Gilbert C. Faure

Plasma Level of a Triggering Receptor Expressed on Myeloid Cells-1: Its Diagnostic Accuracy in Patients with Suspected Sepsis

Context Exposure to bacteria upregulates an immunoglobulin-like molecule on phagocytes called the triggering receptor expressed on myeloid cells-1 (TREM-1). Can TREM-1 help distinguish sepsis from the systemic inflammatory response syndrome (SIRS) without infection? Contribution In this prospective study, 76 critically ill adults with suspected infection had plasma TREM-1 levels measured with a rapid immunoblot technique. Thirty-eight percent of the adults had SIRS, and 62% had sepsis. A TREM-1 level higher than 60 ng/mL increased the odds of sepsis by 8.6 (95% CI, 3.8 to 21.5). Implications Levels of TREM-1 may help diagnose bacterial sepsis in critically ill patients. The Editors Sepsis is a common cause of morbidity and death in intensive care units (1). Clinical and laboratory signs of systemic inflammation, including changes in body temperature, tachycardia, or leukocytosis, are neither sensitive nor specific enough for the diagnosis of sepsis. These signs can also be misleading because critically ill patients often present with the systemic inflammatory response syndrome but no infection (2-4). This issue is of paramount importance, since therapy and outcome differ greatly between patients with and those without sepsis. Moreover, the widespread use of antibiotics for all such patients is likely to increase antibiotic resistance, toxicity, and costs (5). Thus, there is an as-yet-unsatisfied need for clinical or laboratory tools allowing clinicians to distinguish between the systemic inflammatory response syndrome and sepsis. Among the potentially useful markers of sepsis, procalcitonin has been suggested as the most promising (6-8). However, several investigators have questioned the diagnostic and prognostic accuracy of routine procalcitonin measurements, reporting inconsistent and variable results depending on the severity of illness and infection in the patient sample studied (9-11). The triggering receptor expressed on myeloid cells-1 (TREM-1) is a member of the immunoglobulin superfamily, and its expression is upregulated on phagocytic cells in the presence of bacteria or fungi (12). Several experiments by Bouchon and colleagues (13) showed that TREM-1 mediates the acute inflammatory response to microbial products. Human tissues infected with bacteria are infiltrated with neutrophils and macrophages that express high levels of TREM-1. Conversely, TREM-1 is only weakly expressed in samples from patients with noninfectious inflammatory disorders (13). In addition, TREM-1 is shed from the membrane of activated phagocytes and can be found in a soluble form in body fluids. The presence of a soluble form of TREM-1 in samples of bronchoalveolar lavage fluid from mechanically ventilated patients has been shown to be a good indicator of infectious pneumonia (14). In this study, we prospectively investigated the diagnostic value of an assay measuring the plasma level of soluble TREM-1 in distinguishing sepsis from severe systemic noninfectious inflammation among newly admitted critically ill patients with suspected infection. Methods Study Sample All consecutive patients who were newly hospitalized in the medical intensive care unit of a teaching hospital in France between July and September 2003 were prospectively enrolled in the study if they had clinically suspected infection and fulfilled at least 2 criteria of the systemic inflammatory response syndrome (Appendix Table 1) (15). Clinically suspected infection was defined as an explicit statement by the attending physician indicating suspicion of an ongoing infection. In all enrolled patients, diagnostic work-up was performed to identify or rule out infection and antimicrobial therapy was prescribed. Patients were not enrolled if they were older than 80 years of age or were immunocompromised because of treatment with corticosteroids, receipt of bone marrow or organ transplants, leukopenia (leukocyte count < 1.0 109 cells/L) or neutropenia (polymorphonuclear granulocyte count < 0.5 109 cells/L), a hematologic malignant condition, or AIDS. Patients who died or were discharged early (within 12 hours after admission) or presented with complete absence of antimicrobial treatment were also excluded. Patients originated from the emergency department, the general wards, or the operating room. The hospitals institutional review board approved the study, and we obtained informed consent from patients or their relatives before inclusion. Data Collection At admission to the intensive care unit, we recorded the following items for each patient: age; sex; severity of the underlying medical condition, stratified according to the criteria of McCabe and Jackson (16); Simplified Acute Physiology Score II (range, 0 to 194, with a higher score indicating worse condition) (17); Sepsis-related Organ Failure Assessment score (range, 0 to 24; scores for each organ system [respiration, coagulation, liver, cardiovascular, central nervous system, and kidney] ranged from 0 [normal] to 4 [most abnormal]) (18); reason for admission to the intensive care unit; principal diagnosis; vital signs; respiratory variables; and results of routine blood tests and microbiological culture results. Survival or death in the intensive care unit was assessed during a follow-up period as long as 28 days. The attending physician prescribed microbiological tests and antimicrobial therapy according to the usual practice of the intensive care unit, without interference by the research team. Two intensivists retrospectively reviewed all medical records pertaining to each patient and independently classified the diagnosis as the systemic inflammatory response syndrome (that is, no infection), sepsis, severe sepsis, or septic shock at the time of admission, according to established consensus definitions (Appendix Table 1) (15). Agreement about the diagnosis was achieved in all cases. Both intensivists were blinded to plasma soluble TREM-1 values. Measurements of Plasma Levels of Procalcitonin and Soluble TREM-1 Within 12 hours after admission and study enrollment, 5 mL of whole heparinized blood was drawn through an arterial line for measurement of procalcitonin and soluble TREM-1 levels. Plasma was collected by centrifugation at 4 C, separated into aliquots, and stored at 80 C until the day of assay. Plasma procalcitonin concentrations were measured by using an immunoassay with a sandwich technique and a chemiluminescent detection system, according to the manufacturers protocol (LUMITest, Brahms Diagnostica, Berlin, Germany). Plasma soluble TREM-1 levels were assessed as described elsewhere (14). Briefly, 100 L of each plasma sample was dotted on a nitrocellulose membrane, dried, and overcoated in phosphate-buffered saline solution supplemented with 3% bovine serum albumin. The nitrocellulose sheet was then incubated for 60 minutes in the presence of monoclonal antiTREM-1 antibody. After thorough rinsing, the sheet was further incubated for 60 minutes with diluted (1:1000) goat anti-mouse immunoglobulins (Dako, Glostrup, Denmark), washed in phosphate-buffered saline solution supplemented with 20% dimethylsulfoxide, and incubated for 30 minutes with diluted (1:1000) horseradish peroxidaseconjugated streptavidin (Bio-Rad, Cergy, France). The enzyme substrate chromogen Opti-4CN (Bio-Rad) was then added, and color developed in proportion to the amount of soluble TREM-1 bound to the membrane. Each sheet also contained calibration samples of a known concentration of soluble TREM-1 (0 to 5000 ng/mL). Colorimetric determination was achieved by using a reflectance scanner and Quantity One quantitation software (Bio-Rad). Soluble TREM-1 concentration was determined in each sample by plotting the optical densities of the samples to the standard curve. All measurements were performed in duplicate, and results were expressed as mean concentration in ng/mL of plasma. The sensitivity of this technique allows the detection of soluble TREM-1 levels as low as 5 ng/mL, and the entire procedure takes less than 3 hours. The coefficient of variation of the assay was lower than 5%. Statistical Analysis Descriptive results of continuous variables were expressed as the mean (SD). Plasma soluble TREM-1 and procalcitonin levels were expressed as the median and range. Variables were tested for their association with the diagnosis by using the Pearson chi-square test for categorical data and the MannWhitney U test for numerical data. The different groups were compared by using the MannWhitney U test (or nonparametric KruskalWallis test when appropriate) for numerical data and the Pearson chi-square test (or the Fisher exact test when appropriate) for categorical data. The relation between soluble TREM-1 level and clinical or biological features was assessed by using the Spearman correlation test. Receiver-operating characteristic curves were constructed to illustrate various cutoff values of soluble TREM-1, procalcitonin, and C-reactive protein. Sensitivity, specificity, and positive and negative likelihood ratios and their confidence intervals were calculated (19) for the cutoff point that represented the best discrimination, as derived from the areas under the receiver-operating characteristic curves. We used StatView software (Abacus Concepts, Berkeley, California) to complete the analysis. A 2-tailed P value less than 0.05 was considered statistically significant. Role of the Funding Source The funding source had no role in the design, conduct, or reporting of the study or the decision to publish the manuscript. Results Characteristics of the Study Sample From July to September 2003, 98 patients were admitted into our intensive care unit with clinical suspicion of infection. Of these, 22 were not included in the study because of early death, immunocompromised state, age older than 80 years, absence of consent (for 2 patients), or protocol violation (for 5 patients who were not enrolled because an attending physician was unaware of

A Soluble Form of the Triggering Receptor Expressed on Myeloid Cells-1 Modulates the Inflammatory Response in Murine Sepsis

The triggering receptor expressed on myeloid cells (TREM)-1 is a recently discovered receptor expressed on the surface of neutrophils and a subset of monocytes. Engagement of TREM-1 has been reported to trigger the synthesis of proinflammatory cytokines in the presence of microbial products. Previously, we have identified a soluble form of TREM-1 (sTREM-1) and observed significant levels in serum samples from septic shock patients but not controls. Here, we investigated its putative role in the modulation of inflammation during sepsis. We observed that sTREM-1 was secreted by monocytes activated in vitro by LPS and in the serum of animals involved in an experimental model of septic shock. Both in vitro and in vivo, a synthetic peptide mimicking a short highly conserved domain of sTREM-1 appeared to attenuate cytokine production by human monocytes and protect septic animals from hyper-responsiveness and death. This peptide seemed to be efficient not only in preventing but also in down-modulating the deleterious effects of proinflammatory cytokines. These data suggest that in vivo modulation of TREM-1 by sTREM peptide might be a suitable therapeutic tool for the treatment of sepsis.

CHOROID PLEXUS, AGEING OF THE BRAIN, AND ALZHEIMER'S DISEASE

: Choroid plexus tissues are intraventricular structures composed of villi covered by a single layer of ciliated, cuboid epithelium. The plexuses secrete cerebrospinal fluid (CSF), synthesize numerous molecules, carry nutrients from the blood to CSF, reabsorb brain metabolism by-products and participate in brain immunosurveillance. During ageing, atrophy of epithelium occurs along with thickening of basement membranes. Enzymatic activities of epithelial cells decrease significantly. CSF secretion decreases as much as 50%. These modifications are concurrent with subnormal brain activity. In Alzheimers disease, epithelial atrophy, thickening of basement membrane and stroma fibrosis are even more prominent. Ig and C1q deposition along the basement membrane can be frequently detected, suggesting immunological processes. Synthesis, secretory, and transportation functions are significantly altered resulting in decreased CSF turnover, reduced beta-amyloid clearance, and increased glycation phenomena as well as oxidative stress. Such modifications may favour fibrillary transformation of beta-amyloid protein and tau protein polymerisation.

Morphological alterations of the choroid plexus in late-onset Alzheimer's disease.

Abstract Anomalies of the cerebrospinal fluid flow rate and composition that have been reported in patients suffering from Alzheimer’s disease (AD) could be related to alterations of the choroid plexuses (CD). Here we report a photonic and electron morphometric study in which we compared the height of CP epithelial cells and the thickness of their basement membrane on post-mortem samples from AD patients, age-matched controls and two new-borns. Ageing appeared associated with epithelial atrophy and basement membrane thickening, but these features were significantly accentuated in AD. These data suggest that a dramatic alteration of the secretion and filtration could be involved in the multiparametric pathogenesis of late-onset AD.

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

ABSTRACT 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-1β, 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.

Immunological and Nutritional Composition of Human Milk in Relation to Prematurity and Mothers' Parity During the First 2 Weeks of Lactation

BACKGROUND To investigate the effect of prematurity and parity on the dynamics of the major immunologic and nutritional proteins of human milk over the first 2 weeks of lactation. METHODS Microparticle-enhanced nephelometric immunoassays were developed for the quantification of alpha-lactalbumin, beta-casein, serum albumin, lactoferrin, and lysozyme in human milk. These components, immunoglobulin A, and total proteins were assayed in 368 individual samples collected from 74 mothers. RESULTS The dynamics of the major immunologic and nutritional proteins in early lactation presented similar patterns in preterm and term human milks. In comparison with term milk, preterm milk was globally characterized by higher concentrations of immune proteins and lower concentrations of nutritive proteins. These differences were increased by the degree of prematurity, which, however, influenced the absolute and relative protein concentrations differently, depending on the stage of lactation. The protein composition of term milk was similar, whatever the mothers parity. Conversely, the influence of prematurity on the levels of milk proteins during the first days of lactation was even greater in primiparous mothers. CONCLUSIONS This precise description of the composition of preterm and term milk, regarding the main nutritional and immunologic proteins, confirms the influence of both prematurity and parity on milk components and demonstrates the combined effect of these two conditions.

Immunological classification of acute myeloblastic leukemias : Relevance to patient outcome

Immunophenotyping is a major tool to assign acute leukemia blast cells to the myeloid lineage. However, because of the large heterogeneity of myeloid-related lineages, no clinically relevant immunological classification of acute myeloblastic leukemia (AML) has been devised so far. To attempt at formulating such a classification, we analyzed the pattern of expression of selected antigens, on blast cells collected at AML diagnosis. Patients were eligible if they had a first diagnosis of de novo AML and a sufficient number of blast cells for proper immunophenotyping. The relative expression of CD7, CD13, CD14, CD15, CD33, CD34, CD35, CD36, CD65, CD117, and HLA-DR were analyzed by cytometry in a test series of 176 consecutive AML cases. Statistical tools of clusterization allowed to remove antigens with overlapping distribution, leading us to propose an AML classification that was validated in a second AML cohort of 733 patients. We identified five AML subsets (MA to ME) based on the expression of seven antigens within four groups (CD13/CD33/CD117, CD7, CD35/CD36, CD15).-MA and MB-AML have exclusively myeloid features with seldom extramedullary disease and rare expression of lymphoid antigens. No cases of acute promyelocytic leukemia (APL) were observed within MB AML. MC AML have either myeloid or erythroblastic features. MD AML have more frequently high WBC counts than other subsets, which were related to the expression of CD35/CD36 and CD14 and to monoblastic differentiation. ME AML lack CD13, CD33, and CD117 but display signs of terminal myeloid differentiation. Specific independent prognostic factors were related to poor overall survival in each immunological subset: CD34+ (P<3 × 10−4) in MA AML, CD7+ in MB AML, non-APL cases (P<0.03) in MC AML, CD34+ (P<0.002) and CD14+ (P<0.03) in MD AML, CD14+ in ME AML (P<0.01). The inclusion of seven key markers in the immunophenotyping of AML allows a stratification into clinically relevant subsets with individual prognostic factors, which should be considered to define high-risk AML populations.

Elevated synovial expression of triggering receptor expressed on myeloid cells 1 in patients with septic arthritis or rheumatoid arthritis

Objective: To determine whether synovial expression of triggering receptor expressed on myeloid cells 1 (TREM-1) is upregulated in patients with distinct types of inflammatory or non-inflammatory arthritis. Methods: Synovial fluid (SF) samples were analysed for levels of soluble TREM-1 (sTREM; n  =  132), tumour necrosis factor alpha (TNFα, n  =  78) and leucocyte TREM-1 messenger RNA (n  =  48). Synovial tissue from four rheumatoid arthritis (RA) patients, two patients with Crohn’s-associated arthritis, one patient with ankylosing spondylitis and one patient with osteoarthritis were examined for TREM-1 expression by immunohistology, and three of the RA samples were also analysed by Western blotting. Results: Synovial fluid sTREM-1 levels in septic arthritis and RA were similar to each other and were each greater than those in gouty arthritis, non-septic/non-RA inflammatory arthritis and non-inflammatory arthritis. Synovial fluid TNFα and sTREM-1 levels correlated with each other, and sTREM-1 and leucocyte TREM-1 mRNA levels each correlated with SF leucocyte counts. TREM-1 in RA was expressed in situ in synovial tissue by cells of myelomonocytic lineage but was not detectably expressed in control osteoarthritis synovial tissue. Conclusions: Synovial TREM-1 expression is increased in septic arthritis and RA. In patients with acute inflammatory arthritis, elevated SF sTREM-1 levels may point the clinician to a diagnosis of septic arthritis or RA. In RA patients, targeting TREM-1 may have therapeutic benefits by reducing local proinflammatory cytokine and chemokine release.

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.

Monocyte-derived IL-10-secreting dendritic cells in choroid plexus epithelium

Choroid plexuses form an interface between peripheral blood and cerebrospinal fluid. Dendritic-like cells have been reported in a few studies of choroid plexuses in man. Here we used electron microscopy and immunophenotyping to precise the morphologic features and phenotype of these cells. Examination of 10 human choroid plexuses evidenced intra-epithelial dendritic cells with a clear cytoplasm, reniform nucleus and long expansions. These cells express MHC Class II, CD11b, CD14, CD32, CD68 and IL-10, but not CD40, CD80 or CD86, suggesting an immunosuppressive role for these dendritic cells. Their sentinel position could make them participate to the immunological silence of the brain.