Objectives - To evaluate the effects of equine sCD14 and monoclonal antibodies (mAbs) to equine CD14 on LPS-induced TNF° expression of equine peripheral blood mononuclear cells (PBMCs). To determine serum concentrations of soluble (sCD14) in a population of horses with gastrointestinal diseases or other illnesses likely to result in endotoxemia; and identify relationships with clinical data. Animals - Part 1; 10 healthy horses. Part 2; 55 clinical cases and 23 healthy control horses. Procedure - Part 1; PBMCs were incubated with Escherichia coli LPS, CD14 mAb, sCD14, CD14 mAb plus E coli LPS or sCD14 plus E coli LPS. Supernatants were collected at 6 hours and assayed for tumor necrosis factor ° (TNF°) activity. Part 2; Serum sCD14 was measured at admission and then at 24 and 48 hours after admission using a bead-based multiplex assay. Results - Part 1; Pre-incubation with CD14 mAb did not inhibit LPS-induced TNF° protein production in isolated equine monocytes. Use of sCD14 inhibited LPS-induced TNF° protein production in isolated monocytes in a concentration-dependent manner. Part 2; Serum concentration of sCD14 was positively related to duration of clinical signs (P = 0.007), respiratory rate (P=0.04) and band neutrophil count (P = 0.0002). There was no correlation between serum concentration of sCD14 and heart rate, temperature, hematocrit, lactate, white blood cell count, fibrinogen, creatinine, urea nitrogen, glucose and anion gap values. Serum sCD14 did not correlate with outcome at any time point for clinical cases. / Master of Science

The hygiene hypothesis, as originally proposed, postulated an inverse relation between the incidence of infectious diseases in early life and the subsequent development of allergies and asthma. New evidence from epidemiological, biological and genetic studies has significantly enlarged the scope of the hypothesis. It now appears probable that environmental 'danger' signals regulate the pattern of immune responses in early life. Microbial burden in general, and not any single acute infectious illness, is the main source of these signals. The latter interact with a sensitive and complex receptor system, and genetic variations in this receptor system may be an important determinant of inherited susceptibility to asthma and allergies.

The effects of induced subacute ruminal acidosis (SARA) using grain pellet-based (GPI) and alfalfa pellet-based diet models on systemic immunological parameters were evaluated in nonlactating Holstein cows. The systemic immunological parameters analysed in this study included rectal temperature, blood cell leukogram, expression of lipopolysaccharide (LPS) recognition receptors on leukocytic cells, and plasma and serum proteins. Also, blood biochemistry was analysed. There were no significant differences in rectal temperature, blood cell leukogram, expression of LPS recognition receptors and fibrinogen or haptoglobin concentrations between control and SARA induction treatments. Concentrations of serum amyloid A and lipopolysaccharide-binding protein increased while total protein concentrations decreased in response to GPI SARA compared to control. Blood glucose and urea concentrations increased and decreased, respectively, with GPI SARA treatment. Grain pellet-induced SARA resulted in changes to serum proteins and acute phase proteins but did not affect other systemic immunological parameters suggesting a localized inflammatory response was initiated.

The effects of induced subacute ruminal acidosis (SARA) using grain pellet-based (GPI) and alfalfa pellet-based diet models on systemic immunological parameters were evaluated in nonlactating Holstein cows. The systemic immunological parameters analysed in this study included rectal temperature, blood cell leukogram, expression of lipopolysaccharide (LPS) recognition receptors on leukocytic cells, and plasma and serum proteins. Also, blood biochemistry was analysed. There were no significant differences in rectal temperature, blood cell leukogram, expression of LPS recognition receptors and fibrinogen or haptoglobin concentrations between control and SARA induction treatments. Concentrations of serum amyloid A and lipopolysaccharide-binding protein increased while total protein concentrations decreased in response to GPI SARA compared to control. Blood glucose and urea concentrations increased and decreased, respectively, with GPI SARA treatment. Grain pellet-induced SARA resulted in changes to serum proteins and acute phase proteins but did not affect other systemic immunological parameters suggesting a localized inflammatory response was initiated.

Mesenchymale Stammzellen (MSC) sind starke Suppressoren des Immunsystems. Für die immunsupressive Wirkung werden lösliche Faktoren propagiert. In der vorliegenden Arbeit wurde untersucht, ob neben den bekannten Sekretionsfaktoren stromaler Stammzellen wie RANK/RANKL/OPG, TNFα, IFNγ und verschiedene Interleukine, andere Sekretionsfaktoren eine immunmodulatorische Wirkung auf monozytäre Zellen haben. Als Sekretionsprodukt wurde hCyr61 untersucht, das im Überstand von mit TNFα behandelten humanen fetalen Osteoblasten (hFOB) nachgewiesen worden war. Das zur CCN-Familie gehörende Protein hCyr61 ist an der Angiogenese beteiligt, wird im Kallus während der Frakturheilung vermehrt exprimiert und hat, wie zwischenzeitlich nachgewiesen wurde, einen deutlichen Effekt auf die Differenzierung endothelialer Vorläuferzellen aus monozytären Zellen. In den hier beschriebenen Experimenten wurden sowohl monozytäre Zellen aus peripherem Blut (PBMC), als auch THP1-Zellen auf die Veränderung der CD14-, 80-, 83- und 86- Expression unter Stimulation mit und ohne Il-4, GM-CSF und hCyr61 untersucht. Die Expressionsänderung wurde mittels konventioneller PCR, real-time PCR und FACS-Analyse untersucht. In allen drei Nachweismethoden zeigte sich ein Verlust typisch monozytärer Marker wie CD14 unter Stimulation mit hCyr61. Der Verlust an CD14 scheint bei PBMC deutlicher als bei THP1-Zellen, was möglicherweise durch die oben beschriebene endotheliale Differenzierung erklärt wird. THP1-Zellen befinden sich bereits in einer höheren Differenzierungsstufe, sodass sie ihr monozytäres Commitment nicht vollständig verlieren. / Mesenchymal stem cells have a strong suppressive effect on the immune system. Soluble factors are discussed to be responsible for this effect. The aim of this dissertation was to investigate, whether there are other soluble factors with immunomodulatory effects on monocytic cells beside the already known factors like RANK/RANKL/OPG, TNFα, IFNγ and various interleukins. As a soluble factor found in the supernatant of humane fetal osteoblasts stimulated by TNFα, hCyr61 was investigated. This protein belongs to the CCN-family and it takes part in angiogenesis, was found in high concentrations in the callus during the healing process of a fracture and it effects strongly the differentiation of endothelial precursor cells from monocytic cells. The change of the expression of CD14, 80, 83 and 86 was investigated with and without stimulation by Il-4, GM-CSF and hCyr61 in monocytic cells from peripheral blood (PBMC) and THP1-cells. The results were verified by conventional PCR, real-time PCR and FACS analysis. All three methods showed a decrease of CD14 as typical monocyte marker in the presence of hCyr61. The decrease of CD14 in PBMC seems more striking then in THP1, which might be explained by endothelial differentiation mentioned above. THP1 cells have reached a higher level of differentiation, therefore they don´t lose their monocyte commitment anymore.

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2017-06-22T17:47:52Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016CamileWunsch.pdf: 1221188 bytes, checksum: d4f164f1168ed40c958561be1e994dc5 (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2017-06-26T18:42:58Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016CamileWunsch.pdf: 1221188 bytes, checksum: d4f164f1168ed40c958561be1e994dc5 (MD5) / Made available in DSpace on 2017-06-26T18:42:58Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016CamileWunsch.pdf: 1221188 bytes, checksum: d4f164f1168ed40c958561be1e994dc5 (MD5) Previous issue date: 2017-06 / CAPES / Introdução: A doença arterial coronariana (DAC) é a principal causa de morbidade e mortalidade no mundo, sendo caracterizada como uma doença inflamatória crônica, multifatorial, cuja fisiopatologia é a aterosclerose. Considerando a alta herdabilidade da doença, vários polimorfismos genéticos vêm sendo estudados e associados com o surgimento da DAC e, entre eles, destacam-se variantes nos genes CD14, TLR4, NFKB1 e TNFα, os quais regulam a via de sinalização celular do sistema imune inato e desencadeiam o processo inflamatório na DAC. Objetivo: O objetivo principal deste estudo é verificar a possível associação de polimorfismos nos genes CD14 (rs2569190), TLR4 (rs4986790 e rs4986791), NFKB1 (rs28362491) e TNFα (rs1800629 e rs361525) com a DAC. Metodologia: A amostra foi composta por 707 indivíduos adultos submetidos ao exame de cateterismo cardíaco no Hospital Bruno Born, de Lajeado, RS. Todos os indivíduos assinaram um termo de consentimento livre e esclarecido e responderam a um questionário semiestruturado. Os indivíduos foram classificados entre casos e controles, por um médico cardiologista, com base no seguinte critério: presença de estenose, com comprometimento maior do que 50%, em pelo menos uma das artérias coronárias. Foram também coletadas amostras de sangue periférico para análises bioquímicas e moleculares. A extração de DNA foi realizada pelo método de salting out. Os polimorfismos dos genes CD14, TLR4 e TNFα foram genotipados pelo sistema de discriminação alélica TaqMan, em equipamento de reação em cadeia da polimerase (PCR) em Tempo Real (StepOnePlus®). O polimorfismo rs28362491, no gene NFKB1, foi amplificado através da técnica convencional de PCR. Resultados: Identificamos uma associação dos polimorfismos rs2569190, localizado no gene CD14, e rs28362491, no gene NFKB1, com a DAC. Além disso, foram detectados efeitos dos polimorfismos dos genes TLR4 e TNFα nos níveis glicêmicos e dos polimorfismos nos genes TLR4, NFKB1 e TNFα no perfil lipídico. Conclusão: Nossos achados sugerem a participação de polimorfismos nos genes CD14 e NFKB1 no desenvolvimento da DAC na nossa amostra, corroborando evidências prévias do envolvimento de genes do processo inflamatório nessa patologia. / Introduction: Coronary artery disease (CAD) is the main cause of morbidity and mortality in the world, being characterized as a chronic, multifactorial inflammatory disease, whose pathophysiology is atherosclerosis. Considering the high heritability of the disease, several genetic polymorphisms have been investigated and associated with CAD and, among them, there are variants in the CD14, TLR4, NFKB1 and TNFα genes, which regulate cell signaling pathways of the innate immune system and inflammatory process in CAD. Objective: The main objective of this study is to verify the association beteween polymorphisms in the CD14 (rs2569190), TLR4 (rs4986790 and rs4986791), NFKB1 (rs28362491) and TNFα (rs1800629 and rs361525) genes and CAD. Methods: The sample group was composed of 707 adult individuals, recruited at the time when they were bought in for coronary angiography procedures at the Hemodynamic Center of the Hospital Bruno Born, City of Lajeado, Rio Grande do Sul. The individuals were classified between cases and controls by a cardiologist, based on the following criteria: presence of stenosis, greater than 50% of the luminal diameter, in at least one of the coronary arteries. Peripheral blood samples were also collected for biochemical and molecular analyzes. DNA extraction was performed using the salting out method. The polymorphisms in the CD14, TLR4 e TNFα genes were genotyped by Taqman® allelic discrimination assays. The rs28362491 polymorphism in the NFKB1 gene was amplified by polymerase chain reaction (PCR). Results: We identified an association between the polymorphisms rs2569190, located in the CD14 gene, and rs28362491, located in the NFKB1 gene, and CAD. In addition, there were detected significant effects of TLR4 and TNFα gene polymorphisms on glycemic levels and TLR4, NFKB1 and TNFα gene polymorphisms on the lipid profile. Conclusion: Our findings suggest a role for the polymorphisms in CD14 and NFKB1 genes in CAD susceptibility in our sample, corroborating previous evidence of the envolviment of inflamotory process genes in this patology.

Mikroglia sind residente Makrophagen-artige Zellen des Zentralnervensystems (ZNS), die das Gewebe kontinuierlich auf Anzeichen homöostatischer Störungen überwachen. Als die wesentlichen immunkompetenten Effektorzellen im Hirnparenchym exprimieren sie eine Vielzahl von Rezeptoren für pathogen-assoziierte molekulare Strukturmuster (pathogen-associated molecular patterns, PAMPs). Zu diesen Rezeptoren zählt der Toll-like receptor (TLR) 4, der nicht nur Reaktionen der Mikroglia auf bakterielle Infektionen, sondern auch auf Gewebe Schädigungen ermöglicht. Stimulation des TLR4 mit bakteriellem Lipopolysaccharid (LPS) und endogenen schädigung-sassoziierten molekularen Strukturen (damage-associated molecular patterns, DAMPs), die durch Gewebebeeinträchtigung freigesetzt werden, löst sowohl TRIF- als auch MyD88-abhängige Signalkaskaden aus. Die damit induzierte Freisetzung von Zytokinen und Chemokinen rekrutiert und instruiert periphere Immunzellen für eine Protektion und unterstützende Geweberegeneration des ZNS. Wir zeigen hier, dass der TLR4-Korezeptor CD14 ein essenzieller gate keeper für die Generierung von Immunantworten im ZNS ist, die durch LPS oder E. coli-Verabreichung, aber auch durch mechanisches Trauma und ischämischen Schlaganfall ausgelöst werden. In gewissem Gegensatz zu extraneuralen Makrophagen nutzen Mikroglia CD14 zur Erlangung einer extremen Sensitivität gegenüber sehr geringen LPS-Mengen. Gleichzeitig schützt CD14 Mikroglia vor überschießenden Reaktionen auf hohe LPS-Dosen und verhindert dabei insbesondere die exzessive Produktion von CXCL1, eines chemoattraktiven Signals für neutrophile Granulozyten. Entsprechend unterstützt CD14 die ZNS-Rekrutierung von Monozyten und Neutrophilen durch niedrige LPS-Dosen, während es die verstärkte Einwanderung von Neutrophilen durch hohe Dosen von LPS oder E. coli verhindert. Als eine besonders wichtige Funktion beschreiben wir dabei die absolute CD14-Abhängigkeit DAMP-ausgelöster und TLR4-vermittelter Immunreaktionen. CD14-Defizienz (unter cd14-/--Bedingungen) oder CD14 Blockade (durch Antikörper) löschen mikrogliale Reaktionen, die durch Plasma-Fibronektin (als repräsentatives DAMP-Molekül) ausgelöst werden können, komplett aus und beeinträchtigen die Leukozyten-Infiltration nach ZNS-Trauma. Bei einer ischämischen ZNS-Schädigung weisen cd14-/--Mäuse im Gehirn nicht nur weniger Monozyten auf, sondern gleichzeitig ein vergrößertes Infarktvolumen. Wir konnten für Interferon (IFN) b eine Schlüsselfunktion in der CD14-vermittelten Eindämmung der CXCL1-Synthese darstellen, die auf eine negative CD14/TLR4-TRIF-IFNβ-INAR1-Jak- Rückkopplung für MyD88-getriebene Chemokine schließen lässt. Obwohl CD14 somit TLR4-vermittelte Reaktionen auf infektiöse und nicht-infektiöse Agenzien orchestriert, wird seine Expression durch verschiedene TLR-Liganden und Zytokine reguliert. Letztlich unterliegen damit CD14-kontrollierte Funktionen selbst einer komplexen Kontrolle durch ZNS-residente und eingewanderte periphere Zellen. Diese Regulationen können über die Einbeziehung oder den Ausschluss der Kapazitäten des TLR4-Komplexes für eine Schadenserkennung während der ZNS-Reaktionen in unterschiedlichsten pathologischen Szenarien entscheiden.

Ulm, Univ., Diss., 2002.

A molécula CD14 é uma glicoproteína expressa na membrana de leucócitos e células não mieloides, e pode ser encontrada tanto na membrana plasmática ancorada por glicosilfosfatidilinositol (GPI), como na forma solúvel no espaço extracelular. Esta molécula atua como co-receptor associado a receptores semelhantes a Toll (TLR), dentre eles o TLR4, e auxilia na ativação celular após reconhecimento de padrões moleculares associados a patógenos (PAMPs). Macrófagos ativados por produtos microbianos, como pelo lipopolissacarídeo (LPS), liberam citocinas e óxido nítrico (NO) que participam do processo inflamatório e sinalizam para células vizinhas ou distantes sobre a presença de agente agressor ou alterações teciduais. Após a ativação dos macrófagos, também são produzidos eicosanoides (EICs) que são mediadores lipídicos, como as prostaglandinas (PGs), os tromboxanos (TXs) e os leucotrienos (LTs). Os EICs são derivados em parte dos corpúsculos lipídicos (CLs) que são organelas citoplasmáticas funcionais, dinâmicas, ricas em lipídios e enzimas. Resultados anteriores do nosso grupo já demonstraram o envolvimento da molécula CD14 na produção de mediadores lipídicos, sem, contudo, elucidar os mecanismos. Assim, empregando macrófagos derivados da medula óssea (MDMO) estimulados com alta (500 ng/mL) e baixa (10 ng/mL) concentrações de LPS, tivemos como objetivo investigar a participação do CD14 na formação de CLs e na produção de prostanoides. A relevância da molécula CD14 foi ainda investigada na ativação de MDMO pelo ácido araquidônico exógeno (AA) (estímulo não específico). Nossos resultados demonstraram que a molécula CD14 contribui não somente para a formação de CLs e produção de prostanoides, como PGE2 e TXB2, mas também é essencial para a síntese de mediadores inflamatórios, como NO, TNF-? e IL-10, principalmente quando estimulados com baixa concentração de LPS / The CD14 molecule is a membrane-bound glycoprotein expressed by leukocytes and nonmyeloid cells, which can be attached to the plasma membrane by glycosylphosphatidylinositol (GPI) or in its soluble form in the extracellular space. This molecule acts as a co-receptor associated with Toll-like receptors (TLRs), including TLR4, and assists in cell activation after recognition of molecular patterns associated with pathogens (PAMPs). Macrophages activated by microbial products, such as lipopolysaccharide (LPS), release cytokines and nitric oxide (NO) that participate in the inflammatory process and signal to nearby or distant cells about the presence of an aggressive agent or tissue changes. After activation of macrophages, the lipid mediators denominated eicosanoids, such as prostaglandins (PGs), thromboxanes (TXs) and leukotrienes (LTs) are also produced. Eicosanoids are derived in part from lipid droplets (LDs) which are functional, dynamic cytoplasmic organelles, rich in lipids and enzymes. Previous results from our group have already demonstrated the involvement of the CD14 molecule in the production of lipid mediators, without, however, elucidating the mechanisms. Thus, using bone marrow-derived macrophages (BMDM) stimulated with high (500 ng/mL) and low (10 ng/mL) LPS concentrations, we aimed to investigate the participation of CD14 in the formation of LDs and in the production of prostanoids. The relevance of the CD14 molecule was further investigated by the activation of BMDM by exogenous arachidonic acid (AA) (nonspecific stimulus). Our results demonstrated that the CD14 molecule contributes not only to the formation of CLs and the production of prostanoids, such as PGE2 and TXB2, but it is also essential for the synthesis of inflammatory mediators, such as NO, TNF-? and IL-10, especially when stimulated with low LPS concentration

In einer Vielzahl von Arbeiten konnten anti-inflammatorische Eigenschaften von Ethylpyruvat (EP) aufgezeigt werden. An verschiedenen Modellen der Sepsis, des hämorrhagischen Schocks, von Verbrennungsschäden, des Apoplex oder der Ischämie und Reperfusion wurde bei der Behandlung mit EP ein protektiver Effekt sowie eine verminderte Produktion von pro-inflammatorischen Zytokinen nachgewiesen. Als biochemische Grundlage wurde die Interaktion von EP mit dem Transkriptionsfaktor NF-κB identifiziert, die spezifischen Regulationsmechanismen konnten bisher allerdings nicht zufriedenstellend aufgeklärt werden. In dieser Arbeit wurde als eine neue mögliche Erklärung für die anti-inflammatorischen Eigenschaften des EP und weiterer α-oxo-Karbonsäureester die Inhibierung der Glyoxalase I (Glo-I) aufgezeigt. In vitro-Experimente zur Enzymaktivität belegten die Hemmung der Glo-I durch EP, während α-Hydroxy-Karbonsäureester wie L-Ethyllaktat (EL) keine inhibierenden Eigenschaften aufwiesen. Dennoch waren sowohl EP als auch EL und weitere Laktatester in der Lage, die LPS-induzierte Produktion von pro-inflammatorischen Zytokinen wie IL-1β, IL-6, IL-8 und TNF-α von humanen immunkompetenten Zellen zu supprimieren und die Expression von Immunrezeptoren wie HLA-DR, CD14 und CD91 zu modulieren. Somit konnten erstmals anti-inflammatorische Eigenschaften von Laktatestern nachgewiesen sowie eine Verbindung zwischen den Glyoxalase-Enzymen und dem Immunsystem etabliert werden. Diese und weitere Ergebnisse zur Einflussnahme der Karbonsäureester auf die Zellvitalität präsentieren das Glyoxalasesystem als mögliches Ziel neuer Therapiekonzepte für die Immunsuppression und bestätigen dessen Bedeutung für die Entwicklung von Anti-Tumor-Agenzien.