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.

No description available.

A β2-glicoproteína I (β2GPI) é uma proteína de fase aguda, produzida principalmente no fígado e intestino. Os efeitos dessa proteína sobre células mononucleares foram investigados tanto em monócitos humanos de sangue periférico quanto em células promonocíticas humanas da linhagem celular ATCC THP-1. As correlações entre sua concentração plasmática e a intensidade da inflamação sistêmica foram avaliadas em humanos e em um modelo experimental de infecção sistêmica, em ratos. Nenhum efeito da β2GPI foi observado sobre a resposta oxidativa de monócitos de sangue periférico durante a fagocitose de zymosan opsonisado ou de S. aureus, analisada respectivamente por quimiluminescência amplificada por luminol ou por citometria de fluxo. A β2GPI estimulou a viabilidade celular e estimulou a diferenciação dos promonócitos. As células THP-1 tratadas com β2GPI apresentaram adesão aumentada a placas de cultura bem como expressão aumentada de CD54 e CD14. A suplementação com β2GPI foi suficiente para manter a proliferação das células THP-1 em cultura sem a adição de soro por 72h. Não houve correlações entre a concentração plasmática da β2GPI e indicadores clínicos da resposta inflamatória aguda em pacientes sépticos. A concentração da β2GPI não correlacionou com as concentrações plasmáticas de IL-8, SAA e PCR, que foram encontradas elevadas no sangue de pacientes com sepse. A variação da concentração plasmática de β2GPI foi um fenômeno muito precoce no modelo experimental de sepse e translocação bacteriana. Nas primeiras três horas após a indução da sepse endovenosa, a concentração plasmática de β2GPI diminuiu de forma dependente da intensidade de infecção. Sugere-se que efeitos muito precoces de compartimentalização associados ao sangue portal medeiem esta regulação. As concentrações mais baixas de β2GPI foram observadas nos animais expostos à translocação bacteriana através da mucosa intestinal, associada a uma condição inflamatória leve. A derivação da linfa preveniu completamente a diminuição da concentração plasmática de β2GPI. Em conjunto, os resultados revelaram a relevância combinada de via e de intensidade da infecção para o controle da concentração plasmática de β2GPI no início na resposta inflamatória aguda. / The β2-glycoprotein I (β2GPI) is an acute phase protein, produced mainly in the liver and intestine. The effects of this protein upon mononuclear cells were investigated both in monocytes from human peripheral blood, and in the human promonocytic cells from the ATCC THP-1 cell line. The correlations between its plasma concentration and systemic inflammation intensity were evaluated in humans and in ad experimental model of systemic infection in rats. No β2GPI effects were observed upon the oxidative response of blood monocytes during the phagocytosis of opsonized zymosan or S. aureus as analysed by luminol amplified chemiluminescence and flow cytometry. β2GPI enhanced the cellular viability and stimulated the differentiation of the promonocytes. The THP-1 cells treated with β2GPI presented increased adhesion to the plastic of cell culture plates as well as increased expression of CD54 and CD14 antigens. The supplementation with β2GPI was sufficient to support the proliferation of THP-1 cells in serum free culture conditions for 72 h. There were no correlations between the β2GPI plasma concentration and clinical parameters of the acute inflammatory response in septic patients. The β2GPI concentrations didn\'t correlated with the plasma concentrations of IL-8, SAA and C reactive protein, despite these substances were found increased in the blood of patients with sepsis. The β2GPI plasma concentration response was a very early phenomenon in the experimental sepsis and bacterial translocation model. The β2GPI concentration decreased within the first 3h after endovenous sepsis induction, depending on the infection intensity. Very early compartment effects associated with the portal blood are suggested to mediate such regulation. The lowest β2GPI concentrations were found in the animals exposed to bacterial translocation through the intestinal mucosa, associated with a mild inflammatory condition. The lymph derivation completely prevented the plasma β2GPI decrease. Taken together, the results revealed the relevance of both the infection route and intensity to the control of plasma β2GPI concentrations during the acute phase response.

INTRODUÇÃO: Apesar do contínuo progresso no tratamento e suporte clínico a sepse continua sendo uma das principais causas de morbidade e mortalidade nas unidades de terapia intensiva, com desfechos semelhantes ao longo dos últimos 50 anos. A suscetibilidade à infecção grave no recém-nascido é parcialmente devida à imaturidade do sistema imune inato associado à mínima em exposição antigênica in utero e à ação ineficaz das células T efetoras e das célula B. Embora a ativação do sistema imune inato por padrões de reconhecimento (PRR) como os dos receptores Toll-like (TLR) tenham sua importância amplamente reconhecida nos últimos anos, seu comportamento frente a uma infecção in vivo ainda não foi completamente compreendido. Neste trabalho nós analisamos a expressão dos TLR-2 e TLR-4 em células apresentadoras de antígeno em recém-nascidos com e sem sepse. CAUSUÍSTICA E MÉTODO: Trata-se de um estudo prospectivo realizado no período entre fevereiro de 2011 e janeiro de 2013 onde foram incluídos quarenta e cinco recém-nascidos a termo, sem malformação congênita, admitidos na Unidade de Cuidados Intensivos Neonatal do Instituto da Criança-HCFMUSP e divididos em grupos 1 e 2. O grupo 1 consistiu em 27 recém-nascidos com diagnóstico clínico e laboratorial de sepse tardia enquanto que o grupo 2 foi composto por 18 recém-nascidos sem quadro séptico vigente. As citocinas foram determinadas por teste de CBA em sangue periférico. A expressão e MFI dos TLR-2 e TLR-4 foi determinado por imunofenotipagem em APCs e linfócitos no sangue periférico total através de análise pelo citômetro de fluxo BD FACSDiva. RESULTADOS: Os dados clínicos foram semelhantes entre os grupos 1 e 2, exceto para o estado infeccioso. Microrganismos foram identificados em 37 % no grupo 1 e estes tiveram níveis mais elevados de citocinas pró-inflamatórias (IL-8, IL-6, IL-1beta) e de citocina anti-inflamatória (IL-10). Nas células dendríticas, a expressão de TLR-2 e 4 foi semelhante entre os grupos enquanto que houve menor expressão nos pacientes infectados da molécula co-estimuladora CD86 (p < 0,05) e expressão semelhante de CD1a e CD80 em relação aos RN não infectados. No monócito, o MFI para TLR-2 e a freqüência de expressão do TLR-4 foi maior no grupo 1 (p = 0,01). Apesar da frequência de linfócitos totais ter sido mais baixa no grupo 1 (p = 0,002), não foi observada diferença quanto as suas subpopulações exceto em relação a maior frequência de LT efetor no grupo infectado com menor expressão da molécula CD28. Houve maior frequência de LB ativados no grupo 1 enquanto que a população total e as demais subpopulações foram semelhantes em número, moléculas de ativação e na expressão dos TLR-2 e 4 em ambos os grupos. CONCLUSÃO: Este estudo analisou a resposta imune inata no recém-nascido com e sem sepse. As IL-6, IL-8 e IL-10 foram bons indicadores desta doença. Recém-nascidos sépticos, que dependem quase exclusivamente do sistema imune inato, apresentaram pouca resposta in vivo na ativação de células dendríticas e monócitos propiciando uma resposta imune deficiente e maior susceptibilidade à infecção / INTRODUCTION: Despite continuous progress in the clinical treatment and other supportive care therapies, sepsis remains a leading cause of morbidity and mortality in the intensive care unit with similar outcome throughout the past 50 years. The susceptibility to severe infection is partially due to newborn immature innate immune system associated to minimal in utero antigen exposure and effector T and B cell impaired function. Although the importance of pattern recognition domains such as Toll-like receptors (TLR) in the innate immune system activation has been fully acknowledged within the last few years its behavior in front of an in vivo infection scenario is still not completely understood. Here we analyzed the TLR-2 and TLR-4 expression in antigen-presenting cell in healthy and septic newborns. PATIENTS AND METHODS: This prospective study was conducted during the period from February 2011 until January 2013 at Sao Paulo University, Sao Paulo, Brazil. Forty-five term newborns without congenital malformation were included from the Newborn Intensive Care Unit at Children\'s Hospital. As group 1, 27 newborns who had clinical and laboratory diagnostic of late onset sepsis were included while 18 newborns were evaluated in a non-septic status and were included at group 2. Cytokines were measured by cytometric bead array in peripheral blood. TLR-2 and TLR-4 expression and MFI were determined by immunophenotyping at peripheral whole blood in APC cells and lymphocytes and analyzed on a BD FACSDiva flow cytometer. RESULTS: Clinical data was similar between septic and non-septic groups except for the infectious status. Group 1 had microorganisms identified in 37 % septic newborns associated with higher levels of pro-inflammatory (IL-8, IL-6, IL-1beta) and anti-inflammatory interleukins (IL-10). When it comes to dendritic cells, the expression of TLR-2 and 4 was similar between groups whereas there was lower expression of co-molecule CD86 (p < 0,05) and similar expression of CD1a and CD80 between infected and non-infected patients. At monocytes, the MFI for TLR-2 and the frequency of TLR-4 expression was higher in infected newborn (p=0,01). There were lower levels of total lymphocytes in infected patients (p=0,002) but no difference was observed in T cells subtypes frequency except for higher levels of effector T cell in infected group with lower expression of CD28 molecule. Group 1 had higher levels of activated B cell whereas total population and the other subsets were similar in number, activation molecules and TLR-2 and 4 expressions in both groups. CONCLUSION: This study investigated the innate immune response in septic and non-septic newborn. Interleukin levels 6, 8 and 10 were good indicators of sepsis. Septic newborns, which count most exclusively with innate immune system, had little in vivo response at dendritic cell and monocyte activation leading to an impaired immune response and increased susceptibility to infection

INTRODUÇÃO: Apesar do contínuo progresso no tratamento e suporte clínico a sepse continua sendo uma das principais causas de morbidade e mortalidade nas unidades de terapia intensiva, com desfechos semelhantes ao longo dos últimos 50 anos. A suscetibilidade à infecção grave no recém-nascido é parcialmente devida à imaturidade do sistema imune inato associado à mínima em exposição antigênica in utero e à ação ineficaz das células T efetoras e das célula B. Embora a ativação do sistema imune inato por padrões de reconhecimento (PRR) como os dos receptores Toll-like (TLR) tenham sua importância amplamente reconhecida nos últimos anos, seu comportamento frente a uma infecção in vivo ainda não foi completamente compreendido. Neste trabalho nós analisamos a expressão dos TLR-2 e TLR-4 em células apresentadoras de antígeno em recém-nascidos com e sem sepse. CAUSUÍSTICA E MÉTODO: Trata-se de um estudo prospectivo realizado no período entre fevereiro de 2011 e janeiro de 2013 onde foram incluídos quarenta e cinco recém-nascidos a termo, sem malformação congênita, admitidos na Unidade de Cuidados Intensivos Neonatal do Instituto da Criança-HCFMUSP e divididos em grupos 1 e 2. O grupo 1 consistiu em 27 recém-nascidos com diagnóstico clínico e laboratorial de sepse tardia enquanto que o grupo 2 foi composto por 18 recém-nascidos sem quadro séptico vigente. As citocinas foram determinadas por teste de CBA em sangue periférico. A expressão e MFI dos TLR-2 e TLR-4 foi determinado por imunofenotipagem em APCs e linfócitos no sangue periférico total através de análise pelo citômetro de fluxo BD FACSDiva. RESULTADOS: Os dados clínicos foram semelhantes entre os grupos 1 e 2, exceto para o estado infeccioso. Microrganismos foram identificados em 37 % no grupo 1 e estes tiveram níveis mais elevados de citocinas pró-inflamatórias (IL-8, IL-6, IL-1beta) e de citocina anti-inflamatória (IL-10). Nas células dendríticas, a expressão de TLR-2 e 4 foi semelhante entre os grupos enquanto que houve menor expressão nos pacientes infectados da molécula co-estimuladora CD86 (p < 0,05) e expressão semelhante de CD1a e CD80 em relação aos RN não infectados. No monócito, o MFI para TLR-2 e a freqüência de expressão do TLR-4 foi maior no grupo 1 (p = 0,01). Apesar da frequência de linfócitos totais ter sido mais baixa no grupo 1 (p = 0,002), não foi observada diferença quanto as suas subpopulações exceto em relação a maior frequência de LT efetor no grupo infectado com menor expressão da molécula CD28. Houve maior frequência de LB ativados no grupo 1 enquanto que a população total e as demais subpopulações foram semelhantes em número, moléculas de ativação e na expressão dos TLR-2 e 4 em ambos os grupos. CONCLUSÃO: Este estudo analisou a resposta imune inata no recém-nascido com e sem sepse. As IL-6, IL-8 e IL-10 foram bons indicadores desta doença. Recém-nascidos sépticos, que dependem quase exclusivamente do sistema imune inato, apresentaram pouca resposta in vivo na ativação de células dendríticas e monócitos propiciando uma resposta imune deficiente e maior susceptibilidade à infecção / INTRODUCTION: Despite continuous progress in the clinical treatment and other supportive care therapies, sepsis remains a leading cause of morbidity and mortality in the intensive care unit with similar outcome throughout the past 50 years. The susceptibility to severe infection is partially due to newborn immature innate immune system associated to minimal in utero antigen exposure and effector T and B cell impaired function. Although the importance of pattern recognition domains such as Toll-like receptors (TLR) in the innate immune system activation has been fully acknowledged within the last few years its behavior in front of an in vivo infection scenario is still not completely understood. Here we analyzed the TLR-2 and TLR-4 expression in antigen-presenting cell in healthy and septic newborns. PATIENTS AND METHODS: This prospective study was conducted during the period from February 2011 until January 2013 at Sao Paulo University, Sao Paulo, Brazil. Forty-five term newborns without congenital malformation were included from the Newborn Intensive Care Unit at Children\'s Hospital. As group 1, 27 newborns who had clinical and laboratory diagnostic of late onset sepsis were included while 18 newborns were evaluated in a non-septic status and were included at group 2. Cytokines were measured by cytometric bead array in peripheral blood. TLR-2 and TLR-4 expression and MFI were determined by immunophenotyping at peripheral whole blood in APC cells and lymphocytes and analyzed on a BD FACSDiva flow cytometer. RESULTS: Clinical data was similar between septic and non-septic groups except for the infectious status. Group 1 had microorganisms identified in 37 % septic newborns associated with higher levels of pro-inflammatory (IL-8, IL-6, IL-1beta) and anti-inflammatory interleukins (IL-10). When it comes to dendritic cells, the expression of TLR-2 and 4 was similar between groups whereas there was lower expression of co-molecule CD86 (p < 0,05) and similar expression of CD1a and CD80 between infected and non-infected patients. At monocytes, the MFI for TLR-2 and the frequency of TLR-4 expression was higher in infected newborn (p=0,01). There were lower levels of total lymphocytes in infected patients (p=0,002) but no difference was observed in T cells subtypes frequency except for higher levels of effector T cell in infected group with lower expression of CD28 molecule. Group 1 had higher levels of activated B cell whereas total population and the other subsets were similar in number, activation molecules and TLR-2 and 4 expressions in both groups. CONCLUSION: This study investigated the innate immune response in septic and non-septic newborn. Interleukin levels 6, 8 and 10 were good indicators of sepsis. Septic newborns, which count most exclusively with innate immune system, had little in vivo response at dendritic cell and monocyte activation leading to an impaired immune response and increased susceptibility to infection

No description available.

Hintergrund: Die Funktion von Immunzellen hängt von einer konstanten und ausreichenden Energieversorgung ab, die über die OXPHOS in den Mitochondrien und die Glykolyse im Zytosol realisiert wird. Die wichtigsten Substrate dafür sind Sauerstoff und Glukose. Fragestellung: Bei schweren Erkrankungen oder in Entzündungsgebieten ist die zelluläre Energieversorgung stark beeinträchtigt, weil in der Mikroumgebung der Zelle Sauerstoff und Nährstoffe inadäquat bereitgestellt werden. Ziel war herauszufinden, ob und wie humane Immunzellen ihre Lebensfähigkeit und funktionellen Aktivitäten unter solchen Umständen aufrechterhalten. Methoden: Humane CD4+ T-Zellen und CD14+ Monozyten wurden durch MACS aus peripherem Blut gesunder Spender isoliert. Die Sauerstoffverbrauchsmessung mittels Clark-Elektrode war Maß der oxidativen Energiebildung, die mit Myxothiazol und Glukoseentzug gehemmt wurde. Die CD3/CD28-stimulierte T-Zell-Proliferation wurde durchflußzytometrisch mittels CFDA SE analysiert. Basierend auf dem Paraformaldehyd-Saponin-Prozedere wurde die Zytokinsynthese ebenfalls am FACS bewertet, nachdem die T-Zellen in Anwesenheit von Brefeldin A mit PMA/Ionomycin stimuliert wurden. Mit einem käuflichen Testsystem (FACS-Technik) wurde die monozytäre Phagozytose untersucht. Die HIF-1alpha-Expression wurde nach PMA-Ionomycin-Stimulation von Myxothiazol-behandelten T-Zellen auf mRNA- und Proteinebene gemessen. Ergebnisse: Bei Glukoseanwesenheit waren alle untersuchten Immunfunktionen trotz vollständig gehemmter OXPHOS unbeeinträchtigt. Erst bei gleichzeitigem Glukoseentzug, der per sé Proliferation und Phagozytose signifikant beeinträchtigte, waren sie signifikant vermindert. Es wird vermutet, daß T-Zellen die Energieverluste mit einem überschießenden Effekt ihres Sauerstoffverbrauchs und stark angetriebener Glykolyse kompensieren. HIF-1alpha ist dabei nicht entscheidend für die Umschaltung auf anaerobe Energiesynthese. Schlußfolgerung: Die Daten quantifizieren die Energieanforderungen der funktionellen Aktivität in hochgereinigten humanen Immunzellfraktionen. Es wurde nachgewiesen, daß sich Immunzellen unerwartet lange an eine massiv beeinträchtigte Energetik adaptieren können und ihre spezifischen Funktionen aufrechterhalten. / Background: The function of immune cells is dependent upon a constant and adequate supply of energy. Energy is formed via OXPHOS in the mitochondria and via cytosolic glycolysis. Oxygen and glucose are the main substrates for energy synthesis. Objective: In severe diseases or in inflamed areas cellular energy supply is significantly impaired due to inadequate supply of cellular microenvironment with oxygen and nutrients. The aim of this study was to answer the question, whether and how human immune cells maintain viability and functional activity under these circumstances. Methods: Human CD4+ T cells and CD14+ monocytes were isolated by MACS from peripheral blood of healthy donors. The extent of oxidative energy formation was determined via measurement of oxygen consumption using a Clark type electrode. Energy production was restricted in glucose-free cell culture medium and by gradually inhibited OXPHOS using myxothiazol. T cell proliferation was flow-cytometrically analysed using CFDA SE after stimulation with CD3 and CD28 antibodies. Cytokine synthesis was assessed by flow-cytometrical immunofluorescence and the paraformaldehyde-saponin procedure after stimulation of T cells with PMA/ionomycin in the presence of brefeldin A. Phagocytosis of monocytes was measured using a commercial test system (FACS technique). HIF-1alpha expression was assessed by semiquantitative PCR and immunoblot after the stimulation of myxothiazol treated T cells with PMA/ionomycin. Results: In glucose-containing medium all investigated immune functions were unaffected even under complete suppression of OXPHOS. Only when OXPHOS and glycolysis were simultaneously and almost completely suppressed a significant decrease was found. Glucose deprivation per se caused both a significantly reduced proliferation and phagocytosis. It is supposed, that T cells are able to compensate for an energy deficit by an excess of oxygen consumption and strongly induced glycolysis. However, HIF-1alpha was found to be not crucial for switching to anaerobic energy synthesis. Conclusion: These data quantify the energy requirement of functional activity in highly purified human immune cell fractions. An unexpectedly high adaptive potential of immune cells to maintain specific functions even under massively impaired energetic conditions could be demonstrated.

No description available.

A &#946;2-glicoproteína I (&#946;2GPI) é uma proteína de fase aguda, produzida principalmente no fígado e intestino. Os efeitos dessa proteína sobre células mononucleares foram investigados tanto em monócitos humanos de sangue periférico quanto em células promonocíticas humanas da linhagem celular ATCC THP-1. As correlações entre sua concentração plasmática e a intensidade da inflamação sistêmica foram avaliadas em humanos e em um modelo experimental de infecção sistêmica, em ratos. Nenhum efeito da &#946;2GPI foi observado sobre a resposta oxidativa de monócitos de sangue periférico durante a fagocitose de zymosan opsonisado ou de S. aureus, analisada respectivamente por quimiluminescência amplificada por luminol ou por citometria de fluxo. A &#946;2GPI estimulou a viabilidade celular e estimulou a diferenciação dos promonócitos. As células THP-1 tratadas com &#946;2GPI apresentaram adesão aumentada a placas de cultura bem como expressão aumentada de CD54 e CD14. A suplementação com &#946;2GPI foi suficiente para manter a proliferação das células THP-1 em cultura sem a adição de soro por 72h. Não houve correlações entre a concentração plasmática da &#946;2GPI e indicadores clínicos da resposta inflamatória aguda em pacientes sépticos. A concentração da &#946;2GPI não correlacionou com as concentrações plasmáticas de IL-8, SAA e PCR, que foram encontradas elevadas no sangue de pacientes com sepse. A variação da concentração plasmática de &#946;2GPI foi um fenômeno muito precoce no modelo experimental de sepse e translocação bacteriana. Nas primeiras três horas após a indução da sepse endovenosa, a concentração plasmática de &#946;2GPI diminuiu de forma dependente da intensidade de infecção. Sugere-se que efeitos muito precoces de compartimentalização associados ao sangue portal medeiem esta regulação. As concentrações mais baixas de &#946;2GPI foram observadas nos animais expostos à translocação bacteriana através da mucosa intestinal, associada a uma condição inflamatória leve. A derivação da linfa preveniu completamente a diminuição da concentração plasmática de &#946;2GPI. Em conjunto, os resultados revelaram a relevância combinada de via e de intensidade da infecção para o controle da concentração plasmática de &#946;2GPI no início na resposta inflamatória aguda. / The &#946;2-glycoprotein I (&#946;2GPI) is an acute phase protein, produced mainly in the liver and intestine. The effects of this protein upon mononuclear cells were investigated both in monocytes from human peripheral blood, and in the human promonocytic cells from the ATCC THP-1 cell line. The correlations between its plasma concentration and systemic inflammation intensity were evaluated in humans and in ad experimental model of systemic infection in rats. No &#946;2GPI effects were observed upon the oxidative response of blood monocytes during the phagocytosis of opsonized zymosan or S. aureus as analysed by luminol amplified chemiluminescence and flow cytometry. &#946;2GPI enhanced the cellular viability and stimulated the differentiation of the promonocytes. The THP-1 cells treated with &#946;2GPI presented increased adhesion to the plastic of cell culture plates as well as increased expression of CD54 and CD14 antigens. The supplementation with &#946;2GPI was sufficient to support the proliferation of THP-1 cells in serum free culture conditions for 72 h. There were no correlations between the &#946;2GPI plasma concentration and clinical parameters of the acute inflammatory response in septic patients. The &#946;2GPI concentrations didn\'t correlated with the plasma concentrations of IL-8, SAA and C reactive protein, despite these substances were found increased in the blood of patients with sepsis. The &#946;2GPI plasma concentration response was a very early phenomenon in the experimental sepsis and bacterial translocation model. The &#946;2GPI concentration decreased within the first 3h after endovenous sepsis induction, depending on the infection intensity. Very early compartment effects associated with the portal blood are suggested to mediate such regulation. The lowest &#946;2GPI concentrations were found in the animals exposed to bacterial translocation through the intestinal mucosa, associated with a mild inflammatory condition. The lymph derivation completely prevented the plasma &#946;2GPI decrease. Taken together, the results revealed the relevance of both the infection route and intensity to the control of plasma &#946;2GPI concentrations during the acute phase response.

Indiana University-Purdue University Indianapolis (IUPUI) / Juvenile Myelomonocytic Leukemia (JMML) is rare, fatal myeloproliferative disease (MPD) affecting young children, and is characterized by expansion of monocyte lineage cells and hypersensitivity to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) stimulation. JMML is frequently associated with gain-of-function mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase, Shp2. Activating Shp2 mutations are known to promote hyperactivation of the Ras-Erk signaling pathway, but Akt is also observed to have enhanced phosphorylation, suggesting a potential role for Phosphatidylinositol-3-Kinase (PI3K)-Akt signaling in mutant Shp2-induced GM-CSF hypersensitivity and leukemogenesis. Having demonstrated that Class IA PI3K is hyperactivated in the presence of mutant Shp2 and contributes to GM-CSF hypersensitivity, I hypothesized the hematopoietic-specific Class IA PI3K catalytic subunit p110δ is a crucial mediator of mutant Shp2-induced PI3K hyperactivation and GM-CSF hypersensitivity in vitro and MPD development in vivo. I crossed gain-of-function mutant Shp2 D61Y inducible knockin mice, which develop fatal MPD, with mice expressing kinase-dead mutant p110δ D910A to evaluate p110δ’s role in mutant Shp2-induced GM-CSF hypersensitivity in vitro and MPD development in vivo. As a comparison, I also crossed Shp2 D61Y inducible knockin mice with mice bearing inducible knockout of the ubiquitously expressed Class IA PI3K catalytic subunit, p110α. I found that genetic interruption of p110δ, but not p110α, significantly reduced GM-CSF-stimulated hyperactivation of both the Ras-Erk and PI3K-Akt signaling pathways, and as a consequence, resulted in reduced GM-CSF-stimulated hyper-proliferation in vitro. Furthermore, I found that mice bearing genetic disruption of p110δ, but not p110α, in the presence of gain-of-function mutant Shp2 D61Y, had on average, smaller spleen sizes, suggesting that loss of p110δ activity reduced MPD severity in vivo. I also investigated the effects of three PI3K inhibitors with high specificity for p110δ, IC87114, GDC-0941, and GS-9820 (formerly known as CAL-120), on mutant Shp2-induced GM-CSF hypersensitivity. These inhibitors with high specificity for p110δ significantly reduced GM-CSF-stimulated hyperactivation of PI3K-Akt and Ras-Erk signaling and reduced GM-CSF-stimulated hyperproliferation in cells expressing gain-of-function Shp2 mutants. Collectively, these findings show that p110δ-dependent PI3K hyperactivation contributes to mutant Shp2-induced GM-CSF hypersensitivity and MPD development, and that p110δ represents a potential novel therapeutic target for JMML.