Tesis sobre el tema "Triggering receptor"

An effective immune response in mammalian cells relies on a network of molecular interactions to detect and respond to pathogens. The T-cell receptor (TCR) is one of the most important components of this system responsible for the outcome of the immunological response. Paramount to its role is its ability to efficiently signal a productive interaction with a peptide embedded in an MHC molecule. Important aspects of TCR structure and organization are unknown, limiting the current understanding of this process and rendering it highly controversial. The work described in this thesis seeks to define the valency, structural organization and signalling properties of the TCR in order to provide a better framework for thinking about the receptor-triggering problem. The results suggest that a largely monovalent complex diffuses at the surface of T cells, which is able to trigger intracellular signalling in the absence of large structural rearrangements of the extracellular subunits of the TCR. Moreover, a recently proposed mechanism involving conformational rearrangements of the cytoplasmic domains of the complex is shown to fail to explain the regulation of TCR phosphorylation. Steps are also taken toward investigating the role of more subtle conformational rearrangements at atomic resolution. Finally, an investigation of what controls tyrosine phosphorylation of the receptor in resting T lymphocytes led to the development of new approaches to address the role of specific phosphatases. The outcome of this analysis suggested how a finely-tuned balance between kinase and phosphatase activity, at both global and local levels, regulates TCR phosphorylation and T-cell activation.

Non-catalytic tyrosine-phosphorylated receptors (NTRs) are a large group of leukocyte receptors that bind to surface-associated ligands and include both activating and inhibitory members. They contain, or associate with adaptor molecules which contain, tyrosine residues within conserved cytoplasmic motifs that are phosphorylated and dephosphorylated by extrinsic kinases and phosphatases respectively. The mechanism by which NTR-ligand engagement leads to sustained phosphorylation of receptor tyrosinebased motifs and initiation of downstream signalling (termed "receptor triggering"), is as yet unknown. Our hypothesis is that the NTRs signal using the kinetic-segregation (KS) model. The model proposes that large inhibitory phosphatases, but not inner leaflet-bound activating kinases, are segregated in a size-dependent manner from engaged receptors upon ligand binding. This favours kinase activity and drives sustained receptor phosphorylation. To systematically test whether predictions of the KS model hold for all NTRs, we have developed an artificial generic ligand system in which biophysical and biochemical properties of NTR-ligand interactions can be manipulated. These include NTR-ligand dimensions, ligand densities and valency. Our system exploits the interaction between a Strep-Tag II, which is attached to the ectodomain of the NTR, and StrepTactin protein. We first demonstrate that representative NTRs, both activating and inhibitory, can be triggered through ligation of this Strep-Tag II. In agreement with a key KS model prediction, we next show that only ligation by surface-associated ligand leads to triggering, whilst ligation by soluble ligand does not. Additionally, by altering ligand valency and mobility, we provide evidence that receptor clustering can enhance activation. Also in agreement with the KS model, we show that elongation of the artificial ligand dramatically affects activation of a representative NTR. Finally, we provide evidence to suggest that increased receptor clustering may be able to compensate for ligand elongation-mediated defects in NTR signalling. The flexibility of this system will allow us to analyse other NTRs, test further predictions of the model, and investigate the role of other properties of NTR signalling.

Au cours du sepsis, l'expression membranaire du Triggering Receptor Expressed on myeloid cells (TREM)-1 ainsi que la production de sa forme soluble (sTREM-1) sont fortement majorées tant chez la souris que chez l'homme. Ces phénomènes dépendent de la présence de ligands bactériens, ne sont pas reliés à la production de TNF-alpha et découlent d'un mécanisme impliquant P13K. STREM atténue la production de cytokines chez la souris. La modulation de la voie de TREM-1 réduit mais sans totalement l'inhiber la production de cytokines et protège l'animal du décès. L'utilisation d'un peptide modulateur pourrait constituer une thérapeutique intéressante.

Base teórica: A meningite bacteriana é uma causa importante de morbidade e mortalidade na infância. Análise do líquido cefalorraquidiano (LCR) continua a ser a ferramenta de diagnóstico padrão ouro, porém novos biomarcadores para o diagnóstico e prognóstico ainda são necessários. Receptor Desencadeador Expresso nas Células Mielóides Tipo 1 (TREM-1) é um receptor transmembrana expresso em neutrófilos e monócitos, que desempenha um papel importante na modulação da resposta inflamatória. A sua fração solúvel (sTREM-1) também é aumentada na infecção, inflamação ou doenças imunológicas. Neste estudo nós avaliamos, prospectivamente, o valor do TREM-1 como um biomarcador de meningite bacteriana aguda em pacientes pediátricos e sua possível utilização como uma ferramenta de prognóstico neste cenário. Objetivos: O objetivo primário do presente estudo é caracterizar os níveis líquóricos solúveis de TREM-1 (sTREM-1) em pacientes admitidos por suspeita clínica de meningite. Analisamos também os níveis de sTREM-1 nos casos de meningite bacteriana e viral, além de medir a sensibilidade e especificidade deste biomarcador no LCR e estudar se esse biomarcador pode ser um fator associado ao prognóstico em meningite bacteriana aguda. Métodos: Sessenta e um pacientes pediátricos, de 0 a 10 anos foram avaliados quanto à meningite e foram prospectivamente incluídos neste estudo. Na admissão, após a suspeita clínica de meningite foram submetidos à análise do LCR para o diagnóstico e uma amostra do LCR inicial foi utilizado também para análise do sTREM-1. Os pacientes foram acompanhados durante a sua internação com o registro de seu tratamento e desfecho clínico para posterior análise dos dados. Resultados: Dentre os 61 pacientes, 38 (62%) foram negativos para a meningite, 7 (11%) pacientes foram diagnosticados com meningite viral e 16 (27%) pacientes foram diagnosticados com meningite bacteriana aguda e recebeu tratamento direcionado. Sexo (p = 0,15), presença de fatores de risco identificados (p = 0,17), presença de convulsões (p = 0,31), outras complicações clínicas (p = 0,11) e mortalidade (p = 0,66) não diferiram entre os grupos. Anormalidades sensoriais (p <0,0001) e presença de cefaléia (p = 0,003) foram mais prevalentes em pacientes com meningite. Como esperado, a contagem de leucócitos, glicose e proteína no LCR foram significativamente diferentes entre pacientes com meningite e pacientes sem meningite. As concentrações de sTREM-1 no LCR de pacientes com meningite bacteriana foi superior quando comparada com pacientes com meningite viral e com controles (1204,67 pg/ml, 39,34 pg/ml e 12,09 pg/ml, respectivamente; p <0,0001). Quando sTREM-1 foi usado como um determinante de diferenciação entre pacientes com ou sem meningite bacteriana, a análise da área sob a curva ROC foi de 0,95 (IC de 95% = 0,89-1,00; p <0,0001). A presença de fatores de risco para a meningite bacteriana (p = 0,04), anormalidades sensoriais (p <0,0001), contagem de leucócitos no LCR (p = 0,01), níveis de glicose no LCR (p = 0,002), níveis de proteína no LCR (p = 0,032) e os níveis de sTREM-1 no LCR (p = 0,004) foram associados com meningite bacteriana, incluindo os níveis sTREM-1 acima do ponto de corte estabelecido de 68,0 pg/ml (p <0,0001). A meningite bacteriana (p = 0,02) e os valores de sTREM-1 maior do que o ponto de corte (68,0 pg/ml) (p = 0,04) foram associados com sequelas neurológicas graves e morte neste grupo de pacientes. Conclusão: Avaliamos os níveis sTREM-1 de crianças com suspeita clínica de meningite. Os níveis de s-TREM-1 foram aumentados nos casos de meningite bacteriana e correlacionados com o prognóstico. Os nossos resultados sugerem que níveis elevados de sTREM-1 no LCR podem ser utilizados como um biomarcador para o diagnóstico de meningite bacteriana aguda em crianças e que pode ser útil na determinação do prognóstico do paciente nesse cenário.
Background: Bacterial meningitis is an important cause of morbidity and mortality in infancy. Cerebrospinal fluid (CSF) analysis remains the gold standard diagnostic tool, however new biomarkers for diagnosis and prognosis are still required. Triggering receptor expressed on myeloid cells-1 (TREM-1) is a transmembrane receptor expressed on neutrophils and monocytes that plays an important role on the immune response. Its soluble fraction (sTREM-1) is also increased in infection, inflammation or immune diseases. In this study we evaluate the value of sTREM-1 as a biomarker of acute bacterial meningitis in pediatric patients and its possible use as a prognostic tool prospectively. Methods: Sixty-one pediatric patients, from 0 to 10 years of age were evaluated for meningitis and were prospectively included in this study. At admission, following clinical hypothesis of meningitis patients were submitted to CSF analysis for diagnosis and a sample of initial CSF was also used for TREM-1 analysis. Patients were followed during hospitalization and clinical evaluation and treatment outcome were recorded for posterior analysis. Results: Thirty-eight (62%) out of 61 patients were negative for meningitis, 7 (11%) patients were diagnosed with viral meningitis and 16 (27%) patients were diagnosed with and received treatment for acute bacterial meningitis. Sex (p = 0.15), presence of identified risk factors (p = 0.17), presence of seizures (p = 0.31), other clinical complications (p = 0.11), and mortality (p = 0.66) did not differ among groups. Sensorial abnormalities (p<0.0001) and presence of headache (p= 0.003) were more prevalent in patients with meningitis. As expected, leukocyte count, glucose, and protein levels were significantly different between patients with meningitis and patients without meningitis. Concentrations of sTREM-1 in CSF from patients with bacterial meningitis was higher when compared to patients with viral meningitis and with controls (1204.67 pg/ml, 39.34 pg/ml and 12.09 pg/ml, respectively; p<0.0001). When sTREM-1 was used as a determinant to differentiate between patients with or without bacterial meningitis, the analysis of the area under the ROC curve (AUC) was 0.95 (95% CI=0.89-1.00; p<0.0001). Presence of risk factors for bacterial meningitis (p = 0.04), sensorial abnormalities (p<0.0001), CSF leukocyte count (p = 0.01), CSF glucose levels (p = 0.002), CSF protein levels (p = 0.032) and CSF sTREM-1 levels (p = 0.004) were all associated with bacterial meningitis, including sTREM-1 levels above the established cut-off point of 68.0 pg/ml (p<0.0001). Bacterial meningitis (p = 0.02) and values of sTREM-1 higher than the cut-off point (68.0 pg/ml) (p = 0.04) were associated with death and severe neurological disabilities in this patient cohort. Conclusion: We evaluated sTREM-1 levels in CSF of children with clinical hypothesis of meningitis. The sTREM-1 levels were increased in bacterial meningitis and correlated with prognosis. Our results suggest that CSF sTREM- 1 levels can be used as a biomarker for diagnosis of acute bacterial meningitis in children and it might be useful in determining patient’s prognosis in this scenario.

Human immunodeficiency virus (HIV) infection has reached epidemic proportions in South Africa. The availability of highly active anti-retroviral therapy (HAART) prolongs life in HIV-infected persons, who may subsequently present with chronic manifestations of HIV-infection. The respiratory morbidity attendant to HIV-infection, even in the presence of HAART is high, the aftermath of which is lung tissue destruction and bronchiectasis. As a consequence of the political decision not to offer HAART to HIV-infected children, a number of children in South Africa have been left with severe consequences of uncontrolled HIV-infection. Bronchiectasis is one of those and because children with this devastating condition were numerous in the Pretoria region, the author and her colleagues began a Chronic Lung Disease Clinic in that region. This prompted the idea of investigating both the epidemiological profiles of these children and an attempt to intervene with both standard bronchiectasis guideline care and the use of a form of therapy commonly employed in other forms of bronchiectasis. This thesis explores those ideas. Important new and novel findings that were consequent were; that bronchiectasis is diagnosed late in HIV-infected children at a mean age of 6.9 years. The predominant organisms cultured from the airways are Haemophilus influenzae and parainfluenzae in 49% of samples. Pseudomonas aeruginosa (PA), common in cystic fibrosis (CF)-bronchiectasis is an uncommon pathogen in HIV-related bronchiectasis; isolated in only 2% of specimens. Tuberculosis (TB), at least as reported, is a significant antecedent of bronchiectasis, reported in 48.5%of children. A further 21.2% of the patients had received more than two courses of anti-TB treatment. However, proof of TB infection has been lacking. Respiratory morbidity is significant with the mean forced expiratory flow in one second (FEV1) of 53%, in this cohort at the time of presentation. Thirty-six percent of all children were exposed to environmental tobacco smoke, although this was not correlated with disease severity or HIVdisease progression. There is elevation of immunoglobulins in HIV-related bronchiectasis, with a mean IgE of 79 kU/l. This was not, though, associated with HIV disease progression as previously described in adult studies, nor with the presence of allergic bronchopulmonary aspergillosis (ABPA). The elevation in IgE was also not associated with an elevation of T helper-2 mediated cytokines, confirming the lack of association with atopy. The predominant cytokine, identified is interleukin (IL)-8, both systemically and locally (in airway secretions). There was elevation of other T helper-1 driven cytokines, reflecting an ability to mediate adequate inflammatory responses, which was independent of the level of immunosuppression. With the presence of HAART, there was a decline in the pro-inflammatory cytokines over time, which may be attributed to the ongoing effect of HAART that ties in to, or goes beyond the restoration of T cell numbers. Soluble triggering receptor expressed on myeloid cells (sTREM), an innate immune marker, is elevated in children with HIV-related bronchiectasis when compared to a control group of children with cystic fibrosis-related bronchiectasis. sTREM is not associated with the presence of exacerbations and the level of immunosuppression. The use of an anti-inflammatory drug erythromycin also did not impact the sTREM values. There was also no relationship between sTREM and pro and antiinflammatory cytokines and chemokines. Fluorine-18-fluorodeoxyglucose positron emission tomography (18F-FDG PET) could not reliably predict the presence of pulmonary exacerbations. Its diagnostic value was limited to identifying disease activity in acute pneumonia. 18F-FDG PET also had no significant correlation with CRP, inflammatory cytokines or markers of HIV disease activity. In a randomised controlled trial of erythromycin, a cost-effective immunomodulatory drug, compared to placebo, erythromycin was ineffective in reducing the number of pulmonary exacerbations. Erythromycin also failed to demonstrate any effect on systemic and local pro- and anti-inflammatory cytokines/chemokines. With access to anti-retroviral therapy, airway clearance, nutritional rehabilitation and vigilant follow up there was an improvement in pulmonary function parameters and stability of the degree of bronchiectasis that we propose is probably in keeping with an organ system disease modifying effect that may be, an as yet, undefined and undescribed byproduct of HAART.
Thesis (PhD)--University of Pretoria, 2012.
Paediatrics and Child Health
unrestricted

Um die Wirkung herpesviral-kodierter FcgammaRezeptoren auf wirtskodierte zelluläre FcgammaRezeptoren und IgG-vermittelten Effektorfunktionen untersuchen zu können, einen methodisch neuen Ansatz wurde entwickelt, der die Detektion FcgammaR-aktivierender Antikörper ermöglicht. Dieses neuartige Assay beinhaltet die Kokultivierung virusinfizierter Zellen, die mit virusspezifischen IgG-Antikörpern opsoniert sind, mit FcgammaR-zeta BW5147-Transfektanten als Reporterzellen. Diese stabilen Transfektanten exprimieren chimäre Rezeptoren, die aus der extrazellulären Domäne der zellulären FcgammaRezeptoren bestehen, welche mit der TM und intrazellulären Domäne der murinen CD3zeta-Kette fusioniert wurden. Die Aktivierung der CD3zeta-Kette führt zu einer IgG-dosisabhängigen mIL-2 Sekretion, die im ELISA gemessen werden kann. Die FcgammaR-spezifische immune IgG könnte eine wichtige biologische Rolle in der antiviralen Immunabwehr spielen. Herpesviren exprimieren auf der Oberfläche infizierter Zellen viral-kodierte Fc-bindende Glykoproteine. Um zu bestimmen, ob virale FcgammaRezeptoren die IgG-abhängige Aktivierung von wirtskodierten FcgammaRezeptoren beeinflussen können, wurde das oben beschriebene Assay angewandt. Es wurde festgestellt, dass der HCMV-kodierte FcgammaR gp68 die Aktivierung und die nachfolgende Signalkaskade von CD16>CD32=CD64 inhibiert, während der HCMV-kodierte FcgammaR gp34 die Aktivierung von CD16>CD64>CD32 inhibiert. In klarem Kontrast dazu wirkt der HSV-kodierte FcgammaR gE, der CD16 Aktivierung vermindert, CD32 hingegen nur sehr schwach und CD64 gar nicht beeinflußt. Der MCMV-kodierte FcgammaR m138/fcr-1 vermindert die Aktivierung des murinenCD16. Zusammenfassend betrachtet zeigen die ermittelten Daten, dass es sich bei den herpesviral-kodierten FcgammaRezeptoren um hierarchische und redundante Antagonisten der wirtskodierten zellulären FcgammaRezeptoren handelt. Herpesviral-kodierte FcgammaRezeptoren wirken somit der Aktivierung des Immunsystems entgegen.
To study the possible interference of the herpesviral vFcgammaRs with the host FcgammaRs and IgG-mediated effector functions, a new methodological approach to detect FcgammaR activating antibodies was developed. The novel assay comprises the co-cultivation of virus infected cells upon opsonization with immune IgG antibodies and the stably transfected FcgammaR-zeta BW5147 transfectants as responder cells. The transfectants express chimeric receptors bearing the extracellular domain of the host FcgammaRs fused to the transmembrane and tail domains of the murine CD3zeta chain. Triggering the CD3zeta chain is sufficient to elicit IL-2 secretion in a dose dependent manner which is measured in an ELISA. The setup of the new assay provides a defined effector cell population bearing one Fcgamma receptor on the surface, which becomes activated in the presence of immune IgG antibodies bound to the native viral antigens displayed on the surface of infected cells. The assay system allows us to detect and quantify Fc gamma receptor-activating immune IgG in an FcgammaR-specific way, which is thought to have an important biological function in antiviral defense. Several alpha- and beta- herpesviruses express on the surface of infected cells virally encoded Fc binding glycoproteins. The assay described above was applied to determine if the viral FcgammaRs are able to impair IgG-mediated activation of host FcgammaRs. In a systematic approach, the effect on each host FcgammaR by each of the herpesviral FcgammaR was investigated. It was found that HCMV FcgammaR gp68 affects activation and downstream signaling of CD16 > CD32 = CD64, while gp34 attenuates CD16 > CD64 > CD32. In clear contrast, HSV gE impairs CD16 activation and weakly CD32, but has no effect on CD64. Furthemore, MCMV m138/fcr-1 diminishes activation of mouse CD16. Taken together, this data uncover herpesviral FcgammaRs as hierarchical and redundant antagonists precluding host FcgammaRs from triggering immune responses.

博士
國立陽明大學
分子醫學博士學位學程
105
Current understanding of T cell receptor (TCR) triggering is based on the segregation of membrane tyrosine phosphatases having large ectodomains upon engagement between T cells and antigen presenting cells, thereby creating a kinases/phosphatases imbalance to phosphorylate immuno-receptor tyrosine-based activation motifs (ITAMs) in CD3 proteins associated with engaged TCRs. We recently observed that elongated OKT3 anti-CD3 single-chain antibodies (scFv) expressed on 3T3 fibroblasts can effectively trigger TCR signaling, inconsistent with a requirement for CD45 segregation from engaged TCRs. Here, we show that TCR triggering by elongated ligands depends on affinity rather than dimension, and CD45 segregation is not a mandatory step to initiate TCR triggering. We created elongated scFv with defined dimensions expressed on the surface of 3T3 APCs and or distributed over planar lipid bilayers to track CD45 movement by TIRF microscopy. We found that elongated scFv (OKT3-CD43) was unable to segregate CD45 from the ligated TCR microclusters, but inconsistent with the KS model, was able to induce T cell calcium flux, and Zap70 phosphorylation. However, within minutes of triggering, CD45 started to segregate partially from the TCR microclusters or pZap70, indicating that CD45 segregation follows rather than initiates TCR triggering. Using a mutated OKT3 scFv that showed low affinity to CD3ε (OKT3MA), we proved that TCR triggering is more dependent on ligand affinity rather than the ligand dimensions and CD45 physical segregation; short, low affinity scFv (OKT3MA-BGP) was able to trigger TCR similarly to wild type scFv (OKT3-BGP), while elongated low affinity OKT3MA scFv (OKT3MA-CD43) was unable to trigger TCR, nor segregate CD45. Longer follow up of T cells showed proliferation, IL-2 and IFN-γ secretion by the short ligands (high and low affinities) while only high affinity elongated ligand was able to produce productive T cell activation.

I TREM (Triggering Receptor Expressed on Myeloid Cells) sono una famiglia di recettori di membrana con funzione attivatoria e inibitoria, in base alla capacità di associare a DAP12 una proteina adattatrice o alla presenza di un dominio citoplasmatico contenente motivi ITIM (Immunoreceptor tyrosine-based inhibitory motif). I TREM sono espressi da granulociti, monociti, macrofagi, cellule dendritiche, microglia, osteoclasti e piastrine, cellule di origine mieloide coinvolte sia nella risposta immunitaria innata che nell’omeostasi dell’ospite. Identificati come recettori che “triggerano” l’attivazione cellulare, appare oggi chiaro un ruolo modulatorio nella attivazione e differenziazione delle cellule mieloidi. Il TREM-1 funziona da amplificatore della risposta infiammatoria: principalmente espresso da neutrofili e monociti/macrofagi, la sua espressione è upregolata da stimoli microbici. L’ingaggio del TREM-1 con anticorpi attivatori di per se non ha effetti sulla cellula mentre sinergizza con l’attivazione cellulare indotta da “Pattern Recognition Receptors” (PRR)s, quali i Toll-like Receptors e i recettori della famiglia dei NOD. Tale sinergia si esplica in termini di una generale attivazione delle funzioni cellulari, quali la produzione di citochine e chemochine proinfiammatorie e l’attivazione della fagocitosi. A livello intracellulare, TREM-1 attiva il pathway canonico mediato dal motivo ITAM (Immunoreceptor tyrosine based activation motif) contenuto nel dominio citoplasmatico di DAP12. In vivo è stato dimostrato che una forma solubile di TREM-1, agendo da “decoy receptor” ha un ruolo protettivo in modelli animali di infiammazione acuta, quali modelli di endotossemia e di peritonite polimicrobica. Per il TREM-2 sono invece state descritte funzioni diverse, associate anche ad una diversa espressione nei macrofagi, in cellule dendritiche, in osteoclasti e in cellule di microglia. In tali cellule il TREM-2 inibisce la produzione di citochine proinfiammatorie e ne potenzia la fagocitosi, mediando una attivazione alternativa di DAP12 che conduce a effetti anti infiammatori. TREM-2 riveste un ruolo fondamentale nel processo di differenziamento degli osteoclasti sia in vitro che in vivo. Ad oggi non sono stati identificati ligandi per alcun membro della famiglia dei TREM. Con l’utilizzo di una forma solubile del recettore abbiamo identificato alla superficie di neutrofili murini attivati un potenziale ligando di TREM-1. Dopo aver clonato diversi geni selettivamente espressi da questa popolazione cellulare rispetto ad una popolazione di neutrofili negativa per la presenza del ligando, abbiamo constatato che nessuno dei prodotti proteici codificati da questi geni interagisce con TREM-1, lasciando ancora aperta la questione della natura di questo potenziale ligando. Dopo aver invece identificato un potenziale ligando per il TREM-2 espresso alla superficie di diverse linee tumorali, con una approccio biochimico abbiamo isolato da queste cellule un interattore di TREM-2, galectin-1, una lectina specifica per residui di β-lattosio. Abbiamo confermato l’interazione con studi di immunoprecipitazione e di in vitro “binding assay” con Gal-1 immobilizzata su piastra. Inoltre abbiamo dimostrato la specificità dell’interazione tra TREM-2 e Gal- 1 dal momento che i) anticorpi anti Gal-1 e ii) una proteina ricombinante Gal-1 inibiscono il l’interazione di una forma solubile di TREM-2 (TREM-2/Ig) alle cellule tumorali e iii) Gal-1 non interagisce con altri membri della famiglia dei TREM come dimostrato nel saggio in vitro. Nonostante Gal-1 sia una lectina, abbiamo osservato che l’interazione con TREM-2 è mediata da riconoscimenti proteina-proteina. A validare una ruolo funzionale dell’interazione, abbiamo dimostrato che in un modello di osteoclastogenesi in vitro, Gal-1 incrementa notevolmente la formazione di cellule multinucleate positive alla colorazione al TRAP, incrementando inoltre il rapporto nuclei/cellula. Monociti da pazienti affetti dalla sindrome di Nasu-Hakola, difettivi nell’espressione di TREM-2, non sono in grado di differenziare in vitro in osteoclasti maturi e questo difetto di differenziamento non è recuperato dal trattamento con Gal- 1. Questo evidenzia un ruolo della galectin-1, un nuovo interattore di TREM-2, nel processo dell’osteoclastogenesi dipendente da TREM-2.
TREM (triggering receptor expressed on myeloid cell) proteins are a family of cell receptors expressed on myeloid cells that include both activating and inhibiting receptors, based on their ability to interact with the adaptor protein DAP12 or on the presence of a cytoplasmic domain containing ITIM motifs. TREMs are expressed on cells of myeloid origin that function both in the immune system and in remodelling host tissue: these receptors are expressed on granulocytes, monocytes, macrophages, microglia, dendritic cells, osteoclasts, and platelets. Identified as receptors that ‘trigger’ cellular activation, it is now clear that they are modulators of the cellular response, having both positive and negative functions in regulating the activation and differentiation of myeloid cells. They have been proposed to fall into the group of receptors that set signaling “threshold” for cells. TREM-1 functions as an amplifier of inflammation: it is mainly expressed in neutrophils and monocytes/macrophages, in which microbial stimuli strongly upregulate its expression. TREM-1 triggering with activating antibodies does not results in activation by itself, but synergizes with cellular activation induced by Pattern Recognition Receptor, such as TLRs and NOD receptors, in terms of production of inflammatory cytokines and chemokines and induction of phagocytosis and cellular effector function. Intracellularly, TREM-1 engagement activates the canonical activation cascade of protein kinases described for the ITAM containing adaptor DAP12. In vivo, the soluble form of the receptor, potentially acting as a decoy, has inhibiting functions in acute inflammation models, such as endotoxemia and Cecal Ligation and Puncture (CLP). Multiple functions have been described for TREM-2. TREM-2 is expressed broadly by mononuclear phagocytes, including macrophages, microglia and osteoclast precursors. In these cells TREM-2 inhibits TLR-induced cytokine production and potentiates phagocytosis and mediates an alternative activation of DAP12 signaling that leads to anti-inflammatory effects. TREM-2 is fundamental for proper osteoclast differentiation both in vitro and in vivo. So far, no ligand has been identified for any of the members of the family. Using a soluble form of TREM receptors we identified activated neutrophils as TREM-1 ligand expressing cells. We cloned several genes that were selectively expressed by TREM-1L+ neutrophils, but none of the proteins encoded by those genes interacts with TREM-1, leaving an open question on the nature of TREM-1L. Conversely, upon the identification of a candidate ligand for TREM-2 expressed at cell surface of several tumor cell lines, we have isolated a TREM-2 binding protein, galectin-1, a β-lactose lectin, using a biochemical approach. The interaction was confirmed by immunoprecipitation studies and in vitro assay with immobilized Gal-1. TREM-2 binding with GAL-1 is specific since: i) anti Gal-1 antibodies and ii) a recombinant Gal-1 inhibit binding of a soluble form of TREM-2 (TREM-2/Ig) to ligand positive cells; iii) Gal-1 does not interact with other TREM family members as demonstrated by the in vitro binding assay. We also found that Gal-1 interacts with TREM-2 in a protein-protein manner, not involving its carbohydrate recognition domain. In order to validate a functional role of this interaction, we demonstrated that in an in vitro osteoclastogenesis model recombinant Gal-1 enhanced TRAP+ multinucleated cell formation and increases nuclei/cells ratio. Monocytes from TREM-2 deficient patients are unable to differentiate in vitro into mature osteoclasts and addition of the Gal-1 did not rescue the differentiation. This shows the role of the newly identified TREM-2 ligand, galectin-1, in the process of TREM-2-dependent osteoclastogenesis.

碩士
國立陽明大學
生化暨分子生物研究所
99
Inflammation is a well-established risk factor for the development of hepatocellular carcinoma (HCC). Microbiol infections, such as hepatitis B and C viruses, link inflammation to liver tumorigenesis. Toll-like receptors (TLRs) expressed on immune cells have emerged as sensors of pathogen pattern recognition molecules that can detect not only a variety of invading pathogens and microbes, but also endogenous ligands from danger signaling. Based on the roles of TLRs in host defense, the agonists of TLRs have been used as efficient adjuvants in anticancer therapy. However, TLRs are also found to be expressed on various tumor cells, and evidences have suggested that TLRs are able to trigger survival signaling. Here we sought to investigate the mechanism of TLR4-mediated survival signaling in liver cancer. In our results, liver cancer cell lines express different level of TLR4. LPS treatment increased proliferation of Huh7 cells that express higher TLR4, but not in PLC/PRF5 cells with lower TLR4. Additionally, Akt and JNK phosphorylation, Cyclin D1 induction, and I?羠-? degradation were detected following LPS treatment in time- and dose- dependent manner. We also found HCC with LPS pretreatment can resistant to anti-tumor drug. Treatment with low-dose LPS once a week shows a significant promoting effect in Huh7 tumor xenografts model. Briefly, TLR4 expressed on liver cancer acts as a helper, which can provide advantage in growth and survival. Better understanding of these signals and pathways will help us to develop novel therapeutic approaches.

碩士
國立陽明大學
醫學生物技術暨檢驗學系
100
Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease with involvement of multiple organs and is characterized by high level of anti-double-stranded DNA (anti-dsDNA) antibodies in sera. The titers of anti-dsDNA antibodies correlate with disease activity. It is also indicated that bacterial infections are associated with more severe morbidity and higher death rate in lupus. However, the mechanisms of anti-dsDNA antibodies and infections in disease development are still unclear. In the immune system, toll-like receptor (TLR) family and triggering receptor expressed on myeloid cell (TREM) recognize different pathogen structures and activate immune cells to destroy invading pathogens. Recently, TLR9 shows promoting or protective roles in different murine lupus models, but the role of TREM-3 remains unclear in the world. In this study, I investigated the role of TREM-3, and TLR9 in lupus by using 9D7 anti-dsDNA antibody transgenic mice. Although there was no lupus syndrome in TREM-3 knockout (TR3-/-) mice, TR3-/- and 9D7-TR3-/- splenocytes secreted higher concentrations of IL-6 and IL-10 upon in vitro stimulation with TLR ligands (LPS, imiquimod, CpG 1826). CpG 1826 (TLR9 ligand) stimulation induced even significantly higher concentration of IL-10 in 9D7-TR3-/- splenocytes. CpG 2216 (TLR9 ligand) stimulation induced lower concentration of IFN- in TR3-/- and 9D7-TR3-/- splenocytes. I next investigate whether macrophages and B cells interact with each other to secrete more cytokines upon CpG 1826 stimulation since TREM-3 is reported to express on macrophages and B cells secrete 9D7 single chain Ab. Comparing to normal macrophages, TR3-/- macrophages secreted higher concentration of IL-6, IL-12 and IL-10. Co-culture of TR3-/- macropahges with B cells promoted more IL-6, IL-12 and IL-10 secretion. These data indicate that TREM-3 might attenuate TLR signaling and modulate inflammation in immune cells. Soluble form of TREM (sTREM) has been identified in some autoimmune diseases and might have correlation with disease activity. The roles of TREMs in lupus have not been investigated. Therefore, I evaluated sTREM in sera of lupus patients and healthy people. I found that sTREM-2 with elevated levels in lupus patients and showed positive correlation with titers of anti-dsDNA antibodies. The data suggest that TREM involves in progression of SLE.

碩士
國立中興大學
生命科學院碩士在職專班
94
Community-acquired pneumonia (CAP) is a common illness with an incidence rate of approximately 4 to 12 per 1000 adults per year. Because the incidence of CAP increases with age, and with the currently aging population , CAP remains as an important public health problem. Early and appropriate treatment of CAP patients is therefore becoming one of the most important factors to reduce morbidity and mortality. The triggering receptor expressed on myeloid cells (TREM-1) is a member of immunoglobulin superfamily, and its expression on phagocytes is specifically up-regulated by microbial products. The presence of soluble TREM-1 (sTREM-1) in bronchoalveolar-lavage fluid from patients receiving mechanical ventilation may be an indicator of pneumonia. sTREM-1 has been reported as the strongest independent predictor of pneumonia. However, the level of sTREM-1 in plasma and pleural effusion to predict the treatment response is yet to be defined. The aim of this study was to investigate predictive value of PSI score and sTREM-1 on day 1 and day 3 following the clinical treatment. The study would evaluate the cause of treatment failure and the perspective indicator(s), which could help a clinical physician to determine whether to keep on or to discontinue antibiotic treatment and to reduce medical cost. The study was carried out from October, 2004 to June, 2005. Study population included: (1) serum group: patients who had lower respiratory tract infections; (2) pleural effusion group: CAP, TB and lung cancer patients who had recently developed pleural effusion. All patients were treated in the general medical wards. Serum and pleural effusion were centrifuged and the supernatant was frozen at -70C until assay. Duo Set ELISA Development kit (R&D) was used to assay sTREM-1 level in those samples. Following statistical analysis , a significant difference in sTREM-1 level was detected between response and non-response group patients. Also ,a significant difference was found among sTREM-1 levels in normal control, CAP (response and non-response), TB and sever CAP patients (p＜0.001). Moreover, level of sTREM-1 in pleural effusion was higher than that of serum. In particular, in lung cancer patients sTREM-1 level was much higher than that in CAP and TB groups. Since most of pleural effusions was non-inflammatory in lung cancer patients. Therefore, we suspected that lung cancer cell might contain factor(s), which could trigger sTREM-1 Expression in addition to microbial infection, and sTREM-1 may play a different role in disease progression of lung cancer.

Indiana University-Purdue University Indianapolis (IUPUI)
The importance of microglia in neurodegeneration has been highlighted by recent identification of microglial genes associated with increased risk for dementia. Among these, several variants of ‘Triggering Receptor expressed on myeloid cells 2’ (TREM2), confer higher risk for different types of dementia including Alzheimer’s disease and frontotemporal-like dementia with early onset. The mechanism by which alterations in TREM2 predisposes individuals to early dementia and how TREM2 influences proteinopathies, especially tauopathy remains unclear. The first part of this thesis focused on the role of TREM2 in neuronal homeostasis using a novel Trem2 p.Y38C mouse model (Trem2Y38C/Y38C) and mice lacking Trem2 (Trem2-/-). Young adult Trem2Y38C/Y38C and Trem2-/- mice exhibited synaptic impairments with reduced long-term potentiation accompanied by oligodendrocyte/myelin impairments. These pathologies are reminiscent of the clinical manifestation in patients with TREM2 p.Y38C mutation and functional loss of TREM2. Since these alterations were detected in wildtype Trem2Y38C/Y38C and Trem2-/- mice in the absence of any pathological insults, these results demonstrate that TREM2 directly impacts neuronal functions and homeostasis independent of the triggers such as pathological tau. In the second part of the thesis, we addressed the role of TREM2 in tau pathogenesis using aforementioned Trem2Y38C/Y38C and Trem2-/- mouse models crossed to human wildtype tau expressing ‘htau’ mice. Loss of functional TREM2 does not alter the overall phosphorylated tau burden but shifts localization of tau to the interstitial fluid in a tau species and sex-dependent manner. Female htau mice lacking functional TREM2 showed lower insoluble tau (largely intracellular) but higher tau levels in the interstitial fluid (extracellular). Transcriptomic analysis reveal alterations in genes associated with neuroinflammation and microglial phagocytic pathways in htau;Trem2Y38C/Y38C and htau;Trem2-/- mice . These alterations likely suggest compromised uptake and/or clearance of extracellular tau leading to the accumulation of tau in the ISF, which has been shown to be detrimental to the synapses. These results demonstrate that TREM2 is important for microglial, neuronal, and white matter functions and provides unique insights on the aspects of tau pathogenesis impacted by TREM2.

碩士
國立陽明大學
急重症醫學研究所
98
Objectives：To investigate the clinical utility of inflammatory marker triggering receptor expressed on myeloid cells (TREM)-1 at admission for differentiating between typical and atypical bacterial community-acquired pneumonia (CAP). Methods：A prospective, non-interventional study of patients with CAP hospitalized through the emergency department was performed. Surface expression of TREM-1 was analyzed using flow cytometry on peripheral blood cells and soluble TREM-1 (sTREM-1) concentration was determined in plasma. Results：88 patients with clinical suspicion of CAP were eligible. The causative pathogen was identified in 39 patients (44.3%). After excluding four mixed pneumonia, 21 typical and 14 atypical bacterial infections were enrolled. Patients with typical bacterial CAP demonstrated increased TREM-1 surface expression on monocytes and neutrophils. Median plasma sTREM-1 levels at admission were 65.2 pg/mL (range, 17.6 to 138.1 pg/ml) in patients with typical CAP and 25.9 pg/mL (range, 11.5 to 54.8 pg/mL) in patients with atypical CAP (P &lt; 0.001). sTREM-1 had good discriminative value to differentiate typical from atypical pathogens with an area under the receiver operating characteristic curve of 0.87 (95% confidence interval, 0.75 - 0.98). At a cutoff levels of 44.2 pg/mL, sTREM-1 yielded a sensitivity of 81%, a specificity of 79%, a positive likelihood ratio of 3.79, and a negative likelihood ratio of 0.24. Conclusions：In newly admitted patients with CAP, determination of the TREM-1 levels may provide useful additional diagnostic information on the bacterial etiology. Key Words：atypical pneumonia; biological marker; community-acquired pneumonia; diagnosis; triggering receptors expressed on myeloid cells (TREM)-1.

碩士
國立陽明大學
微生物及免疫學研究所
105
Atherosclerosis is a chronic inflammatory disorder and have been showed involved in many disease. Recent studies have shown that foam cells play an important role in the development of atherosclerosis. The foam cells are associated with macrophages and the oxidized low-density lipoprotein (ox-LDL). Our lab had shown that triggering receptor expressed on myeloid cells type II (TREM-2) was a crucial receptor for foam cell formation. These unpublished data suggested that the formation of foam cells was reduced in ox-LDL treated macrophages lacking TREM-2. Otherwise, CD36 have been also reported to involve in foam cells formation when the macrophage uptake ox-LDL. Interestingly, our preliminary result suggested that losing TREM-2 induced a lower level gene expression of CD36, but not SA-A. We first investigated the surface CD36 level. Our data implicated that TREM-2 deficiency led to a low level of CD36 expression in macrophage. We then analyzed the PPAR-γ and found that TREM-2 depleted BMDM express a lower level of PPAR-γ protein, but not mRNA level. We further confirmed that a higher PPAR-γ protein expression in the WT BMDM compared to TREM-2 depleted BMDM after treat with the serum or ox-LDL. We also used anti-CD36 antibody to block the binding between CD36 and ox-LDL. These data suggested that TREM-2 involved in the CD36 mediated PPAR-γ signals pathway. Since some papers suggested that phosphorylated ERK involved in the PPAR-γ activity because the PPAR-γ may act as a substrate for ERK activity, we at last confirmed that a lower level of phosphorylation ERK in the WT BMDM compared to TREM-2 depleted BMDM after treat with the serum or ox-LDL. We will use the U0126 to dissect the recover assay. In conclusion, we showed that losing TREM-2 may led CD36 downregulation, and these may due to a lower PPAR-γ expression since TREM-2 depleted BMDM have a higher phosphorylation ERK.

碩士
國立陽明大學
微生物及免疫學研究所
103
Inflammatory bowel disease (IBD) is a chronic inflammatory disorder that mainly affects the gastrointestinal tract. The major types in IBD are Crohn’s disease (CD) and ulcerative colitis (UC). Previous studies showed that triggering receptor expressed on myeloid cells (TREM)-1, a surface pattern recognition receptor (PRR), was up-regulated on inflamed intestinal macrophages, and the levels of soluble TREM-1 inpatient sera were positively correlated with the disease activity of IBD patients. Interestingly, we revealed that the TREM-1 KO mice were not protected from DSS-induced colitis. Oppositely, mice with TREM-1 deficiency showed a great loss of body weight, increased lethality and increased IL-13 gene expression after DSS treatment. In the present study, we further examined whether IL-13 contributes to the augment of DSS-induced colitis in TREM-1 KO mice, and characterized the IL-13 producing cell in the inflamed colon of DSS-induced colitis. We showed that IL-13 was up-regulated in DSS-treated TREM-1 KO mice. And the IL-13 was mainly produced by innate lymphoid cells-2 (ILC2s). However, neutralizing IL-13 with a recombinant soluble mIL13Rα2.Fc fusion protein could not suppress DSS-induced intestinal inflammation and intestinal damage in TREM-1 KO mice. In summary, we verified that IL-13 producing ILC2s were up-regulated in DSS-treated TREM-1 KO colons. However, whether IL-13 plays a crucial role in DSS-induced colitis remains unclear. More experiments are needed to characterize the role of IL-13 in experimental IBD in TREM-1 KO mice.

碩士
國立陽明大學
生理學研究所
100
According to the United States Renal Data System (USRDS), the prevalence of end stage renal disease (ESRD) is the highest in Taiwan in 2009. Accumulation of leukocytes, particularly macrophages, lymphocytes and neutrophils, in the tubulointerstitium plays a pivotal role in the development of chronic renal inflammatory disease and causes structural and functional injury. It has been reported that triggering receptor expressed on myeloid cells-2 (TREM-2) is a negative immune regulator of macrophage transition in wound healing. We found TREM-2 was upregulated in renal tissues of unilateral ureteral obstruction (UUO) mice. We further conducted a murine UUO model in TREM-2 deficiency (TREM-2-/-) mice to assess the pathologic role of TREM-2 in kidney injury. Our data showed that TREM-2-/- mice displayed significantly more tubular injury and interstitial fibrosis than wild-type (WT) mice did 14 days after UUO. F4/80+ cell infiltration was comparable between WT and TREM-2-/- UUO mice. The expression of inducible nitric oxide synthase and IL-6, makers of M1 macrophage, were significantly elevated in TREM2-/- UUO mice, but other markers of M1 and M2 macrophages showed inconsistent results. Intriguingly, a significant increase of neutrophil infiltration was observed in TREM2-/- mice 14 days after UUO. As our expectation, the tubular injury was significantly ameliorated after neutrophil depletion in TREM-2-/- in 14 days of UUO, indicating that neutrophils play an important role in late stage of obstruction nephropathy when lost TREM-2. Correspondingly, the mRNA expression of interleukin-17 (IL-17), a widely recognized inflammatory cytokine for neutrophil recruitment, was upregulated in TREM2-/- UUO mice. To investigate whether TREM-2 and UUO tissue lysate mediate the differentiation of naïve lymphocytes into IL-17-producing T lymphocyte, we co-cultured WT and TREM-2-/- bone marrow-derived dendritic cells (BMDCs) with CD4+ T cells respectively. Current result showed that IL-17 expression was upregulated in the TREM-2-/- group after stimulated with UUO tissue lysate. To further investigate the role of TREM-2 in Th17 activation, we cultured BMDCs alone with administration of UUO tissue lysate but the IL-17 related cytokine, IL-6 and IL-23 showed no significant difference between WT and TREM-2-/- group. Taken collectively, although TREM-2 doesn’t involved in mediating IL-17 related cytokines release from BMDCs, TREM-2 does play a role in downregulation of Th17 development and IL-17-mediated neutrophil infiltration, therefore attenuates the tubulointerstitium injury in UUO WT mice.

碩士
國立陽明大學
微生物及免疫學研究所
102
Chronic kidney disease (CKD) has been one of the leading cause of death in Taiwan with the complications such as cardiovascular disease, hyperlipidemia, anemia and metabolic bone disease. Understanding the regulatory mechanism involved in renal inflammation remains urgent for drug and therapeutic development. Unilateral ureteral obstruction (UUO) model is one of the recent developed nephritis models which mimic the inflammatory condition and renal pathology in CKD with accelerated pathologies on renal fibrosis and tubular atrophy. Previously we have revealed that the expression of triggering receptor expressed on myeloid cells (TREM)-2, a surface pattern recognition receptor (PRR), was up-regulated in affected kidneys and the pathology was more severe in TREM-2 deficient mice than that in wild type mice following UUO, suggested that TREM-2 might play a protective role during renal inflammation. Neutrophil increment in the UUO kidneys was evidenced by immunohistochemical staining, and was proved to contribute to the renal injury in TREM-2 deficient mice. In the present study, we further obtained the increment of Th17 and the up-regulation of related cytokines which might contribute to the augment of neutrophil recruitment and enhanced renal pathology in UUO-treated TREM-2 deficient mice. Due to the expression preference of TREM-2 on dendritic cells (DC) surface, we hypothesized that activation and recruitment of DC in the inflamed kidneys, which lead to Th17-neutrophil path activation, are under the regulation of TREM-2. Accordingly, adoptive transferring of TREM-2 deficient DCs, compared to WT ones, into UUO-treated WT mice significantly exacerbated the IL-17 secretion from CD4(+) T cells obtained in the obstructive kidneys, further verified the pivotal role of DC-expressed TREM-2 on modulating Th17 response during the renal inflammation.

碩士
國立陽明大學
生理學研究所
103
Renal fibrosis is the common pathologic finding of end-stage renal disease (ESRD) and is characterized by interstitial extracellular matrix (ECM) and myofibroblast accumulation. Unilateral ureteral obstruction (UUO) is a widely used renal fibrosis model which is characterized by infiltrating macrophages and the different stages of obstructed nephropathy. Previously, we reported that triggering receptor expressed on myeloid cells 1 (TREM-1), an important inflammatory regulator, is up-regulated in the affected renal tissues of UUO mice. TREM-1 is critical for regulating M1 macrophage polarization upon UUO. Depleting TREM-1, accompanied with enhanced macrophage M2 polarization, ameliorates the UUO renal pathology. MicroRNAs are short, endogenous, single-stranded RNA molecules that can silence gene expression via the specific binding to the complementary sequences inside target genes. Whether TREM-1 could regulate miRNA involved in UUO remains unknown. We performed microarray analysis to identify the differences in miRNA profiles between WT and Trem1-/- kidneys with UUO-induced nephritis. Among the identified candidates, miR-21 was significantly up-regulated upon UUO in WT kidneys in comparison with sham process ones. TREM-1 deficiency further enhanced the expression level of miR-21, accordingly reduces the expression levels of miR-21 targets such as PDCD4, PTEN, and IL-12a in the Trem1-/- UUO kidneys. Surprisingly, depleting TREM-1, instead of affecting the expressions of miR-21 and its targets in macrophages directly, significantly promoted the production of IL-10 (a central inhibitory cytokine which can limits the immune responses) in the cultured bone marrow-derived macrophages (BMDM) via modulating the CRTC3 nuclear translocation. IL-10 could synergistically promote the pro-fibrinogenic cytokine TGFb1 production from TNF-a-treated macrophages and renal tubular NRK-52E cells, and consequently enhance the miR-21 expression in the stressed renal tubular cells. Furthermore, overexpressing miR-21 in the tubular NRK-52E cells, instead of down-regulation of inflammation and cell apoptosis as previous reports, could sensitize tubule cells to H2O2-mediated cell death. Moreover, TREM-1 depletion significantly reduced the induction of iNOS and also ameliorated the H2O2-induceded ROS generation by macrophages, subsequently leading to the reduction in ROS-dependent apoptosis of renal tubular cells upon UUO nephritis. In the present study, we revealed that TREM-1 could indirectly regulate the level of miR-21 through IL-10. We also found that TREM-1 and miR-21 may synergistically enhance the ROS-mediated tubular cell death, resulting in the exacerbation of UUO nephritis.

碩士
高雄醫學大學
醫學研究所碩士班
95
英文摘要 Background: Early diagnosis of serious bacterial infection (SBI) in young infants is a difficult problem by using clinical symptoms and signs. The goal of this study is to evaluate to diagnostic value of newly discovered inflammatory mediators: soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) or CXC chemokine IP-10 level for early diagnosis of SBI in infants younger than 4 months of age. Methods: We enrolled pediatric patients who were less than 4 months of age with a suspicion to have SBI and admitted in neonatal intensive care unit or complete nursing unit of pediatric department of Kaohsiung Medical University hospital. Peripheral blood was drawn for measurement of complete blood count, CRP, sTREM-1 or IP-10 levels at admission. Positive blood, CSF, or urine culture was considered to have SBI. Soluble TREM-1 and IP-10 were detected by commercial ELISA kits. Results: There were 118 patients to have sTREM-1 measurement. The SBI group (n=39) have higher plasma sTREM-1 level than non-SBI group (n=79) (299.8±555.4 v.s. 15.4±19.7，p=0.003 after adjusting age by ANCOVA analysis). Plasma sTREM-1 level higher than 55.2 ng/mL was more accurate than WBC count, absolute neutrophils counts, IT ratio, and CRP for indicating SBI in infants.[sensitivity 64.1% (95% CI, 55%-73%); specificity 97% (95% CI, 94%-100%); positive likelihood ratio 21.3; negative likelihood ratio 0.37; diagnostic odds ratio 57.5]。Sixty patients were collected to have measurement of IP-10. Plasma IP-10 level had significantly increase in SBI group [320.1±497.9 v.s. 11.6±23.7, p=0.016, after adjusting age by ANCOVA analysis] 。Plasma IP-10 level higher than 48.2 ng/mL had best diagnostic accuracy for indicating SBI. [sensitivity 81% (95% CI, 71%-90%); specificity 95% (95% CI 89%-100%); positive likelihood ratio 15.9，negative likelihood ratio 0.2; diagnostic odds ratio 79.3]。 Conclusion: In infants who were less than 4 months old, plasma sTREM-1 or IP-10 level might play a potential role in early identification of serious bacterial infection.

碩士
國立陽明大學
醫學生物技術暨檢驗學系
102
Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease. Patient’s immune system attacks his own cells and tissues and results in inflammation and tissue damage. Patients are characterized by antinuclear antibodies (ANAs) in serum, especially anti-double-strand DNA antibodies (anti-dsDNA). TREM-1 (Triggering Receptors Expressed on Myeloid cells-1) has been demonstrated to synergize with TLR (Toll-Like Receptors) in amplifying the inflammatory response and play an important role in many bacterial infectious diseases and aseptic inflammatory diseases. TREM-1 blockade had been shown to reduce the inflammation in septic mice and mice with collagen-induced arthritis (CIA). Furthermore, levels of soluble form of TREM-1 (sTREM-1) were found elevated in sera of SLE patients. In our preliminary study, TREM-1 expression has increased significantly in spleen of lupus mice (B6.Lpr) and also has a positive correlation with lupus symptoms, suggesting that TREM-1 may be involved in the progression of SLE. In this study, we established TREM-1-/--Lpr mice by breeding B6.Lpr mice with TREM-1-/- mice to investigate the role of TREM-1 in SLE. First, we found the survival rate of TREM-1-/--Lpr mice were significantly lower than control groups. Lupus symptoms of TREM-1-/--Lpr mice became more severe nearly at 42-weeks including spleen weight, lymph node weight, proteinuria level, BUN levels, anti-dsDNA and anti-Sm antibodies titers. WBC and lymphocytes of TREM-1-/--Lpr mice was significantly increased at 24-weeks, but was compensable at the age of 42-weeks. Peripheral immature T cell (CD3+CD4-CD8-T cell) of TREM-1-/--Lpr mice were greatly increased compared to B6.Lpr mice at 42-weeks. Furthermore, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), plasma cells, T cells, B cells, immature T cells and immature B cells (T1 B) were increased enormously in spleen and lymph node of TREM-1-/--Lpr mice at 42-weeks. In addition, concentrations of BAFF (B-cell activating factor), IFN-, IL-2, IFN-, TNF, IL-4, IL-6, IL-10 and IL-17 were elevated in serum of TREM-1-/--Lpr mice. These data indicated that TREM-1-/--Lpr mice represented more severe clinical symptoms of SLE. Immature T and immature B cells of TREM-1-/--Lpr mice were increased significantly in peripheral blood, spleen and lymph node, suggesting that TREM-1 may modulate the development of autoreactive lymphocytes in SLE.

碩士
國立陽明大學
熱帶醫學研究所
93
Host innate responses to bacterial infections are primarily mediated by neutrophils, monocytes and macrophages. Stimulation of these cells via signaling pathway initiates secretion of pro-inflammatory mediators, which promote the elimination of infectious agents and the induction of tissue repair. However, excessive inflammation owing to bacterial infections can lead to tissue damage and septic shock. Inflammatory responses to microbial products are amplified by a pathway mediated by triggering receptor expressed on myeloid cells (TREM)-1. TREM-1 is an activating receptor expressed at high levels on neutrophils and monocytes/macrophages that infiltrate human tissues infected with bacteria. Furthermore, it is upregulated on peritoneal neutrophils of patients with microbial sepsis and mice with experimental lipopolysaccaride (LPS)-induced shock. These results indicated a critical function of TREM-1 in acute inflammatory responses to bacteria and implicated TREM-1 as a potential therapeutic target for septic shock. In this study, we investigated the expressions of mTrem-1 and interrelated cytokine mRNA gene from splenocytes of mice infected with Schistosoma mansoni at early, acute and chronic inflammatory phase respectively. The results showed that mTrem-1 mRNA and protein were significantly expressed on the first week after mice infected with S. mansoni and reached second peak at week 9 post-infection. We further used flow cytometry to analyze the production of mTrem-1 protein, the data showed that the protein increased gradually by week, and reached the maximum at week 5. In addition, mTrem-1 mRNA have shown no expression in liver tissue, either during the early stage, or during the acute and chronic stages in which a great quantity of eggs have produced. It’s also been shown that the expression of mTrem-1 mRNA is surpressed when SEA was given for stimulation in J774A.1 (murine macrophages cell line) via in vitro culture. Moreover, the receptor expression of the cell surface has also decreased due to the impact, which indicates that the eggs of this parasite can subjectively inhibit the expression of mTrem-1 on macrophages by their secreted substances, so as to lower the inflammatory attack of macrophages on the eggs. These results indicated the expression profiles of mTrem-1 are significantly related to the inflammatory responses of mice infected with S. mansoni. The anti-inflammatory patterns of SEA might be useful in finding more effective anti-inflammatory candidate targets.

博士
國立陽明大學
臨床醫學研究所
102
Klebsiella pneumoniae liver abscess (KPLA) is prevalent in Eastern Asia. Liver abscess can develop after translocation of K. pneumoniae from a patient’s bowel into the liver via the portal circulation. Capsular serotype K1 of K. pneumoniae is thought to be the major virulence determinant responsible for KPLA and the associated invasive syndrome. Diabetes is a well-known risk factor for KPLA. However, other host factors for the development of KPLA have rarely been addressed in the literature. In the current work, we aimed to investigate the antibiotics use and the innate immunity mediated by Triggering Receptor Expressed on Myeloid cells 1 (TREM-1) in the pathogenesis of KPLA. K. pneumoniae strains obtained from patients with liver abscess in Taiwan were susceptible to nearly all antibiotics tested, except for ampicillin, which is consistent with natural resistance of K. pneumoniae. Treatment with ampicillin and amoxicillin changes the ecology of the bowel flora and may lead to overgrowth of K. pneumoniae and predisposition to KPLA. TREM proteins are a recently discovered family of cell surface receptors broadly expressed on the myeloid cells of human and mouse origins. Though the initial findings established TREM-1 as an amplifier of the systemic inflammatory response syndrome associated with sepsis, the exact role of TREM-1 in vivo in response to various bacterial infectious disease models remains unknown. In the first part of my work, an animal model was used to investigate the association between ampicillin/amoxicillin use and KPLA. In the animal study, ampicillin or sterile water was administered orogastrically in serotype K1 K. pneumoniae-colonized mice and the outcome was compared. Ampicillin administration predisposed K. pneumoniae-colonized mice to increased bacterial burden, liver abscess and necrosis, and lethality. We further designed a case-control study using the National Health Insurance Research Database and showed that the adjusted OR associating the use of ampicillin/amoxicillin within the past 30 days of KPLA development was 3.5 (95% CI= 2.5–5.1). We concluded that the use of ampicillin/amoxicillin may lead to overgrowth of K. pneumoniae in the intestine, therefore predisposing the hosts to KPLA. In the second part of my work, another animal model was used to characterize the role of TREM-1 in KPLA. The survival, bacterial burden in tissues, inflammatory cytokines, and histology findings between the wild-type and Trem-1-knockout (KO) mice after oral inoculation of capsular type K1 K. pneumoniae were compared. Translocation of K. pneumoniae to mesenteric lymph nodes and liver was examined, and intestinal permeability, antimicrobial peptide expression and the clearance of K. pneumoniae in the small intestine were determined. In the absence of TREM-1, mice with KPLA showed increased K. pneumoniae dissemination, more pronounced liver and systemic inflammation, and reduced survival. Impaired bacterial clearance in the small intestine causing enhanced K. pneumoniae translocation rendered Trem-1-KO mice more susceptible to K. pneumoniae oral infection. In conclusion, TREM-1-mediated bacterial clearance in the small intestine is an important immune response against K. pneumoniae. TREM-1 deficiency enhances K. pneumoniae translocation in the small intestine and increases the mortality rate of mice with KPLA.