7 days of incubation, the Sub2 and Sub3 of the monocytes induced approximately 10 % higher proliferation rates of T cells than the Sub1. The resulting frequency of CD25+ T cells in these two co-culture settings was also 10 % higher. The proliferation analysis after 14 days again showed for the Sub3 ca. 10-15 % more proliferating T cells than for the other populations. At this, the frequency of CD25 expression was equal in all co-cultures. In the secretion assay all three subpopulations induced a Th1 response, but the range of IFN-γ-positive T cells was somewhat higher for the CD16+ monocytes. Under LPS stimulation the CD14+/CD16+ monocytes produced the highest amounts of TNF-α followed by the CD14++/CD16+. The CD14++/CD16- showed the lowest amounts of TNF-α. In co-culture with activated T cells the CD14++/CD16+ monocytes demonstrated the strongest TNF-α secretion from all subpopulations. After LPS stimulation, a higher level of IL-1β was measured for both CD16+ populations than for the classical monocytes. In the LPS and the Zymosan stimulated cultures, the IL-6 secretion of the CD14++/CD16- subpopulation was higher than in the two other fractions. Under all three stimuli the highest levels of IL-8 were detected for the CD14++/CD16- monocytes followed by the CD14++/CD16+. The lowest IL-8 production was found by the CD14+/CD16+. The IL-10 secretion of the CD14++/CD16- and CD14++/CD16+ monocytes was increased compared to the CD14+/CD16+ subpopulation after LPS and Zymosan stimulation. The in vitro generated immature dendritic cells from the different monocyte subsets showed a differentiated morphology. The DCs of the CD14+/CD16+ monocytes had the strongest HLA-DR expression compared to the other two Sub-DC populations. No differences in the HLA-DR intensity were found between the Sub1- and Sub2-DCs. The rate of CD11c-positive cells was significantly higher in the Sub1-DCs than in the Sub2-DCs. However, the Sub3-DCs expressed no CD11c altogether. The proliferation assay over 7 days showed no significant differences between the subpopulations. Nevertheless, a tendency for lower levels of proliferating or CD25-positive T lymphocytes was seen in T cells co-cultured with the Sub2-DC. After 14 days, the ratio of proliferating T cells was 10 % higher with the Sub1-DCs than with the Sub2-DCs. The Sub1-DC co-culture yielded ca. 10 % more CD25+ T cells than the other two. The secretion assay revealed for all Sub-DCs a Th1 response of the T cells, with no differences in the amount of IFN-γ-positive T cells between the Sub-DC cultures. The results illustrate on one hand the proinflammatory potential (e.g. TNF-α secretion, higher proliferation), on the other hand the antiinflammatory effect (IL-10 secretion) of CD14++/CD16+ monocytes. A role in the regulation of inflammatory and infectious diseases seems to be possible for this subpopulation. The subpopulation 2 can be regarded as an independent fraction of monocytes. The functional differences between the analyzed monocyte subpopulations are further underscored following differentiation into immature dendritic cells. Considering the increased proportion of CD16+ monocytes in various autoimmune diseases and their clear subdivision in a CD14++/CD16+ and a CD14+/CD16+ subpopulation, new investigations about the clinical relevance are warranted. The findings obtained in the work presented could be in the basis for these future clinical studies.

Thesis (MScMedSc)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: South Africa is ranked amongst the top tuberculosis (TB) burden countries in the world and the Western Cape has a particularly high incidence of the disease. Previous studies have showed that several genes may play crucial roles in susceptibility to TB. In this study, we investigated the role of three genes previously associated with susceptibility to TB and progression to disease. These genes were Surfactant protein D (SFTPD), Macrophage receptor with collagenous structure (MARCO) and CD14. The proteins from these genes bind M. tuberculosis and are involved in the uptake of the bacteria into macrophages. The study investigated the role of ten polymorphisms from SFTPD and MARCO within a South African Coloured (SAC) population, where tuberculosis is highly prevalent. A casecontrol study design was used and polymorphisms were genotyped with Taqman® genotyping assays and amplification refractory mutation system polymerase chain reaction (ARMS-PCR). The results were analysed for association with disease, linkage disequilibrium, haplotypes and gene-gene interactions. Allele and genotype frequencies were also determined which allowed for comparisons to other populations. Five SNPs were associated with TB: two in SFTPD (rs1923537; rs2255326) and three in MARCO (rs1318645; rs3943679; rs2119112). The associated SNPs were located in regions other than exons and the effects of polymorphisms in these regions are not well understood but studies in other genes have shown them to play a functional role. Gene-gene interaction analysis showed that polymorphisms interacted with each other within and between genes, illustrating the importance of epistasis and the complexity of the genetic influences on TB. In addition to the case-control association studies, the role of the rs2569190 promoter SNP in CD14 was assessed. Gene-expression analysis was conducted with qPCR and a reporter gene assay and results from both of these approaches showed that individuals with the TT genotype had a twofold greater expression level than individuals with the CC genotype. Previously, the TT genotype has been associated with stronger promoter activity and expression of soluble CD14 in serum. Since the TT genotype was present at a higher frequency in the control group, we speculate that greater expression of CD14 may contribute to a more TB resistant phenotype. The work presented in this study illustrates the importance of the host genetic component of TB. Genetic studies will eventually revolutionize the current treatment regime as the identification of vulnerable individuals and populations will aid in the development of personalised medicines. / AFRIKAANSE OPSOMMING: Suid-Afrika is een van die top tuberkulose (TB) lande in die wêreld en die Wes-Kaap het veral ‘n hoë insidensie van die siekte. Vorige studies het gewys dat verskeie gene bydra to die vatbaarheid vir tuberkulose. In hierdie studie het ons drie gene, wat voorheen vir vatbaarheid vir tuberkulose en progressie na die siekte ondersoek is, bestudeer. Hierdie gene is Surfactant protein D (SFTPD), Macrophage receptor with collagenous structure (MARCO) en CD14. Die proteïene van hierdie gene bind M. tuberculosis en is betrokke in die opname van die bakterieë in die makrofages. Hierdie studie het tien polimorfismes van SFTPD en MARCO in die Suid-Afrikaanse Kleurlingbevolking (SAK), wat ‘n hoë TB insidensie het, getoets. Pasiënt-kontrole assosiasie studies is gedoen en polimorfismes is gegenotipeer met Taqman® genotiperingsisteem en die amplifikasie refraktoriese mutasie sisteem polimerase ketting reaksie (ARMS-PCR). Die resultate is geanaliseer vir assosiasies met TB, koppelings disekwilibrium, haplotipes en geen-geen interaksies. Alleel en genotype frekwensies is ook bepaal en vergelyk met die van ander bevolkings. Vyf enkel nukleotied polimorfismes (ENPs) is met TB geassosieer: twee in SFTPD (rs1923537; rs2255326) en drie in MARCO (rs1318645; rs3943679; rs2119112). Die geassosieerde ENPs was nie in eksons nie. Die effek van polimorfismes in areas anders as eksons word nie goed verstaan nie, maar studies het bewys dat hulle wel ‘n funksionele rol kan hê. Geen-geen interaksie analise het gewys dat polimorfismes interaksies met mekaar binne sowel as tussen gene gehad het, wat die belangrikheid van epistase en die kompleksiteit van genetiese invloede op TB illustreer. Tesame met die pasiënt-kontrole assosiasie studies is die rol van die rs2569190 promoter ENP in CD14 ook ondersoek. Geenuitdrukkingsanalise is gedoen met qPKR en rapporteerder geen toetse. Die resultate van beide hierdie benaderings het gewys dat individue met die TT genotipe twee keer soveel uitdrukkingsvlakke gehad het as individue met die CC genotipe. Die TT genotipe is voorheen geassosieer met sterk promoter aktiwiteit en die uitdrukking van oplosbare CD14 in serum. Aangesien die TT genotipe meer in die kontrolegroep gevind is, spekuleer ons dat die hoër uitdrukking van CD14 kan bydra tot ‘n meer TB weerstandbiedende fenotipe. Hierdie werk illustreer die belangrikheid van die gasheer genetiese komponent in TB. Genetiese studies sal in die toekoms die huidige behandeling regime revolusioneer, aangesien die identifikasie van individue en bevolkings met ‘n hoë risiko om TB te ontwikkel sal bydra tot die ontwikkeling van persoonlike medisynes. / The National Research Foundation (NRF); the South African Medical Research Council (MRC); Stellenbosch University and the Harry Crossley Foundation.

AIM: to determine if a relationship exists between TLR-2, TLR-4, TLR-9, or CD14 polymorphisms and risk for developing chronic periodontal disease in an African-American population. This is the first study conducted to determine role of SNPs in TLR genes and CD14 gene in a periodontally-diseased African-American population. Additionally, this is the first study to assess the role of TLR-9 polymorphism in periodontitis patients. METHODS: A total of 130 subjects were involved in the study. The chronic periodontitis (CP) group contained 73 subjects, and the healthy control (NP) group 57subjects. Genotyping was performed in TLR2 (G2408A), TLR4 (A896G),TLR9 (T1486C) and CD14 (C260T) genes by TaqMan® allelic discrimination using Assay-by-DesignSM SNP Genotyping Assays (Applied Biosystems). Accuracy of genotyping was confirmed by known DNA samples of each genotype and by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analyses on selected samples. Fisher’s exact test and chi-square analyses were performed to compare genotype and allele frequencies. Within disease groups, we investigated whether SNPs were related to disease severity by step-wise logistic regression adjusted for age, gender, and smoking status. RESULTS: There was a significant difference in the distribution of specific TLR9 (T1486C) genotypes between the periodontally diseased group versus the control group. Expression of TT genotype was more prevelant in periodontally-diseased individuals compared to periodontally-healthy subjects (p<0.0001) whereas individuals expressing C allele of the TLR9 SNP (CC&CT) were more frequently found in the control group after adjusting for age, gender, and smoking status (p<0.0001) There was no statistically significant difference in the distribution of genotypes between groups for any other TLRs or CD14 polymorphism. CONCLUSION: Based on findings of this study, homozygocity for the T allele of TLR 9 polymorphism was related to chronic periodontal disease susceptibility in African Americans. Additionally, presence of the C allele at TLR-9 appeared to confer resistance to periodontal destruction. Our results showed that specific SNPs in TLR-2, -4 and CD14 genes are not related to periodontitis in African Americans.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 49 pages. Includes Vita. Includes bibliographical references.

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Wong, Yun Sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 142-162). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.vi / Acknowledgement --- p.ix / Statement of Work --- p.x / Table of Contents --- p.xi / List of Tables --- p.xiv / List of Figures --- p.xvi / Glossary of Terms and Abbreviations --- p.xviii / Chapter Section I: --- Introduction --- p.1 / Chapter Chapter 1: --- General Overview of Asthma --- p.2 / Chapter 1.1 --- Asthma definition and its phenotype --- p.2 / Chapter 1.2 --- Asthma epidemiology and its prevalence in past decades --- p.4 / Chapter 1.3 --- Hygiene hypothesis and asthma development --- p.8 / Chapter 1.4 --- Asthma pathogenesis and innate immunity --- p.12 / Chapter 1.5 --- The environmental factors and genetic makeup in relation with asthma --- p.17 / Chapter Chapter 2: --- Study Plan and Obj ective --- p.21 / Chapter Section II: --- Literature Review --- p.24 / Chapter Chapter 3: --- Indoor Environmental factors of Asthma --- p.25 / Chapter 3.1 --- Overview of the indoor environmental factors --- p.25 / Chapter 3.2 --- House dust endotoxin --- p.27 / Chapter 3.2.1 --- Determinants of endotoxin exposure in home environment --- p.21 / Chapter 3.2.2 --- Protective role of endotoxin in allergy and asthma development --- p.29 / Chapter 3.2.3 --- Deleterious effect of endotoxin exposure in asthma: the dark side --- p.31 / Chapter 3.3 --- Allergen --- p.34 / Chapter 3.3.1 --- Allergens: an update --- p.34 / Chapter 3.3.2 --- Determinants of allergens in home environment --- p.35 / Chapter 3.3.3 --- Allergens avoidance: environmental intervention --- p.36 / Chapter 3.4 --- Nitrogen dioxide --- p.40 / Chapter 3.4.1 --- Determinants of indoor nitrogen dioxide and its relation with gas cooking --- p.40 / Chapter 3.4.2 --- The adverse effects of nitrogen dioxide on respiratory symptoms --- p.41 / Chapter 3.4.3 --- Reactive nitrogen species and nitrosative stress in asthma --- p.42 / Chapter Chapter 4: --- CD14 Single Nucleotide Polymorphisms and Asthma --- p.45 / Chapter 4.1 --- Overview of CD14 receptor --- p.45 / Chapter 4.2 --- Action of CD14 receptor in endotoxin response --- p.47 / Chapter 4.3 --- Relation of CD14 with asthma --- p.48 / Chapter 4.3.1 --- Associations between CD14 polymorphisms and asthma phenotypes --- p.48 / Chapter 4.3.2 --- Endotoxin switch concept: from gene to gene - environment --- p.52 / Chapter Section III: --- Study Core --- p.55 / Chapter Chapter 5: --- Methodology in indoor environment investigation and its result --- p.57 / Chapter 5.1 --- Study Population --- p.57 / Chapter 5.2 --- Home Visiting Protocol --- p.60 / Chapter 5.2.1 --- The International Study of Asthma and Allergies in Childhood (ISAAC) --- p.60 / Chapter 5.2.2 --- ISAAC questionnaire --- p.61 / Chapter 5.2.3 --- House dust collection procedures --- p.62 / Chapter 5.2.4 --- Indoor nitrogen dioxide measurements --- p.65 / Chapter 5.2.4.1 --- Ogawa passive sampler --- p.65 / Chapter 5.2.4.2 --- Preparation and measurement procedures --- p.66 / Chapter 5.2.4.3 --- Indoor nitrogen dioxide quantification --- p.67 / Chapter 5.3 --- House dust extraction --- p.69 / Chapter 5.4 --- House dust endotoxin measurement --- p.70 / Chapter 5.5 --- Allergen measurement --- p.72 / Chapter 5.6 --- Statistical Analysis --- p.75 / Chapter 5.7 --- Results --- p.77 / Chapter 5.7.1 --- Demographic data and subjects characteristics --- p.77 / Chapter 5.7.2 --- "Dust weight, endotoxin and allergen levels and their determinants in household" --- p.82 / Chapter 5.7.3 --- Indoor NO〕2levels and its determinant in household --- p.95 / Chapter 5.7.4 --- Associations between indoor environmental factors and respiratory health --- p.96 / Chapter 5.7.4.1 --- Clinical symptoms --- p.96 / Chapter 5.7.4.2 --- Exhaled NO levels --- p.101 / Chapter 5.7.4.3 --- Spirometric indices --- p.103 / Chapter Chapter 6： --- Methodology in genotyping CD14 polymorphisms and its result --- p.105 / Chapter 6.1 --- Study population --- p.105 / Chapter 6.2 --- Serum Total and allergen-specific IgE measurement --- p.106 / Chapter 6.3 --- CD14 Genotyping s --- p.107 / Chapter 6.3.1 --- Genotyping promoter SNPs ofCD14/-159 and -1359 --- p.107 / Chapter 6.3.2 --- Genotyping promoter SNP of CD14/-1619 --- p.109 / Chapter 6.3.3 --- Validation of genotyping by sequencing --- p.111 / Chapter 6.4 --- Statistical Analysis --- p.112 / Chapter 6.5 --- Results --- p.113 / Chapter 6.5.1 --- Subjects characteristics and clinical features. --- p.113 / Chapter 6.5.2 --- Associations between CD14 SNPs and asthma phenotypes --- p.114 / Chapter Chapter 7: --- Discussion --- p.120 / Chapter 7.1 --- Influence of indoor factors on asthmatic children --- p.120 / Chapter 7.2 --- CD14 polymorphisms in modifying asthma phenotypes --- p.135 / Chapter Chapter 8: --- Conclusion and Further Works --- p.138 / References --- p.141 / Appendix 1 Questionnaire / Appendix 2 Publications

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