Combined use of multiparametric flow cytometry and cytomorphology to enhance detection of neuroblastoma metastatic cells in bone marrow.

INTRODUCTION: In the context of neuroblastoma (NB), the screening for bone marrow (BM) metastasis is a recurrent issue for hematology laboratory routine practice. Detection of low tumor burden using light microscopy is often difficult. In this regard, our objective was to evaluate the performance of multiparametric flow cytometry (FC) for detecting NB metastatic cells in BM. METHODS: We applied a new FC multiparametric panel allowing the analysis of the co-expression of 5 surface markers: GD2 (disialoganglioside 2), CD9, CD56, CD81, and CD90, on CD45-negative BM cell populations, and compared results with BM biopsy immunohistochemistry, which is the reference method. RESULTS: In spike-in tests, the multiparametric FC successfully detected NB cells mixed in peripheral blood mononuclear cells to a level of 0.01%. FC analysis was performed on 45 sets of BM aspirates sampled from 21 children, either at diagnosis or during follow-up. Combining multiparametric FC with light microscopy improved NB metastasis detection, with a higher sensitivity (76.9% vs 61.5%) and a higher specificity (94.4% vs 77.8%) as compared to light microscopy alone. At the time of diagnosis, multiparametric FC detected NB metastatic cells in all cases. CONCLUSION: These results illustrate the performance of multiparametric FC analysis to detect metastatic BM infiltration of NB. This is of particular interest in an emergency context, since when combined with light microscopy, it enhances the detection of metastatic invasion within a short timeframe, allowing an adapted and rapid clinical management.

Standardization of Flow Cytometric Immunophenotyping for Hematological Malignancies: The FranceFlow Group Experience.

Flow cytometry is broadly used for the identification, characterization, and monitoring of hematological malignancies. However, the use of clinical flow cytometry is restricted by its lack of reproducibility across multiple centers. Since 2006, the EuroFlow consortium has been developing a standardized procedure detailing the whole process from instrument settings to data analysis. The FranceFlow group was created in 2010 with the intention to educate participating centers in France about the standardized instrument setting protocol (SOP) developed by the EuroFlow consortium and to organise several rounds of quality controls (QCs) in order to evaluate the feasibility of its application and its results. Here, we report the 5 year experience of the FranceFlow group and the results of the seven QCs of 23 instruments, involving up to 19 centers, in France and in Belgium. The FranceFlow group demonstrates that both the distribution and applicability of the SOP have been successful. Intercenter reproducibility was evaluated using both normal and pathological blood samples. Coefficients of variation (CVs) across the centers were <7% for the percentages of cell subsets and <30% for the median fluorescence intensities (MFIs) of the markers tested. Intracenter reproducibility provided similar results with CVs of <3% for the percentages of the majority of cell subsets, and CVs of <20% for the MFI values for the majority of markers. Altogether, the FranceFlow group show that the 19 participating labs might be considered as one unique laboratory with 23 identical flow cytometers able to reproduce identical results. Therefore, SOP significantly improves reproducibility of clinical flow in hematology and opens new avenues by providing a robust companion diagnostic tool for clinical trials in hematology. © 2019 International Society for Advancement of Cytometry.

Usefulness of Flow Cytometric Mepacrine Uptake/Release Combined with CD63 Assay in Diagnosis of Patients with Suspected Platelet Dense Granule Disorder.

Dense granule disorder is one of the most common platelet abnormalities, resulting from dense granule deficiency or secretion defect. This study was aimed to evaluate the clinical usefulness of the flow cytometric combination of mepacrine uptake/release assay and CD63 expression detection in the management of patients with suspected dense granule disorder. Over a period of 5 years, patients with abnormal platelet aggregation and/or reduced adenosine triphosphate (ATP) secretion suggestive of dense granule disorder were consecutively enrolled. The flow cytometric assays were systematically performed to further investigate dense granule functionality. Among the 26 included patients, 18 cases showed impaired mepacrine uptake/release and reduced CD63 expression on activated platelets, consistent with δ-storage pool deficiency (SPD). Another seven patients showed decrease in mepacrine release and CD63 expression but mepacrine uptake was normal, indicating secretion defect rather than δ-SPD. Unfortunately, ATP secretion could not be measured in 7 out of the 26 patients due to insufficient sample and/or severe thrombocytopenia. This test combination provides a rapid and effective method to detect the heterogeneous abnormalities of platelet dense granule by distinguishing between storage and release defects. This combination is particularly advantageous for severely thrombocytopenic patients and pediatric patients in which only minimal sample is required.

Validation of a single-platform method for hematopoietic CD34+ stem cells enumeration according to accreditation procedure.

INTRODUCTION: Stem cells for autologous and allogenic transplantation are obtained from several sources including bone marrow, peripheral blood or cord blood. Accurate enumeration of viable CD34+ hematopoietic stem cells (HSC) is routinely used in clinical settings, especially to monitor progenitor cell mobilization and apheresis. The number of viable CD34+ HSC has also been shown to be the most critical factor in haematopoietic engraftment. The International Society for Cellular Therapy actually recommends the use of single-platform flow cytometry system using 7-AAD as a viability dye. AIM: In a way to move routine analysis from a BD FACSCaliburTM instrument to a BD FACSCantoTM II, according to ISO 15189 standard guidelines, we define laboratory performance data of the BDTM Stem Cell Enumeration (SCE) kit on a CE-IVD system including a BD FACSCanto II flow cytometer and the BD FACSCantoTM Clinical Software. InterQCTM software, a real time internet laboratory QC management system developed by VitroTM and distributed by Becton DickinsonTM, was also tested to monitor daily QC data, to define the internal laboratory statistics and to compare them to external laboratories. METHODS: Precision was evaluated with BDTM Stem Cell Control (high and low) results and the InterQC software, an internet laboratory QC management system by Vitro. This last one drew Levey-Jennings curves and generated numeral statistical parameters allowing detection of potential changes in the system performances as well as interlaboratory comparisons. Repeatability, linearity and lower limits of detection were obtained with routine samples from different origins. Agreement evaluation between BD FACSCanto II system versus BD FACSCalibur system was tested on fresh peripheral blood, freeze-thawed apheresis, fresh bone marrow and fresh cord blood samples. RESULTS: Instrument's measure and staining repeatability clearly evidenced acceptable variability on the different samples tested. Intra- and inter-laboratory CV in CD34+ cell absolute count are consistent and reproducible. Linearity analysis, established between 2 and 329 cells/µl showed a linear relation between expected counts and measured counts (R2=0.97). Linear regression and Bland-Altman representations showed an excellent correlation on samples from different sources between the two systems and allowed the transfer of routine analysis from BD FACSCalibur to BD FACSCanto II. CONCLUSIONS: The BD SCE kit provides an accurate measure of the CD34 HSC, and can be used in daily routine to optimize the enumeration of hematopoietic CD34+ stem cells by flow cytometry. Moreover, the InterQC system seems to be a very useful tool for laboratory daily quality monitoring and thus for accreditation.

Development of a new method for identification and quantification in cerebrospinal fluid of malignant cells from breast carcinoma leptomeningeal metastasis.

BACKGROUND: The diagnosis of leptomeningeal metastasis (LM) in patients with solid tumors remains difficult. The usual diagnostic methods of cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium enhanced MRI of the entire neuraxis lack both specificity and sensitivity. The Veridex CellSearch® technology has been designed for the detection of circulating tumor cells (CTC) in blood from cancer patients and validated for the follow-up and prognosis of breast, prostate, colorectal, and lung cancer. Our aim was to adapt this technology for the detection and the enumeration of tumor cells in the CSF of breast cancer patients presenting with LM. METHODS: On the occasion of a randomized phase III study evaluating the role of the intrathecal treatment in LM from breast cancer (DEPOSEIN, EudraCT N°: 2010-023134-23), the CellSearch® technology was adapted to direct enrichment, enumeration and visualization of tumor cells in 5 mL CSF samples, collected on CellSave® Preservative Tubes and analyzed within 3 days after CSF sampling. RESULTS: Sixteen CSF of 8 patients with primary breast cancer presenting with LM were studied. EpCAM+/cytokeratin + cells with typical morphology could be observed and enumerated sequentially with reproducible results in low or elevated numbers in 8 patients. CONCLUSION: This methodology, established on a limited volume of sample and allowing delayed processing, could prove of great interest in the diagnosis and follow-up of cancer patients with LM, especially to appreciate the efficacy of chemotherapy.

Increased gingival crevicular fluid levels of soluble triggering receptor expressed on myeloid cells (sTREM) -1 in severe periodontitis.

AIM: This study was designed to evaluate the presence of a new regulator of innate immunity in periodontitis: the soluble form of triggering receptor on myeloid cells-1 (sTREM-1) in gingival crevicular fluid (GCF). MATERIAL AND METHODS: GCF was collected at four sites, three pathological and one healthy from 17 patients with periodontitis, and at one healthy site from 23 control patients. An enzyme-linked immunosorbent assay (ELISA) kit was used to quantify sTREM-1 levels in collected crevicular fluid. Recorded clinical parameters were probing pocket depth (PPD), bleeding upon probing, tooth mobility, plaque index (PlI), and gingival index (GI). RESULTS: The mean sTREM-1 level in collected fluid was significantly higher in pathological sites than in healthy sites from either periodontal or control patients: 353.9 pg/ml, 50.2 pg/ml and 25.4 pg/ml respectively. Soluble TREM-1 concentration was significantly correlated with PPD. The sTREM-1 levels increased with the augmentation of the PlI and GI scores and levelled off at score 2 for both indexes. In multivariate analysis, periodontal pocket depth and smoking status were statistically associated with highest sTREM-1 concentrations. CONCLUSION: sTREM-1 was detected in crevicular fluid and its concentration was higher in pathological sites. It could be a marker of periodontal tissue destruction.

Soluble TREM-like transcript-1 regulates leukocyte activation and controls microbial sepsis.

The triggering receptor expressed on myeloid cells (TREM)-1 plays a crucial role during the onset of sepsis by amplifying the host immune response. The TREM-like transcript-1 (TLT-1) belongs to the TREM family, is selectively expressed on activated platelets, and is known to facilitate platelet aggregation through binding to fibrinogen. In this study, we show that a soluble form of TLT-1 is implicated in the regulation of inflammation during sepsis by dampening leukocyte activation and modulating platelet-neutrophil crosstalk. A 17-aa sequence of the TLT-1 extracellular domain (LR17) is responsible for this activity through competition with the TREM-1 ligand. Whereas early or late LR17 treatment of septic mice improves survival, treml-1(-/-) animals are highly susceptible to polymicrobial infection. The present findings identify platelet-derived soluble TLT-1 as a potent endogenous regulator of sepsis-associated inflammation and open new therapeutic perspectives. We anticipate soluble TLT-1 to be important in regulating leukocyte activation during other noninfectious inflammatory disorders.

Combination biomarkers to diagnose sepsis in the critically ill patient.

RATIONALE: Although the outcome of sepsis benefits from the prompt administration of appropriate antibiotics on correct diagnosis, the assessment of infection in critically ill patients is often a challenge for clinicians. In this setting, simple biomarkers, especially when used in combination, could prove useful. OBJECTIVES: To determine the usefulness of combination biomarkers to diagnose sepsis. METHODS: Three hundred consecutive patients were enrolled to construct a biologic score that was next validated in an independent prospective cohort of 79 critically ill patients from another center. MEASUREMENT AND MAIN RESULTS: Plasma concentrations of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and procalcitonin (PCT) were assayed, and the expression of the high-affinity immunoglobulin-Fc fragment receptor I (FcγRI) CD64 on neutrophils (polymorphonuclear [PMN] CD64 index) in flow cytometry was measured. A "bioscore" combining these biomarkers was constructed. Serum concentrations of PCT and sTREM-1 and the PMN CD64 index were higher in patients with sepsis compared with all others (P < 0.001 for the three markers). These biomarkers were all independent predictors of infection, the best receiver-operating characteristic curve being obtained for the PMN CD64 index. The performance of the bioscore, better than that of each individual biomarker, was externally confirmed in the validation cohort. CONCLUSIONS: This prospective study, including inceptive and validation cohorts of unselected intensive care unit patients, demonstrates the high performance of a bioscore combining the PMN CD64 index together with PCT and sTREM-1 serum levels in diagnosing sepsis in the critically ill patient.

Plasma soluble triggering receptor expressed on myeloid cells-1 in Crohn's disease.

BACKGROUND: No definite conclusions can be drawn from available data on the accuracy of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) to assess disease activity in Crohn's disease. AIMS: Plasma sTREM-1 levels were correlated with disease activity markers in Crohn's disease. METHODS: 191 consecutive patients from a single referral centre (Nancy IBD cohort) were prospectively enrolled between June 1, 2005 and December 12, 2008. Plasma sTREM-1 levels were also assessed amongst 20 healthy controls. RESULTS: The sTREM-1 was detectable in 87 Crohn's disease patients (46%). Plasma sTREM-1 level was higher in Crohn's disease patients (interquartile range, 0-356) than in healthy controls (interquartile range, 0-15.1; P=0.01). It was neither correlated with Crohn's disease activity index (r=0.05, P=0.56), C-reactive protein (r=0.06, P=0.53), nor with albumin (r=-0.041, P=0.66). Crohn's disease activity index, C-reactive protein and albumin median levels were similar between patients with positive sTREM-1 levels and those with undetectable sTREM-1 levels. Azathioprine (P=0.06), infliximab (P=0.68) and methotrexate (P=0.56) did not influence sTREM-1 levels. CONCLUSION: Plasma sTREM-1 does not appear to be an accurate marker of disease activity in Crohn's disease and cannot be recommended for assessing disease activity in these patients.

Serum sTREM-1 (soluble triggering receptor expressed on myeloid cells-1) associates negatively with embryo quality in infertility patients.

PROBLEM: Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is a useful biomarker of infection and inflammation. METHOD OF STUDY: We studied serum and follicular fluid sTREM-1 in infertile patients (N = 110) utilizing enzyme-linked immunosorbent assay. RESULTS: Serum and follicular sTREM-1 were in good correlation (Pearson's correlation 0.56, P < 0.0001) with higher values in follicular fluid (140.4 ± 34.4 and 115.6 ± 35.1 pg/mL, t-test, P < 0.0001). Endometriosis associated with lower follicular and serum sTREM-1 compared with male factor infertility patients (age-adjusted r = -25.7 pg/mL, P = 0.018; r = -22.1 pg/mL, P = 0.030). No associations between follicular or serum sTREM-1 and clinical parameters were found, except higher serum sTREM-1 associated with lower embryo quality in all patients (adjusted r = -0.3%, P = 0.033), with a cutoff value between 111.5 and 113.3 pg/mL (OR = 0.38, P = 0.048; OR = 0.34, P = 0.028) predicting that more than 39% of embryos would be with good quality. CONCLUSION: Serum sTREM-1 could represent a prognostic marker for female fecundity, probably indicating impaired inflammatory reaction of immune system.

Follicular proinflammatory cytokines and chemokines as markers of IVF success.

Cytokines are key modulators of the immune system and also contribute to regulation of the ovarian cycle. In this study, Bender MedSystems FlowCytomix technology was used to analyze follicular cytokines (proinflammatory: IL-1ß, IL-6, IL-18, IFN-γ, IFN-α, TNF-α, IL-12, and IL-23;, and anti-inflammatory: G-CSF), chemokines (MIP-1α, MIP-1ß, MCP-1, RANTES, and IL-8), and other biomarkers (sAPO-1/Fas, CD44(v6)) in 153 women undergoing in vitro fertilization (IVF). Cytokine origin was studied by mRNA analysis of granulosa cells. Higher follicular MIP-1α and CD44(v6) were found to correlate with polycystic ovary syndrome, IL-23, INF-γ, and TNF-α with endometriosis, higher CD44(v6) but lower IL-ß and INF-α correlated with tubal factor infertility, and lower levels of IL-18 and CD44(v6) characterized unexplained infertility. IL-12 positively correlated with oocyte fertilization and embryo development, while increased IL-18, IL-8, and MIP-1ß were associated with successful IVF-induced pregnancy.

The relationship between CD4+CD25+CD127- regulatory T cells and inflammatory response and outcome during shock states.

INTRODUCTION: Although regulatory T lymphocytes (Tregs) have a pivotal role in preventing autoimmune diseases and limiting chronic inflammatory conditions, they may also block beneficial immune responses by preventing sterilizing immunity to certain pathogens. METHODS: To determine whether naturally occurring Treg cells have a role in inflammatory response and outcome during shock state we conducted an observational study in two adult ICUs from a university hospital. Within 12 hours of admission, peripheral whole blood was collected for the measurement of cytokines and determination of lymphocyte count. Sampling was repeated at day three, five and seven. Furthermore, an experimental septic shock was induced in adult Balb/c mice through caecal ligation and puncture. RESULTS: Forty-three patients suffering from shock (26 septic, 17 non septic), and 7 healthy volunteers were included. The percentage of Tregs increased as early as 3 days after the onset of shock, while their absolute number remained lower than in healthy volunteers. A similar pattern of Tregs kinetics was found in infected and non infected patients. Though there was an inverse correlation between severity scores and Tregs percentage, the time course of Tregs was similar between survivors and non survivors. No relation between Tregs and cytokine concentration was found. In septic mice, although there was a rapid increase in Treg cells subset among splenocytes, antibody-induced depletion of Tregs before the onset of sepsis did not alter survival. CONCLUSIONS: These data argue against a determinant role of Tregs in inflammatory response and outcome during shock states.

Triggering receptor expressed on myeloid cells-1 as a new therapeutic target during inflammatory diseases.

The Triggering Receptor Expressed on Myeloid cells (TREM)-1 is a recently identified molecule involved in monocytic activation and inflammatory response. It belongs to a family related to Natural Killer cell-receptors and is expressed on neutrophils, mature monocytes and macrophages. The engagement of TREM-1 synergizes with several Toll Like Receptors (TLR) and/or NOD Like Receptors (NLR) activation in amplifying the inflammatory response mediated by microbial components or danger signals. The implication of TREM-1 during experimental models of acute or chronic inflammatory conditions, as well as during cancer, begins to understand. Furthermore, the modulation of the TREM-1 signaling pathway by the use of small synthetic peptides derived from its extracellular moiety confers interesting survival advantages during experimental murine septic shock and protects from organ damage during other inflammatory diseases. This review summarizes the recent advances on TREM-1 biology and highlights the promises of its therapeutic modulation.

Effects of the TREM 1 pathway modulation during hemorrhagic shock in rats.

The triggering receptor expressed on myeloid cells (TREM) 1, a receptor expressed on the surface of neutrophils and monocytes/macrophages, synergizes with the Toll-like receptors in amplifying the inflammatory response mediated by microbial components. Because the pathogenesis of severe blood loss-induced excessive inflammation and multiple organ failure implies leukocyte activation and bacterial translocation, we hypothesized that the TREM-1 pathway modulation would prove beneficial in this setting. Wistar rats were subjected to a 1-h period of hemorrhagic shock and then reperfused with shed blood and ringer lactate for 1 h. At the time of reperfusion, animals were administered with LP17 (a synthetic soluble TREM-1 decoy receptor), a control peptide, or a vehicle (isotonic sodium chloride solution). Plasma concentration of TNF-alpha, IL-6, and soluble TREM-1 were measured by enzyme-linked immunosorbent assay. Lung permeability was assessed by the weight-dry ratio and fluorescein isothiocyanate-labeled albumin lung-blood ratio. Organ dysfunction was appreciated by measuring plasma aspartate aminotransferase and urea concentrations. Bacterial translocation was estimated by blood, mesenteric lymph nodes, and spleens culture. Hemorrhagic shock associated with cardiovascular collapse, lactic acidosis, systemic inflammatory response, and organ dysfunction that was partly prevented by LP17 administration. Hemorrhagic shock induced a marked increase in lung permeability that was also prevented by TREM-1 modulation. Finally, LP17 improved survival. Thus, the early modulation of the TREM-1 pathway by means of a synthetic peptide may be useful during severe hemorrhagic shock in rats in preventing organ dysfunction and improving survival.

Effects of the TREM-1 pathway modulation during mesenteric ischemia-reperfusion in rats.

OBJECTIVES: The triggering receptor expressed on myeloid cells (TREM)-1, a receptor expressed on the surface of neutrophils and monocytes/macrophages, synergizes with the Toll-like receptors in amplifying the inflammatory response mediated by microbial components. Because the pathogenesis of ischemia-reperfusion-induced gastrointestinal tissue injury and multiple organ failure implies leukocyte activation and bacterial translocation, we hypothesized that the TREM-1 pathway modulation would prove beneficial in this setting. DESIGN: Animal study. SETTING: Research laboratory. SUBJECTS: Adult male Wistar rats (250-300 g). INTERVENTIONS: Rats were subjected to intestinal ischemia-reperfusion induced by occlusion of the superior mesenteric artery during 60 mins and reperfused for 180 mins. At the time of reperfusion, animals were administered with LP17 (a synthetic TREM-1 inhibitor), a control peptide, or a vehicle (normal saline). Plasma concentrations of tumor necrosis factor-alpha, interleukin-6, and soluble TREM-1 were measured by enzyme-linked immunosorbent assay. Hepatic activation of the transcriptional factor nuclear factor-kappaB was assessed by electrophoretic mobility shift assay. Hepatic oxidant-antioxidant balance was estimated by measurement of lipid peroxidation and catalase activity. Ileal mucosal permeability was estimated by fluorescein dextran-4 clearance and bacterial translocation by mesenteric lymph nodes culture. MEASUREMENTS AND MAIN RESULTS: Ischemia-reperfusion was associated with cardiovascular collapse, lactic acidosis, and systemic and hepatic inflammatory response that were partly prevented by LP17 administration. Liver lipid peroxidation and catalase depletion were attenuated by LP17. Ischemia-reperfusion induced a marked increase in ileal mucosal permeability and an associated bacterial translocation that was also prevented by TREM-1 modulation. LP17 delayed mortality. CONCLUSIONS: The modulation of the TREM-1 pathway by the means of a synthetic peptide may be useful during acute mesenteric ischemia.

High-mobility group box 1 protein plasma concentrations during septic shock.

OBJECTIVE: To investigate plasma high-mobility group box 1 protein (HMGB1) concentration and its relationship with organ dysfunction and outcome in septic shock patients. DESIGN AND SETTING: Prospective, noninterventional study. Medical adult intensive care unit at a university hospital in France. PATIENTS: 42 critically ill patients with septic shock. METHODS: Arterial blood was drawn within 12 h of admission for the measurement of plasma HMGB1 concentration by ELISA. Repeated sampling was performed on days 3, 7, and 14. RESULTS: Median HMGB1 concentration was 4.4 ng/ml (IQR 1.2-12.5) at admission, with no difference between survivors and nonsurvivors. A positive correlation was observed between HMGB1 and SOFA score and lactate, and procalcitonin concentrations. There was a progressive but statistically nonsignificant decline in HMGB1 concentration among the survivors, while nonsurvivors showed an increase in HMGB1 level between days 1 and 3. SOFA score and lactate and procalcitonin concentrations did not vary significantly between days 1 and 3. When measured on day 3, HMGB1 discriminated survivors from nonsurvivors with 66% sensitivity and 67% specificity, and concentration greater than 4 ng/ml was associated with an odds ratio of death of 5.5 (95% CI 1.3-23.6).

Growth arrest-specific protein 6 plasma concentrations during septic shock.

INTRODUCTION: The product of growth arrest-specific gene 6 (Gas6) is a vitamin K dependent protein that is secreted by leucocytes and endothelial cells in response to injury and participates in cell survival, proliferation, migration and adhesion. Our purpose was to investigate plasma Gas6 concentration and its relation to organ dysfunction in patients with septic shock. METHODS: Forty-five patients with septic shock admitted to a medical adult intensive care unit were enrolled. Plasma Gas6 concentration was determined using enzyme-linked immunosorbent assay at days 1, 3, 7 and 14. RESULTS: The median (interquartile range) Gas6 concentration was 51 (5 to 95) pg/ml at admission. A positive correlation (Spearman rank-order coefficient [rs] = 0.37, P = 0.01) was found between Gas6 level and Sepsis-related Organ Failure Assessment score. Patients requiring renal support had higher Gas6 concentration that those without need for haemofiltration (76.5 [52 to 164] pg/ml versus 10.5 [1.5 to 80.5] pg/ml; P = 0.04). Moreover, there was a positive correlation between Gas6 and aspartate transaminase (rs = 0.42, P = 0.006) and between Gas6 and prothrombin time (rs = 0.45, P = 0.02). Although there was a progressive decline in Gas6 concentration in survivors (analysis of variance, P = 0.01), nonsurvivors exhibited persistently elevated Gas6. However, the two populations diverged only after day 7 (P = 0.04). CONCLUSION: Plasma concentrations of Gas6 correlate with disease severity, especially with renal and hepatic dysfunction, in septic shock.

TREM-1 promotes survival during septic shock in mice.

Triggering receptor expressed on myeloid (TREM)-1 is integral to the inflammatory response occurring during septic shock, although its precise function has yet to be determined. Here we show that in vivo silencing of TREM-1 using siRNA duplexes in a fecal peritonitis mouse model resulted in a blunted inflammatory response and increased mortality. This was associated with impaired bacterial clearance related to marked inhibition of the neutrophil oxidative burst. By contrast, TREM-1-silenced mice were highly resistant to a lethal endotoxin challenge, while partial silencing of TREM-1 in the bacterial peritonitis model produced a significant survival benefit. These data highlight the crucial role of the TREM-1 pathway in mounting an adequate inflammatory and cytotoxic response to polymicrobial sepsis, and both the therapeutic promise and potential risks of its modulation.

Modulation of the triggering receptor expressed on myeloid cells-1 pathway during pneumonia in rats.

BACKGROUND: Triggering receptor expressed on myeloid cells-1 (TREM-1) is a cell-surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. The aim of the present study was to investigate the effect of the modulation of the TREM-1 pathway during experimental pneumonia in rats. METHODS: Adult male Wistar rats were intratracheally inoculated with Pseudomonas aeruginosa (PAO1 strain) and randomly treated or not treated with an analogue synthetic peptide derived from the extracellular moiety of TREM-1 (LP17). RESULTS: P. aeruginosa induced a severe pneumonia associated with signs of severe sepsis within the first 24 h. In septic rats, LP17 improved hemodynamic status, attenuated the development of lactic acidosis and hypoxemia, modulated lung and systemic inflammatory responses and coagulation activation, reduced lung histological damage, and improved survival. CONCLUSIONS: The modulation of the TREM-1 pathway by the use of such synthetic peptides as LP17 appears beneficial during P. aeruginosa pneumonia in rats in attenuating lung and systemic inflammatory responses.

Modulation of the triggering receptor expressed on the myeloid cell type 1 pathway in murine septic shock.

The triggering receptor expressed on myeloid cell type 1 (TREM-1) is a cell surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. To investigate the effect of the modulation of the TREM-1 pathway during experimental murine sepsis, we used analogue synthetic peptides derived from the extracellular moiety of TREM-1. The TREM-1 ligand was expressed on both peritoneal and peripheral neutrophils during experimental peritonitis in mice. The TREM-1 peptides inhibited the recognition by TREM-1 of its ligand and protected endotoxinic mice from death. In septic rats, the TREM-1 peptides improved the hemodynamic status, attenuated the development of lactic acidosis, modulated the production of such proinflammatory cytokines as tumor necrosis factor alpha and interleukin-1beta, and improved survival. The protective effect of these peptides on arterial pressure could partly be explained by a decreased production of nitric oxide. These data suggest that in vivo modulation of TREM-1 might be a suitable therapeutic tool for the treatment of sepsis.