PET/TC de cuerpo completo para la detección de metástasis de melanoma coroideo

El melanoma coroideo es el tumor maligno ocular primario más frecuente en los adultos y las metástasis más frecuentes son a nivel hepático con un mal pronóstico a pesar de los tratamientos. Estas metástasis se han descrito hasta en el 50 % de los melanomas coroideos. Estudios recientes muestran que la ecografía hepática y las analíticas hepáticas tienen baja sensibilidad en el diagnóstico de las metástasis hepáticas de los melanomas de úvea. Exponemos el caso de un paciente con historia de melanoma coroideo. La exploración preoperatoria, incluyendo TC de abdomen, analítica sanguínea y enzimas hepáticos resultó normal. En un PET/TC realizado mostró lesiones en el hígado y pulmones. El PET/TC de cuerpo entero mostró metástasis de un melanoma uveal. El PET/TC es una herramienta útil para la detección de las metástasis hepáticas y extrahepáticas.Choroidal melanoma is the most common primary intraocular cancer in adults. Metastases are most commonly found in the liver and are rapidly fatal despite aggressive therapy. These metastases have been reported in the follow-up to 50 % of uveal melanoma. However, recent reports have shown that liver ultrasonography and liver function tests have low sensitivity in the diagnosis of metastatic uveal melanoma. The study reports on a patient with a history of choroidal melanoma. Preoperative medical evaluation, including CT imaging of the abdomen, complete blood count and liver enzymes proved negative. A PET/ CT was requested and showed lesions in the liver and lungs. Whole-body PET/CT revealed melanoma metastases. The PET/CT is a sensitive tool for the detection and localization of hepatic and extrahepatic metastatic choroidal melanoma

The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation

Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation

Leukocyte- and Platelet-Derived Microvesicle Interactions following In Vitro and In Vivo Activation of Toll-Like Receptor 4 by Lipopolysaccharide

BACKGROUND: Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS). METHODOLOGY/PRINCIPAL FINDINGS: Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens. CONCLUSIONS/SIGNIFICANCE: Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood

Potentiation of Epithelial Innate Host Responses by Intercellular Communication

The epithelium efficiently attracts immune cells upon infection despite the low number of pathogenic microbes and moderate levels of secreted chemokines per cell. Here we examined whether horizontal intercellular communication between cells may contribute to a coordinated response of the epithelium. Listeria monocytogenes infection, transfection, and microinjection of individual cells within a polarized intestinal epithelial cell layer were performed and activation was determined at the single cell level by fluorescence microscopy and flow cytometry. Surprisingly, chemokine production after L. monocytogenes infection was primarily observed in non-infected epithelial cells despite invasion-dependent cell activation. Whereas horizontal communication was independent of gap junction formation, cytokine secretion, ion fluxes, or nitric oxide synthesis, NADPH oxidase (Nox) 4-dependent oxygen radical formation was required and sufficient to induce indirect epithelial cell activation. This is the first report to describe epithelial cell-cell communication in response to innate immune activation. Epithelial communication facilitates a coordinated infectious host defence at the very early stage of microbial infection

Down-regulation of endothelial TLR4 signalling after apo A-I gene transfer contributes to improved survival in an experimental model of lipopolysaccharide-induced inflammation

The protective effects of high-density lipoprotein (HDL) under lipopolysaccharide (LPS) conditions have been well documented. Here, we investigated whether an effect of HDL on Toll-like receptor 4 (TLR4) expression and signalling may contribute to its endothelial-protective effects and to improved survival in a mouse model of LPS-induced inflammation and lethality. HDL cholesterol increased 1.7-fold (p < 0.005) and lung endothelial TLR4 expression decreased 8.4-fold (p < 0.005) 2 weeks after apolipoprotein (apo) A-I gene transfer. Following LPS administration in apo A-I gene transfer mice, lung TLR4 and lung MyD88 mRNA expression, reflecting TLR4 signalling, were 3.0-fold (p < 0.05) and 2.1-fold (p < 0.05) lower, respectively, than in LPS control mice. Concomitantly, LPS-induced lung neutrophil infiltration, lung oedema and mortality were significantly attenuated following apo A–I transfer. In vitro, supplementation of HDL or apo A–I to human microvascular endothelial cells-1 24 h before LPS administration reduced TLR4 expression, as assessed by fluorescent-activated cell sorting, and decreased the LPS-induced MyD88 mRNA expression and NF-κB activity, independently of LPS binding. In conclusion, HDL reduces TLR4 expression and signalling in endothelial cells, which may contribute significantly to the protective effects of HDL in LPS-induced inflammation and lethality

Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers

Pattern recognition underpins innate immunity; the accurate identification of danger, including infection, injury, or tumor, is key to an appropriately targeted immune response. Pathogen detection is increasingly well defined mechanistically, but the discrimination of endogenous inflammatory triggers remains unclear. Tenascin-C, a matrix protein induced upon tissue damage and expressed by tumors, activates toll-like receptor 4 (TLR4)-mediated sterile inflammation. Here we map three sites within tenascin-C that directly and cooperatively interact with TLR4. We also identify a conserved inflammatory epitope in related proteins from diverse families, and demonstrate that its presence targets molecules for TLR detection, while its absence enables escape of innate immune surveillance. These data reveal a unique molecular code that defines endogenous proteins as inflammatory stimuli by marking them for recognition by TLRs

O-Antigen Delays Lipopolysaccharide Recognition and Impairs Antibacterial Host Defense in Murine Intestinal Epithelial Cells

Although Toll-like receptor (TLR) 4 signals from the cell surface of myeloid cells, it is restricted to an intracellular compartment and requires ligand internalization in intestinal epithelial cells (IECs). Yet, the functional consequence of cell-type specific receptor localization and uptake-dependent lipopolysaccharide (LPS) recognition is unknown. Here, we demonstrate a strikingly delayed activation of IECs but not macrophages by wildtype Salmonella enterica subsp. enterica sv. (S.) Typhimurium as compared to isogenic O-antigen deficient mutants. Delayed epithelial activation is associated with impaired LPS internalization and retarded TLR4-mediated immune recognition. The O-antigen-mediated evasion from early epithelial innate immune activation significantly enhances intraepithelial bacterial survival in vitro and in vivo following oral challenge. These data identify O-antigen expression as an innate immune evasion mechanism during apical intestinal epithelial invasion and illustrate the importance of early innate immune recognition for efficient host defense against invading Salmonella

TLR4 signalling in pulmonary stromal cells is critical for inflammation and immunity in the airways

Inflammation of the airways, which is often associated with life-threatening infection by Gram-negative bacteria or presence of endotoxin in the bioaerosol, is still a major cause of severe airway diseases. Moreover, inhaled endotoxin may play an important role in the development and progression of airway inflammation in asthma. Pathologic changes induced by endotoxin inhalation include bronchospasm, airflow obstruction, recruitment of inflammatory cells, injury of the alveolar epithelium, and disruption of pulmonary capillary integrity leading to protein rich fluid leak in the alveolar space. Mammalian Toll-like receptors (TLRs) are important signalling receptors in innate host defense. Among these receptors, TLR4 plays a critical role in the response to endotoxin

Gene profiling of the erythro- and megakaryoblastic leukaemias induced by the Graffi murine retrovirus

<p>Abstract</p> <p>Background</p> <p>Acute erythro- and megakaryoblastic leukaemias are associated with very poor prognoses and the mechanism of blastic transformation is insufficiently elucidated. The murine Graffi leukaemia retrovirus induces erythro- and megakaryoblastic leukaemias when inoculated into NFS mice and represents a good model to study these leukaemias.</p> <p>Methods</p> <p>To expand our understanding of genes specific to these leukaemias, we compared gene expression profiles, measured by microarray and RT-PCR, of all leukaemia types induced by this virus.</p> <p>Results</p> <p>The transcriptome level changes, present between the different leukaemias, led to the identification of specific cancerous signatures. We reported numerous genes that may be potential oncogenes, may have a function related to erythropoiesis or megakaryopoiesis or have a poorly elucidated physiological role. The expression pattern of these genes has been further tested by RT-PCR in different samples, in a Friend erythroleukaemic model and in human leukaemic cell lines.</p> <p>We also screened the megakaryoblastic leukaemias for viral integrations and identified genes targeted by these integrations and potentially implicated in the onset of the disease.</p> <p>Conclusions</p> <p>Taken as a whole, the data obtained from this global gene profiling experiment have provided a detailed characterization of Graffi virus induced erythro- and megakaryoblastic leukaemias with many genes reported specific to the transcriptome of these leukaemias for the first time.</p

Expression of toll-like receptors 2 and 4 in subjects with asthma by total serum IgE level

Emerging data suggest that innate immunity may play a role in asthma, particularly the toll-like receptors (TLRs). Some studies pointed to an involvement of TLRs 2 and 4 in the pathogenesis of allergic asthma, and other studies related TLRs to IgE. However, there are not any studies that have comprehensively evaluated the expression of TLRs 2 and 4 in inflammatory cells, in peripheral blood and induced sputum specimens from asthmatic patients, according to their total serum IgE. We studied 44 asthmatic patients (15 with high total serum IgE and 29 with normal total serum IgE). On a single visit, all patients underwent: induced sputum, pulmonary function tests, determination of exhaled nitric oxide fraction, venipuncture for blood analysis and skin prick allergy tests. The induced sputum cellularity was analyzed by flow cytometry, where expression of TLRs 2 and 4 was studied using fluorochrome-conjugated monoclonal antibodies. Asthmatic patients with high total serum IgE showed, a higher percentage of macrophages expressing TLR4 (42.99 % ± 22.49) versus asthmatic patients with normal total serum IgE (28.84 % ± 15.16) (P = 0.048). Furthermore, we observed a correlation (but weak) between the percentage of macrophages expressing TLR4 in induced sputum and the total serum IgE level (R = 0.314; P = 0.040). Asthmatic subjects with high total serum IgE show increased macrophage expression of TLR4 in induced sputum. This outcome may result from a link between innate immunity and IgE-mediated, adaptive immune responses in asthma, and point to TLR4 as a potential therapeutic target