Dynamical modeling of pro- and anti-inflammatory cytokines in the early stage of septic shock.

A dynamical model of the pathophysiological behaviors of IL18 and IL10 cytokines with their receptors is tested against data for the case of early sepsis. The proposed approach considers the surroundings (organs and bone marrow) and the different subsystems (cells and cyctokines). The interactions between blood cells, cytokines and the surroundings are described via mass balances. Cytokines are adsorbed onto associated receptors at the cell surface. The adsorption is described by the Langmuir model and gives rise to the production of more cytokines and associated receptors inside the cell. The quantities of pro and anti-inflammatory cytokines present in the body are combined to give global information via an inflammation level function which describes the patient's state. Data for parameter estimation comes from the Sepsis 48 H database. Comparisons between patient data and simulations are presented and are in good agreement. For the IL18/IL10 cytokine pair, 5 key parameters have been found. They are linked to pro-inflammatory IL18 cytokine and show that the early sepsis is driven by components of inflammatory character.

[Nuclear magnetic resonance based metabolic phenotyping for patient evaluations in operating rooms and intensive care units]. / Phénotypage métabolique par résonance magnétique nucléaire pour l'évaluation périopératoire et en réanimation.

Metabolic phenotyping consists in the identification of subtle and coordinated metabolic variations associated with various pathophysiological stimuli. Different analytical methods, such as nuclear magnetic resonance, allow the simultaneous quantification of a large number of metabolites. Statistical analyses of these spectra thus lead to the discrimination between samples and the identification of a metabolic phenotype corresponding to the effect under study. This approach allows the extraction of candidate biomarkers and the recovery of perturbed metabolic networks, driving to the generation of biochemical hypotheses (pathophysiological mechanisms, diagnostic tests, therapeutic targets…). Metabolic phenotyping could be useful in anaesthesiology and intensive care medicine for the evaluation, monitoring or diagnosis of life-threatening situations, to optimise patient managements. This review introduces the physical and statistical fundamentals of NMR-based metabolic phenotyping, describes the work already achieved by this approach in anaesthesiology and intensive care medicine. Finally, potential areas of interest are discussed for the perioperative and intensive management of patients, from newborns to adults.

Association between discordant immunological response to highly active anti-retroviral therapy, regulatory T cell percentage, immune cell activation and very low-level viraemia in HIV-infected patients.

The mechanisms sustaining the absence of complete immune recovery in HIV-infected patients upon long-term effective highly active anti-retroviral therapy (HAART) remain elusive. Immune activation, regulatory T cells (T(regs)) or very low-level viraemia (VLLV) have been alternatively suspected, but rarely investigated simultaneously. We performed a cross-sectional study in HIV-infected aviraemic subjects (mean duration of HAART: 12 years) to concomitantly assess parameters associated independently with inadequate immunological response. Patients were classified as complete immunological responders (cIR, n = 48) and inadequate immunological responders (iIR, n = 39), depending on the CD4(+) T cell count (> or < 500/mm(3)). Clinical and virological data (including very low-level viraemia) were collected. In parallel, immunophenotyping of CD4(+) lymphocytes, including T(reg) subsets, and CD8(+) T cells was performed. Percentages of activated CD4(+) T cells, T(regs), effector T(regs) and terminal effector T(regs) were found to be significantly elevated in iIR. Neither the percentage of activated CD8(+) T cells nor VLLV were found to be associated with iIR. In the multivariate analysis, nadir of CD4(+) T cell count and percentage of T(regs) were the only two parameters associated independently with iIR [odds ratio (OR) = 2·339, P = 0·001, and OR = 0·803, P = 0·041]. We present here the largest study investigating simultaneously the immune response to long-term HAART, activation of CD4(+) and CD8(+) T cells, T(reg) percentages and very low-level viraemia. Causative interactions between T(regs) and CD4(+) T cells should now be explored prospectively in a large patients cohort.

[Reversing ICU-acquired immunosuppression: an innovative biomarker-guided therapeutic strategy for decreasing sepsis mortality and nosocomial infection rate]. / Biomarqueurs et thérapeutiques individualisées dans la prise en charge de l'immunosuppression acquise en réanimation.

Septic syndromes (systemic inflammatory response associated with infection) remain a major although largely under-recognized health care problem and represent the first cause of mortality in intensive care units. Regarding immune response, it is now agreed that sepsis induces an anti-inflammatory process, acting as a negative feedback. This inhibitory mechanism becomes deleterious as nearly all immune functions are rapidly compromised. The magnitude and persistence over time of this immunosuppression is correlated with nosocomial infections and mortality. Decreased HLA-DR expression on monocytes/increased percentage of regulatory T cells are biomarkers identifying patients at risk who could benefit from immunotherapy. This review attempts to integrate these new facts into an up-to-date account of sepsis pathophysiology.

[Low monocytic HLA-DR expression and risk of secondary infection]. / Diminution de l'expression monocytaire de HLA-DR et risque d'infection hospitalière.

OBJECTIVES: The aim of this bibliographic review is to evaluate the usefulness of the measurement of HLA-DR expression on circulating monocytes (mHLA-DR) in predicting the development of nosocomial infections and unfavourable outcome in critically ill patients. DATA SOURCE: References obtained from the medical database PubMed in English and in French were reviewed. The keywords included separately or in combination were: HLA-DR antigens, sepsis, trauma, injuries, wounds, burns, stroke, pancreatitis, postoperative, prognostic, immunity, monocytic. DATA EXTRACTION: Data in selected articles were reviewed, clinical and basic science research relevant information were extracted. DATA SYNTHESIS: Low mHLA-DR expression appears as a marker for monocytic dysfunctions and immunosuppression, temporarily present in the majority of critically ill patients admitted to the ICU (sepsis, trauma injuries, postoperative, burns, pancreatitis and stroke). The decrease in mHLA-DR expression is a predictor of septic complications in all these clinical conditions. However, no predictive threshold value could be determined regarding unfavourable outcome. CONCLUSION: The monitoring of mHLA-DR expression could be a biomarker to detect ICU patients at high risk of developing secondary nosocomial infections. Those patients could probably benefit of preemptive strategies to prevent these infections.

Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls. II. Technical issues.

Cellular basophil activation tests (BAT) such as histamine or sulfidoleukotriene-release tests for allergy diagnosis have been available for some time, but expression of basophil-activation markers such as CD63 and CD203c detected by flow cytometry has attracted particular attention in recent years. Not only the potential but also the possible pitfalls of flow-cytometric BAT have been stressed recently. Some authors have suggested that the technical problems are still such that BAT should only be performed in specialist laboratories. In an earlier review based on our clinical experience obtained over several years, we showed that, even using different protocols, reproducible and meaningful clinical results can be obtained. In this paper, we review the current knowledge in relation to several technical issues and show that flow-cytometric BAT already represents a major advance in the field of in vitro allergy diagnosis. We conclude that there are no serious technical justifications for depriving allergic patients of clinically indicated BAT tests, which can be performed reliably by any laboratory with the appropriate experience in allergy diagnosis and flow cytometry.

Analysis of superantigenic toxin Vbeta T-cell signatures produced during cases of staphylococcal toxic shock syndrome and septic shock.

Most clinical isolates of Staphylococcus aureus harbour genes encoding superantigenic toxins that bind the Vbeta domain of T-cells, but little information is available concerning superantigenic toxin production during staphylococcal toxic shock syndrome (TSS) and septic shock. This prospective study investigated 14 patients with staphylococcal TSS or septic shock; the toxin gene profile of each isolate was determined and flow-cytometry was used to identify the discriminant Vbeta signature (DVbetaS) of each superantigenic toxin in vitro. Attempts were also made to identify in-vivo production of superantigenic toxin DVbetaS in patients' blood. The DVbetaS identified in vitro were: toxic shock syndrome toxin (TSST)-1, Vbeta 2; staphylococcal enterotoxin (SE), Vbeta 9, Vbeta 22; SEB, Vbeta 3, Vbeta 14, Vbeta 17; SED, Vbeta 1, Vbeta 8; egc, Vbeta 5.3, Vbeta 7.1, Vbeta 9, Vbeta 23; and SElK, Vbeta 5.1. The DVbetaS of TSST-1 and SEB were detected in patients with menstrual and non-menstrual TSS, respectively, whereas no Vbeta signature was detected during septic shock. All patients with septic shock (but only one patient with TSS) had lymphopenia and/or impaired cellular immunity. Detection of a superantigenic toxin DVbetaS may help to show which toxin is produced during staphylococcal TSS, thus confirming the diagnosis and hastening the administration of anti-toxin therapy. In contrast, this approach failed to demonstrate superantigenic toxin involvement in cases of septic shock. In this latter condition, a superantigenic toxin may not be produced by S. aureus, or its production may occur without expansion of targeted T-cells because of T-cell apoptosis and/or anergy.

Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls.

For the diagnosis of allergy, cellular basophil activation tests (BAT), e.g. histamine or sulfidoleukotriene release tests, have long been introduced, but the expression of basophil activation markers such as CD63 and CD203c detected by flow cytometry has attracted more recent attention. A recent opinion paper in this Journal has stressed not only the potential but also the possible pitfalls of flow-cytometric BAT. We have applied clinical validation of various BAT in various ways for several years, and our experience shows that these new technologies have more potentials and perspectives than pitfalls. A comprehensive review of clinically validated studies on allergy to aeroallergens, insect venoms, latex, food allergens and drugs, e.g. myorelaxants, beta-lactams, pyrazolones and non-steroidal anti-inflammatory drugs, as well as chronic urticaria shows clearly that even with different protocols, reproducible and meaningful results can be obtained. Although the available technologies may still be optimized and better standardized, there are no serious reasons to deprive allergic patients of clinically indicated BAT, which can be performed reliably by any laboratory with allergy and flow-cytometric capacity and expertise.

[Preliminary results in standardization of flow cytometry protocols for monocytic HLA-DR measurement and their application in the follow up of septic shock]. / Standardisation de la mesure de l'antigène HLA-DR monocytaire par cytométrie en flux: résultats préliminaires et application dans le suivi des chocs septiques.

In septic shock, the diminished HLA-DR expression on monocytes has been proposed as a marker of immunoparalysis that correlates with an increased risk for fatal outcome. The present study was designed to determine whether some differences in protocol procedures could lead to discrepant results in HLA-DR measurement. After establishing a reliable protocol, the second objective was to illustrate the immunoparalysis in patients with septic shock. HLA-DR measurement on monocytes was determined by means of flow cytometry in 54 healthy donors and 16 patients with septic shock. We demonstrated that storage temperature, storage duration before staining and red cells lysis constitute crucial steps in HLA-DR measurement. The precision results with coefficients of variation below 5%, were quite convincing for a manual immunoassay. At 48 hours after diagnosis of septic shock, we found severely decreased percentages of monocytes expressing HLA-DR in septic patients (24 +/- 4%, mean +/- SEM) in comparison with healthy donors (90 +/- 1%), p < 0.001). Furthermore, the persistence of a low level of monocytic HLA-DR (less than 50 %) at day 9 after admittance was associated with patients who died. This study illustrates the state of immunoparalysis in patients with septic shock and supports the potential interest in measuring HLA-DR expression on monocytes. However, multicenter studies are now needed to validate this parameter. Based on our analytical results, we conclude that a critical issue in such studies will be the capacity in each center to perform standardized measurement of HLA-DR. It should be remembered that this determination requires the definition of a common analytical procedure between laboratories participating in the trial.

Marked improvement of the basophil activation test by detecting CD203c instead of CD63.

BACKGROUND: The flow cytometric basophil activation test by detection of CD63 expression has been developed as an alternative method for in vitro diagnosis of IgE-mediated reactions to various allergens. Despite promising initial studies, the test remains disappointing in terms of sensitivity. CD203c has recently been demonstrated as a specific activation marker of basophils that is rapidly up-regulated after allergen challenge in sensitized patients. OBJECTIVE: The goal of the present study was to compare basophil activation tests by using either CD203c or CD63 in the diagnosis of immediate-type allergy to latex. METHODS: Twenty-seven patients (health care workers of our institution) who developed clinical features evocative of allergy after contact with latex were included and classified into two groups. Group 1 (n = 16) comprised true allergic patients who presented with typical signs of immediate allergic reaction associated with a positive skin test (prick test). Group 2 (n = 11) consisted of patients whose clinical history was not typical and had negative skin test. Twelve healthy subjects were also studied as controls. We compared the sensitivity of two triple-staining flow cytometric protocols measuring basophil activation after latex stimulation: CD45-IgE-CD63 and CD45-IgE-CD203c. RESULTS: The CD203c protocol showed a higher sensitivity than the CD63 protocol (75% vs. 50%). In comparison, latex-specific IgE sensitivity was found to be 69%. Furthermore, the magnitude of the basophil response was significantly higher with CD203c in comparison with CD63. Specificity was 100% for both protocols. CONCLUSION: Due to superior gating of basophils and a higher range of activation in response to allergen, the basophil activation test is markedly improved by use of CD203c instead of CD63.

Procalcitonin as an acute phase marker.

Procalcitonin is a 14-kDa protein encoded by the Calc-1 gene along with calcitonin and katacalcin. The function and regulation of this protein are quite different from those of the other gene products. Blood concentrations of procalcitonin are increased in systemic inflammation, especially when this is caused by bacterial infection. Studies of its behaviour in patients with bacterial sepsis have led to the proposal that it may be a useful marker of systemic bacterial infection, with greater specificity and sensitivity than acute phase proteins such as C-reactive protein.

Prostaglandin D2 is a potent chemoattractant for human eosinophils that acts via a novel DP receptor.

Prostaglandin D2 (PGD2) is released following exposure of asthmatics to allergen and acts via the adenylyl cyclase-coupled receptor for PGD2 (DP receptor). In this study, it is reported that human eosinophils possess this receptor, which would be expected to inhibit their activation. In contrast, it was found that prostaglandin D2 is a potent stimulator of eosinophil chemotaxis, actin polymerization, CD11b expression, and L-selectin shedding. These responses are specific for eosinophils, as neutrophils display little or no response to prostaglandin D2. They were not due to interaction with receptors for other prostanoids, as prostaglandins E2 and F(2alpha), U46619 (a thromboxane A2 analogue), and carbaprostacyclin (a prostacyclin analogue) displayed little or no activity. Furthermore, they were not shared by the selective DP receptor agonist BW245C and were not prevented by the selective DP receptor antagonist BWA868C, indicating that they were not mediated by DP receptors. In contrast, the prostaglandin D2 metabolite 13,14-dihydro-15-oxoprostaglandin D2 induced eosinophil activation but did not stimulate DP receptor-mediated adenosine 3',5'-cyclic monophosphate (cAMP) formation. These results indicate that in addition to the classic inhibitory DP1 receptor, eosinophils possess a second, novel DP2 receptor that is associated with PGD2-induced cell activation. These 2 receptors appear to interact to regulate eosinophil responses to PGD2, as blockade of DP1 receptor-mediated cAMP production by BWA868C resulted in enhanced DP2 receptor-mediated stimulation of CD11b expression. The balance between DP1 and DP2 receptors could determine the degree to which prostaglandin D2 can activate eosinophils and may play a role in eosinophil recruitment in asthma.

Human gammadelta T cells express a higher TCR/CD3 complex density than alphabeta T cells.

The aim of our study was to compare CD3 expression on gammadelta T cells and alphabeta T cells in human patients. The antigen density of TCR and CD3 on both subsets was assessed by a quantitative method in eight patients. In parallel, we developed and validated a reliable direct tricolor staining protocol that we tested on samples from hospitalized and healthy individuals (n = 60). Our results demonstrate that human gammadelta T cells constitutively express approximately twofold more of the TCR/CD3 complex than alphabeta T cells. We suggest that this enhanced expression of the TCR/CD3 complex could contribute to the higher reactivity of gammadelta T cells compared to alphabeta T cells. These clinical laboratory results confirm the fundamental data described elsewhere. gammadelta T cells deserve further clinical investigations to understand their precise role in human immunity.

[Standardization and automation of HLA B27 typing by flow cytometry: validation and comparison with microlymphocytotoxicity]. / Standardisation et automatisation du typage HLA B27 par une technique cytofluorimétrique: validation par comparaison à la microlymphocytoxicité.

One of the strongest known association between human leukocyte antigen (HLA) phenotype and disease is that of ankylosing spondylitis and HLA-B27. Thus, the determination of HLA-B27 status is an useful tool in the diagnosis of ankylosing spondylitis. To date, the 2 reference methods for HLA typing (microlymphocytotoxicity and molecular biology techniques), are costly in terms of both technician time and materials, and require a great deal of experience. In total, these techniques are not well-suited for routine application in clinical immunology laboratories. Use of flow cytometry has recently been applied for HLA-B27 typing. Nevertheless, it requires an extensive validation protocol. We developed a flow cytometry technique as standardized as possible (whole blood, automated lysing system, automated photomultiplier voltage calibration, definition of thresholds stable with time) and validated our results by comparison with microlymphocytotoxicity. In total, 326 samples were analyzed. We found 99% of concordant results between the 2 techniques, and neither false positive results nor false negative results with flow cytometry could be observed. These results illustrate the reliability of the protocol. It should be remembered that reference technique remains necessary to confirm the few results (< 1%) found in "grey zone" by flow cytometry. Standardization of flow cytometry techniques, as described in this work for HLA B27, seems to be a reasonable goal for the next decade in clinical immunology laboratories.

The clinical usefulness of the measurement of cytokines.

The utilization of accurate and sensitive methods for the measurement of cytokines in body fluids is prerequisite for the proper use of these mediators in clinical practice. Many factors contribute to the complexity of cytokine quantitation. Bioassays historically preceded immunoassays, which are now very popular, but there is a need for standardization. Nevertheless, due to the local effects of cytokines, the study of their blood levels is of limited value for an understanding of the pathophysiology of these mediators. This explains the development of alternative approaches to assess the ability of cells to produce cytokines. These include the Enzyme-Linked Immuno Spot Assay (ELISPOT), the measurement of cell-associated cytokines by flow cytometry, and the study of cytokine secretion by isolated peripheral blood mononuclear cells or by whole blood test. All these techniques, associated with a local detection of cytokines by immunohistochemistry or in situ hybridization and reverse transcriptase polymerase chain reaction, appear to be complementary tools for a better understanding of the biology of cytokines. Selected examples of possible clinical applications related to infectious diseases, cancer, autoimmune diseases, allergy, transplantation and preclinical evaluation of drugs and biotechnology products are given.

Procalcitonin and calcitonin gene-related peptide decrease LPS-induced tnf production by human circulating blood cells.

The pathogenesis of septic shock is mainly due to unregulated tumour necrosis factor-alpha (TNF-alpha) production. Procalcitonin (PCT) and calcitonin gene-related peptide (CGRP) are alternative transcription products of the calcitonin gene. Since high PCT levels have been described in human sepsis, and since CGRP inhibits TNF synthesis in rats, we examined the role of these peptides in the regulation of the inflammatory response during septic shock. LPS-induced TNF production was assessed using a human whole blood model. In this model, PCT (10(-7) M) and CGRP (10(-6) M) significantly inhibit TNF production by 27 and 24 % respectively. The effect of CGRP was reversed by CGRP 8-37 (10 microM), an antagonist of CGRP receptor. No effect on interleukin (IL)-1, IL-6 and IL-8 was found. This is the first description of an anti-inflammatory role for PCT and CGRP in humans.

Factors that predict chemotherapy-induced myelosuppression in lymphoma patients: role of the tumor necrosis factor ligand-receptor system.

PURPOSE: To analyze factors that predict the occurrence of chemotherapy-induced myelosuppression and, in particular, the role of the tumor necrosis factor (TNF) ligand-receptor system in lymphoma patients at the beginning of their treatment. PATIENTS AND METHODS: We investigated the predictive factors for myelosuppression after the first course of chemotherapy in a cohort of 101 consecutive, previously untreated lymphoma patients receiving regimens that include doxorubicin and cyclophosphamide. Plasma samples were tested at baseline by enzyme-linked immunosorbent assay for TNF and its soluble receptors. Univariate and multivariate analyses were performed with a forward regression procedure that included all of the parameters that were found to be significant in the univariate analysis. The dose of chemotherapy and the prophylactic treatment with granulocyte colony-stimulating factor were deliberately included in this model. RESULTS: Sixty-seven patients experienced World Health Organization (WHO) grade 4 neutropenia, and 37 patients experienced febrile neutropenia, which was responsible for WHO grade 2 through 4 infections in 23 patients. In multiparametric regression analysis, the occurrence of grade 4 neutropenia was associated with high doses of cyclophosphamide (odds ratio ¿OR, 19.8; P =.008) and high levels of soluble p75-R-TNF (OR, 8.52; P =.001). The duration of grade 4 neutropenia for more than 5 days was associated with the lack of hematopoietic growth factor administration (OR, 6.76; P =.004) and high levels of soluble p75-R-TNF (OR, 5.84; P =.0023). The occurrence of febrile neutropenia was associated with high doses of cyclophosphamide (OR, 4.7; P =.007), altered performance status (OR, 18.8; P <.0001) and high levels of soluble p75-R-TNF (OR, 3.49; P =.029). CONCLUSION: This study indicates that in addition to the dose of chemotherapy and the administration of hematopoietic growth factors, poor performance status and high p75-R-TNF levels can predict the occurrence of chemotherapy-induced myelosuppression in lymphoma patients. This model may help in selecting patients for prophylactic growth factor administration.