Valérie Faivre

Monocytic HLA-DR expression in intensive care patients: interest for prognosis and secondary infection prediction.

Objectives: To test early measurement of human leukocyte antigen‐DR expression on circulating monocytes (mHLA‐DR) as prognostic marker, and the trend of mHLA‐DR recovery for the prediction of late secondary infection risk in a large intensive care unit population. Design: Prospective, observational study over 16 mos. Setting: Intensive care unit in a tertiary teaching hospital. Inclusion criteria: Simplified Acute Physiology Score II >15, age >18 yrs. Measurements and Main Results: The mHLA‐DR was measured by flow cytometry within the first 3 days and twice a week until discharge. We used a logistic regression model for outcome prediction, and a competing risk approach to test the relationship between mHLA‐DR recovery (log (mHLA‐DR) slope) and incidence of secondary infection. A total of 283 consecutive patients suffering from various pathologies were monitored (Simplified Acute Physiology Score II = 39, Sepsis‐related Organ Failure Assessment of 5 on day 0). Early mHLA‐DR was decreased in the whole population, however, more deeply in sepsis (p < .0001). Low mHLA‐DR was associated with mortality in the whole population (p = .003), as in subgroups (nonseptic, neurologic, and septic), but not when adjusted on Simplified Acute Physiology Score II. In patients with a length of stay of >7 days (n = 70), the lower the slope of mHLA‐DR recovery, the higher the incidence of the first secondary infection (adjusted on early mHLA‐DR, p = .04). Conclusions: For a given severity, mHLA‐DR proved not to a predictive marker of outcome, but a weak trend of mHLA‐DR recovery was associated with an increased risk of secondary infection. Monitoring immune functions through mHLA‐DR in intensive care unit patients therefore could be useful to identify a high risk of secondary infection.

Levosimendan restores both systolic and diastolic cardiac performance in lipopolysaccharide-treated rabbits: comparison with dobutamine and milrinone.

Objective:Current treatment strategies for severe septic conditions (i.e., intravenous fluids, vasopressors, and cardiac inotropes) reestablish fluid balance and improve cardiac systole but do not address diastolic dysfunction. Our study aimed to fully characterize both systolic and diastolic abnormalities of sepsis-associated heart failure and to identify treatment that would support full-cycle cardiac improvement. Design:Endotoxin-injected rabbits, an animal model of abnormal cardiac function in human sepsis, were used to delineate cardiac abnormalities and to examine effects of drug treatments on heart systolic and diastolic function (n = 30); saline-injected animals served as comparators (n = 17). As treatment, three inotropes commonly used for treatment of cardiac failure were infused for 45 mins in separate animal groups—milrinone, dobutamine, and levosimendan. Measurements:Variables of left ventricular systolic and diastolic function were assessed with a pressure conductance catheter. Measurements were made before and after endotoxin/saline injection and before and after inotrope treatment. Results:Pressure–volume analyses of the left ventricle showed marked impairment in systolic function and in all indices of diastolic function (isovolumic relaxation time constant, left ventricular end-diastolic pressure, and end-diastolic pressure–volume relationship) in endotoxin-treated rabbits. The inotropes, milrinone, dobutamine, and levosimendan, could each partially or completely restore systolic function in the lipopolysaccharide-treated rabbits. However, only levosimendan therapy led to additional beneficial effects on left ventricular relaxation and diastolic function. Conclusions:Cardiac failure in severe sepsis results from impairments in both systolic and diastolic functions. Treatment with the calcium sensitizer levosimendan improved both systolic and diastolic cardiac functions in septic animals, but cyclic adenosine monophosphate–dependent inotropes milrinone and dobutamine only improved systolic function.

Systemic and renal macro- and microcirculatory responses to arginine vasopressin in endotoxic rabbits*

Objective:Arginine vasopressin is being used increasingly to treat vasodilatory hypotension, although little is known of its effects on regional perfusion. Arginine vasopressin hemodynamic effects in physiology are mainly mediated through the V1a receptor on blood vessels. To investigate this further, we studied the effect of arginine vasopressin on systemic and renal blood flow in anesthetized, ventilated rabbits given either intravenous saline or endotoxin, and the impact of blocking V1a receptors. Design:Prospective, randomized, controlled study. Setting:Animal research laboratory. Subjects:Male White New Zealand rabbits. Interventions:Measurement was made of mean arterial blood pressure, aortic and renal blood flow velocities (pulsed Doppler), and renal cortical and medullary flow (laser Doppler). Measurements and Main Results:In a first series of animals, incremental intravenous boluses of arginine vasopressin ranging from 1 to 1000 ng were administered 90 mins postendotoxin or saline. In control rabbits (n = 9), increasing doses of arginine vasopressin elevated mean arterial blood pressure but reduced both aortic and renal blood flow velocity and renal cortical flow (p < .05). In endotoxic animals (n = 6), arginine vasopressin produced a similar increase in mean arterial blood pressure although aortic flow was maintained while renal blood flow velocity increased, mostly in its diastolic component (p < .05). Pretreatment with the V1a receptor antagonist in a second series of animals blunted all the effects observed in both control (n = 5) and endotoxic (n = 6) animals, suggesting that arginine vasopressin acted mainly through V1a subtype in this early phase of sepsis. Conclusions:Preservation of renal blood flow with arginine vasopressin during endotoxemia, in particular to the cortex, suggests it could be a promising agent for hemodynamic support during septic shock.

Cardiac and Renal Effects of Levosimendan, Arginine Vasopressin, and Norepinephrine in Lipopolysaccharide-treated Rabbits

Background:Because sepsis-induced myocardial dysfunction related to sepsis is at least partially related to a decrease in cardiac myofilament response to calcium, the use of the new myofilament-calcium sensitizer, levosimendan, has been proposed. In addition, arginine vasopressin is increasingly proposed as a vasopressor in septic patients, although data on its effects on cardiac function are still scarce. The aim of the current study was to assess, invasively and noninvasively, whether levosimendan, arginine vasopressin, and norepinephrine, either alone or combined, may modify sepsis-induced myocardial dysfunction and renal hemodynamics. Methods:Thirty-six hours after lipopolysaccharide or saline administration, rabbits were studied either after slight sedation for echocardiography or after general anesthesia with sodium pentobarbital for the following measurements: aortic flow velocity and maximum acceleration of blood flow in the ascending aorta and renal macrocirculation and microcirculation. Results:Levosimendan improved, within 30 min of administration, both maximum acceleration of blood flow by 20 ± 12% (n = 8; P < 0.05) and left ventricular shortening fraction by a similar extent. Furthermore, low doses of arginine vasopressin markedly deteriorated cardiac function via an afterload-independent mechanism, even when animals were pretreated with levosimendan, whereas norepinephrine showed no detrimental effects on cardiac function. The study also showed that norepinephrine often improved renal medullary blood flow, whereas arginine vasopressin consistently decreased it. Conclusion:Levosimendan and norepinephrine both exert beneficial effects in endotoxemic animals and should be further explored in human sepsis trials.

Exocytosis acts as a modulator of the ILT4-mediated inhibition of neutrophil functions

Significance Neutrophils are key components of inflammatory responses and immune defense against pathogens. Neutrophil functions are tightly controlled by surface receptors. Our study shows that the engagement of immunoglobulin-like transcript 4 (ILT4) inhibitory receptor impairs phagocytosis and respiratory burst of neutrophils. Moreover, we found that following induction of neutrophil granule exocytosis, ILT4 expression increases as a result of the rapid translocation of an intracellular pool to the cell surface. This increase of ILT4 expression enhances the ILT4-mediated inhibition of neutrophil activity. Finally, ILT4 up-regulation on neutrophils is impaired in sepsis patients. These results reveal a unique mechanism of regulation of neutrophil functions through ILT4 and its exocytosis that may have implications in inflammatory disorders and immune responses. Neutrophils play a major role in inflammatory responses and immune defense against pathogens. Even though expression of inhibitory receptors has been reported on neutrophils, their role remains poorly defined. Here we show that primary human neutrophils expressed immunoglobulin-like transcript 4 (ILT4) inhibitory receptor and that this expression was induced during differentiation of the myelomonoblast PLB-985 cell line into “neutrophil-like” cells. Functional assays indicated that human leukocyte antigen G, the preferred ligand of ILT4, inhibited the phagocytic function of neutrophils. ILT4 engagement also impaired reactive oxygen species production induced through CD32a and both receptors were found colocalized into neutrophil lipid rafts. Moreover, neutrophil degranulation induced through inflammatory stimuli increased ILT4 expression as a result of the rapid translocation of an intracellular pool to the cell surface. Consequently to this ILT4 up-regulation, the human leukocyte antigen G-mediated inhibition of neutrophil phagocytic function was enhanced. Finally, we found that ILT4 up-regulation induced on healthy donor neutrophils following stimulation was impaired in presence of plasma from patients with sepsis. Similarly, ILT4 up-regulation was inhibited in neutrophils from septic patients. Altogether, our results reveal a unique mechanism of regulation of neutrophil functions through ILT4 and its exocytosis that may have implications in inflammatory disorders.

Human Monocytes Differentiate into Dendritic Cells Subsets that Induce Anergic and Regulatory T Cells in Sepsis

Background Sepsis is a multifactorial pathology with high susceptibility to secondary infections. Innate and adaptive immunity are affected in sepsis, including monocyte deactivation. Methodology/Principal Findings To better understand the effects of alterations in monocytes on the regulation of immune responses during sepsis, we analyzed their differentiation in dendritic cell (DC). Cells from septic patients differentiated overwhelmingly into CD1a−negative DC, a population that was only a minor subset in controls and that is so far poorly characterized. Analysis of T cell responses induced with purified CD1a−negative and CD1a+ DC indicated that (i) CD1a−negative DC from both healthy individuals and septic patients fail to induce T cell proliferation, (ii) TGFβ and IL-4 were strongly produced in mixed leukocyte reaction (MLR) with control CD1a−negative DC; reduced levels were produced with patients DC together with a slight induction of IFNγ, (iii) compared to controls, CD1a+ DC derived from septic patients induced 3-fold more Foxp3+ T cells. Conclusion/Significance Our results indicate a strong shift in DC populations derived from septic patients’ monocytes with expanded cell subsets that induce either T cell anergy or proliferation of T cells with regulatory potential. Lower regulatory cytokines induction on a per cell basis by CD1a−negative dendritic cells from patients points however to a down regulation of immune suppressive abilities in these cells.

ALVEOLAR NEUTROPHIL OXIDATIVE BURST AND β2 INTEGRIN: EXPRESSION IN EXPERIMENTAL ACUTE PULMONARY INFLAMMATION ARE NOT MODIFIED BY INHALED NITRIC OXIDE

It was recently proposed that nitric oxide (NO) inhalation interferes with polymorphonuclear neutrophil (PMN) activation status during acute pulmonary inflammation, although variable results have been observed considering timing of NO administration, species, and model differences. After intratracheal administration of lipopolysaccharide (LPS) in rats, we characterized pulmonary inflammatory reaction (lung wet, dry, and wet to dry weights) and, using flow cytometry, the activation status (H2O2 production and β2 integrin CD11b/CD18 expression) of PMN obtained from blood and from bronchoalveolar lavage (BAL). Eight hours after LPS injection, rats received for an additional 10 h, at a same Fio2 (85%), either 15 parts per million NO or the same gas flow of nitrogen. We found that 18 h after LPS, lung wet, dry, and wet-to-dry weights, H2O2 production, and CD11b/CD18 expression were increased. PMN obtained from BAL were highly activated as evidenced by an already maximal expression of the β2 integrin CD11b/CD18, whereas the high H2O2 production at basal state could be further enhanced after ex vivo stimulation. Blood PMN were not different from control cells at basal state; however, their increased capacity to be stimulated ex vivo suggested an in vivo priming effect of intratracheal LPS. In conclusion, inhaled NO, given with a high Fio2, in the presence of this established endotoxinic lung injury did not reverse the markers of PMN activation studied nor lung edema formation in this rat model.

Elevated production of radical oxygen species by polymorphonuclear neutrophils in cerebrospinal fluid infection

BackgroundCentral nervous system infection is a daily concern in neurointensive care; however, diagnosis remains difficult because classical criteria based on cerebrospinal fluid (CSF) analysis are difficult to interpret in post-trauma or neurosurgery patients after recent bleeding. A rapid, specific, sensitive test to diagnose CSF infection would help streamline therapeutic decisions in clinical practice and limit the risk of multiresistant bacteria. We hypothesized that polymorphonuclear neutrophil (PMN) phenotype and radical oxygen species (ROS) production in CSF may be specific to the presence of infection.MethodsThis study included 30 patients with suspected CSF infection with ventricular hemorrhage requiring external ventricular drainage, and 13 patients after trauma or surgery. Criteria for evaluating CSF infection included positive culture and > 100 leukocytes/mm3. Analysis of PMN phenotype was performed using flow cytometry (CD16, CD11b, and CD62L). ROS production was analyzed through luminometry (luminol).ResultsInfected CSF exhibited higher production of ROS compared with noninfected CSF. PMNs in CSF exhibited low CD16 and high annexin V expression, suggesting apoptosis.ConclusionsMeasurement of ROS production may discriminate infected from noninfected CSF. This simple test would be easy to employ in clinical practice to improve CSF infection management.

Downregulation of Blood Monocyte HLA-DR in ICU Patients Is Also Present in Bone Marrow Cells

Background The downregulation of blood monocyte HLA-DR expression also occurs in tissue infiltrative cells in a context of acute clinical inflammation, especially sepsis. This context favors the development of secondary infections and results from various mechanisms. Little is known about HLA-DR expression on bone marrow (BM) cells of the monocyte lineage, the source of circulating monocytes. This study analyzed the BM HLA-DR expression in ICU patients compared to BM monocytes from non-ICU patients and to blood monocytes of control healthy donors. A potential dysfunction of myeloid differentiation was investigated in a sub-population of these ICU patients to characterize the phenotype of the immature forms of monocytes and granulocytes in BM. Methods and Findings BM and blood were drawn from 33 ICU and 9 non-ICU patients having a BM analysis to precise the etiology of abnormal low count in blood cells. The data were compared with blood cells of 28 control donors. Flow cytometry was used for both HLA-DR expression and phenotyping of immature forms of monocytes and granulocytes. HLA-DR expression was downregulated in both blood and BM monocyte in ICU patients compared to BM of non-ICU patients and blood of control donors. Amplitude of HLA-DR downregulation was comparable in septic and non-septic ICU patients. The phenotype of immature forms of monocytes and granulocytes in BM (n = 11) did not show abnormal myeloid (monocyte + granulocyte) differentiation. Conclusion The downregulation of HLA-DR in BM monocyte lineage is present in ICU patients without major changes in myeloid cells. It may result from a regulation mediated by soluble and/or neuro-endocrine factors present in BM cell microenvironment.

Multicenter testing of the rapid quantification of radical oxygen species in cerebrospinal fluid to diagnose bacterial meningitis.

Purpose Meningitis is a serious concern after traumatic brain injury (TBI) or neurosurgery. This study tested the level of reactive oxygen species (ROS) in cerebrospinal fluid (CSF) to diagnose meningitis in febrile patients several days after trauma or surgery. Methods Febrile patients (temperature > 38°C) after TBI or neurosurgery were included prospectively. ROS were measured in CSF within 4 hours after sampling using luminescence in the basal state and after cell stimulation with phorbol 12-myristate 13-acetate (PMA). The study was conducted in a single-center cohort 1 (n = 54, training cohort) and then in a multicenter cohort 2 (n = 136, testing cohort) in the Intensive Care and Neurosurgery departments of two teaching hospitals. The performance of the ROS test was compared with classical CSF criteria, and a diagnostic decision for meningitis was made by two blinded experts. Results The production of ROS was higher in the CSF of meningitis patients than in non-infected CSF, both in the basal state and after PMA stimulation. In cohort 1, ROS production was associated with a diagnosis of meningitis with an AUC of 0.814 (95% confidence interval (CI) [0.684–0.820]) for steady-state and 0.818 (95% CI [0.655–0.821]) for PMA-activated conditions. The best threshold value obtained in cohort 1 was tested in cohort 2 and showed high negative predictive values and low negative likelihood ratios of 0.94 and 0.36 in the basal state, respectively, and 0.96 and 0.24 after PMA stimulation, respectively. Conclusion The ROS test in CSF appeared suitable for eliminating a diagnosis of bacterial meningitis.