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As highlighted by the last international consensus definition for sepsis and septic shock (sepsis-3), sepsis comes from a complex relationship between a pathogen and a dysregulated host response. To date, the treatment of sepsis is based on antimicrobial treatment, source control, and organ support. Extracorporeal blood purification therapies have been proposed as adjuvant therapies to modulate the dysregulated inflammatory response. These therapies aim mostly at removing inflammatory mediators (cytokines) and endotoxins from the blood. However, so far, they failed to clearly demonstrate an improvement in patient survival when evaluated in randomized trials. Recently, new devices directly targeting the primary determinants of sepsis, e.g., the pathogen itself and the host immune cells, have been developed. This short review aimed at presenting new blood purification devices that have recently been developed to target pathogens and immune cells. For each, we will present the mechanism of action of the therapy and discuss the related literature., (© 2022 S. Karger AG, Basel.)

Introduction: We analysed blood DNAemia of TTV and four herpesviruses (CMV, EBV, HHV6, and HSV-1) in the REAnimation Low Immune Status Marker (REALISM) cohort of critically ill patients who had presented with either sepsis, burns, severe trauma, or major surgery. The aim was to identify common features related to virus and injury-associated pathologies and specific features linking one or several viruses to a particular pathological context., Methods: Overall and individual viral DNAemia were measured over a month using quantitative PCR assays from the 377 patients in the REALISM cohort. These patients were characterised by clinical outcomes [severity scores, mortality, Intensive Care Unit (ICU)-acquired infection (IAI)] and 48 parameters defining their host response after injury (cell populations, immune functional assays, and biomarkers). Association between viraemic event and clinical outcomes or immune markers was assessed using χ 2 -test or exact Fisher's test for qualitative variables and Wilcoxon test for continuous variables., Results: The cumulative incidence of viral DNAemia increased from below 4% at ICU admission to 35% for each herpesvirus during the first month. EBV, HSV1, HHV6, and CMV were detected in 18%, 12%, 10%, and 9% of patients, respectively. The incidence of high TTV viraemia (>10,000 copies/ml) increased from 11% to 15% during the same period. Herpesvirus viraemia was associated with severity at admission; CMV and HHV6 viraemia correlated with mortality during the first week and over the month. The presence of individual herpesvirus during the first month was significantly associated (p < 0.001) with the occurrence of IAI, whilst herpesvirus DNAemia coupled with high TTV viraemia during the very first week was associated with IAI. Herpesvirus viraemia was associated with a lasting exacerbated host immune response, with concurrent profound immune suppression and hyper inflammation, and delayed return to immune homeostasis. The percentage of patients presenting with herpesvirus DNAemia was significantly higher in sepsis than in all other groups. Primary infection in the hospital and high IL10 levels might favour EBV and CMV reactivation., Conclusion: In this cohort of ICU patients, phenotypic differences were observed between TTV and herpesviruses DNAemia. The higher prevalence of herpesvirus DNAemia in sepsis hints at further studies that may enable a better in vivo understanding of host determinants of herpesvirus viral reactivation. Furthermore, our data suggest that EBV and TTV may be useful as additional markers to predict clinical deterioration in ICU patients., Competing Interests: FM, VM, AP, KB-P, and JT are employees of bioMérieux SA, an in vitro diagnostic company. LD was employed by IVIDATA. BM was employed by company Soladis Inc. FV, GM, AP, TR, and JT are employees of Hospices Civils de Lyon. FM, LD, BM, VM, AP, FV, GM, AL, TR, KB-P, and JT work in a joint research unit, co-funded by the Hospices Civils de Lyon and bioMérieux. LT is an employee of and hold stock and shares in GlaxoSmithKline. LQ is an employee of Sanofi Pasteur. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mallet, Diouf, Meunier, Perret, Reynier, Leissner, Quemeneur, Griffiths, Moucadel, Pachot, Venet, Monneret, Lepape, Rimmelé, Tan, Brengel-Pesce and Textoris.)

Objectives: Decreased expression of human leukocyte antigen-DR on monocytes (mHLA-DR) is recognized as the most appropriate marker for the monitoring of immune alterations in septic patients and critically ill subjects. Its measurement has been established for years by flow cytometry, but remains under-used due to pre-analytical constraints. The objectives of the present work were to develop a rapid and robust one-step protocol., Methods: A novel, simplified protocol has been developed to measure mHLA-DR in whole blood using flow cytometry. It is a one-step procedure that includes red cell lysis, antibodies, and fixative reagents. It has been compared to the standardized routine protocol in two consecutive cohorts of septic shock patients (n = 37). Finally, the protocol was applied to a few subjects in point-of-care settings, by collecting capillary blood from fingerpricks., Results: Strong correlation was observed between the one-step method and routine protocol in 24 patients. After testing several stabilizing agents, the procedure was further optimized by adding a low-dose formaldehyde to the stain and lyse solution. This improved method was tested in a second cohort of 13 patients, and again strongly correlated to the routine protocol. Finally, the fingerprick and venous puncture samples were shown to provide similar results., Conclusions: The present work demonstrates the feasibility of a bedside protocol for flow cytometry measurement of mHLA-DR in critically ill subjects. This helps overcome pre-analytical constraints previously identified, which have limited wider use of this biomarker in intensive care units. In addition, preliminary results from fingerprick samples are promising., Competing Interests: The authors report no conflicts of interest., (Copyright © 2020 by the Shock Society.)

Sepsis is a worldwide health priority characterized by the occurrence of severe immunosuppression associated with increased risk of death and secondary infections. Interleukin 10 (IL-10) is a potent immunosuppressive cytokine which plasma concentration is increased in septic patients in association with deleterious outcomes. Despite studies evaluating IL-10 production in specific subpopulations of purified cells, the concomitant description of IL-10 production in monocytes and lymphocytes in septic patients' whole blood has never been performed. In this pilot study, we characterized IL-10 producing leukocytes in septic shock patients through whole blood intracellular staining by flow cytometry. Twelve adult septic shock patients and 9 healthy volunteers were included. Intracellular tumor necrosis factor-α (TNFα) and IL-10 productions after lipopolysaccharide stimulation by monocytes and IL-10 production after PMA/Ionomycine stimulation by lymphocytes were evaluated. Standard immunomonitoring (HLA-DR expression on monocytes, CD4+ T lymphocyte count) of patients was also performed. TNFα expression by stimulated monocytes was reduced in patients compared with controls while IL-10 production was increased. This was correlated with a reduced monocyte HLA-DR expression. B cells, CD4+, and CD4- T lymphocytes were the three circulating IL-10 producing lymphocyte subsets in both patients and controls. No difference in IL-10 production between patients and controls was observed for B and CD4- T cells. However, IL-10 production by CD4+ T lymphocytes significantly increased in patients in parallel with reduced CD4+ T cells number. Parameters reflecting altered monocyte (increased IL-10 production, decreased HLA-DR expression and decreased TNFα synthesis) and CD4+ T lymphocyte (increased IL-10 production, decreased circulating number) responses were correlated. Using a novel technique for intracellular cytokine measurement in whole blood, our results identify monocytes and CD4+ T cells as the main IL-10 producers in septic patients' whole blood and illustrate the development of a global immunosuppressive profile in septic shock. Overall, these preliminary results add to our understanding of the global increase in IL-10 production induced by septic shock. Further research is mandatory to determine the pathophysiological mechanisms leading to such increased IL-10 production in monocytes and CD4+ T cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fabri, Kandara, Coudereau, Gossez, Abraham, Monard, Cour, Rimmelé, Argaud, Monneret and Venet.)

Sepsis is one of the leading causes of in-hospital mortality. In some patients, sepsis-induced immunosuppression is associated with increased risk of death and secondary infections. In oncology, myeloid-derived suppressor cells (MDSCs) have been described to inhibit various immune functions. Monocytic MDSCs (M-MDSCs) represent a subtype of MDSCs. The objectives of the present study was to determine by flow cytometry the % M-MDSCs (among total monocyte population) in a cohort of septic shock patients and to assess its association with deleterious outcomes: 28-day mortality and occurrence of nosocomial infections. The cohort included 301 patients. They presented with immune alterations usually found 3-4 days after the onset of shock: lymphopenia (median T CD4: 362/μL, quartiles: 235-591/μL) and low monocytic HLA-DR expression (median: 4,944 AB/C, quartiles: 3,104-8,266 AB/C). From admission until the end of the first week, % M-MDSCs was significantly increased in patients compared with healthy donors (p < 0.01). In early samples, no association with deleterious outcomes was identified. However, after one week, patients who were going to die or to develop nosocomial infections presented with significantly higher % M-MDSCs than non-survivors and non-infected patients (p < 0.01). These associations remained significant in multivariate analyses, odds ratio of 4.4 (p = 0.001) regarding 28-day mortality and 2.4 (p = 0.013) regarding occurrence of nosocomial infections. In conclusion, % M-MDSCs was markedly increased after septic shock. One week-persistence of an increased proportion of M-MDSCs was associated with unfavorable outcomes.

The complexity of sepsis pathophysiology hinders patient management and therapeutic decisions. In this proof-of-concept study we characterised the underlying host immune response alterations using a standardised immune functional assay (IFA) in order to stratify a sepsis population. In septic shock patients, ex vivo LPS and SEB stimulations modulated, respectively, 5.3% (1/19) and 57.1% (12/21) of the pathways modulated in healthy volunteers (HV), highlighting deeper alterations induced by LPS than by SEB. SEB-based clustering, identified 3 severity-based groups of septic patients significantly different regarding mHLA-DR expression and TNFα level post-LPS, as well as 28-day mortality, and nosocomial infections. Combining the results from two independent cohorts gathering 20 HV and 60 patients, 1 cluster grouped all HV with 12% of patients. The second cluster grouped 42% of patients and contained all non-survivors. The third cluster grouped 46% of patients, including 78% of those with nosocomial infections. The molecular features of these clusters indicated a distinctive contribution of previously described genes defining a "healthy-immune response" and a "sepsis-related host response". The third cluster was characterised by potential immune recovery that underlines the possible added value of SEB-based IFA to capture the sepsis immune response and contribute to personalised management.

Background: Critical illness such as sepsis is a life-threatening syndrome defined as a dysregulated host response to infection and is characterized by patients exhibiting impaired immune response. In the field of diagnosis, a gap still remains in identifying the immune profile of critically ill patients in the intensive care unit (ICU)., Methods: A new multiplex immune profiling panel (IPP) prototype was assessed for its ability to semiquantify messenger RNA immune-related markers directly from blood, using the FilmArray System, in less than an hour. Samples from 30 healthy volunteers were used for the technical assessment of the IPP tool. Then the tool was clinically assessed using samples from 10 healthy volunteers and 20 septic shock patients stratified using human leukocyte antigen-DR expression on monocytes (mHLA-DR)., Results: The IPP prototype consists of 16 biomarkers that target the immune response. The majority of the assays had a linear expression with different RNA inputs and a coefficient of determination (R2) > 0.8. Results from the IPP pouch were comparable to standard quantitative polymerase chain reaction and the assays were within the limits of agreement in Bland-Altman analysis. Quantification cycle values of the target genes were normalized against reference genes and confirmed to account for the different cell count and technical variability. The clinical assessment of the IPP markers demonstrated various gene modulations that could distinctly differentiate 3 profiles: healthy volunteers, intermediate mHLA-DR septic shock patients, and low mHLA-DR septic shock patients., Conclusions: The use of IPP showed great potential for the development of a fully automated, rapid, and easy-to-use immune profiling tool. The IPP tool may be used in the future to stratify critically ill patients in the ICU according to their immune status. Such stratification will enable personalized management of patients and guide treatments to avoid secondary infections and lower mortality., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Decreased monocytic (m)HLA-DR expression is the most studied biomarker of sepsis-induced immunosuppression. To date, little is known about the relationship between sepsis characteristics, such as the site of infection, causative pathogen, or severity of disease, and mHLA-DR expression kinetics., Methods: We evaluated mHLA-DR expression kinetics in 241 septic shock patients with different primary sites of infection and pathogens. Furthermore, we used unsupervised clustering analysis to identify mHLA-DR trajectories and evaluated their association with outcome parameters., Results: No differences in mHLA-DR expression kinetics were found between groups of patients with different sites of infection (abdominal vs. respiratory, p = 0.13; abdominal vs. urinary tract, p = 0.53) and between pathogen categories (Gram-positive vs. Gram-negative, p = 0.54; Gram-positive vs. negative cultures, p = 0.84). The mHLA-DR expression kinetics differed between survivors and non-survivors (p < 0.001), with an increase over time in survivors only. Furthermore, we identified three mHLA-DR trajectories ('early improvers', 'delayed or non-improvers' and 'decliners'). The probability for adverse outcome (secondary infection or death) was higher in the delayed or non-improvers and decliners vs. the early improvers (delayed or non-improvers log-rank p = 0.03, adjusted hazard ratio 2.0 [95% CI 1.0-4.0], p = 0.057 and decliners log-rank p = 0.01, adjusted hazard ratio 2.8 [95% CI 1.1-7.1], p = 0.03)., Conclusion: Sites of primary infection or causative pathogens are not associated with mHLA-DR expression kinetics in septic shock patients. However, patients showing delayed or no improvement in or a declining mHLA-DR expression have a higher risk for adverse outcome compared with patients exhibiting a swift increase in mHLA-DR expression. Our study signifies that changes in mHLA-DR expression over time, and not absolute values or static measurements, are of clinical importance in septic shock patients.

Background: Flow cytometry is a powerful analytical technique. However, it requires time-consuming, multi-step sample procedure. A new protocol was developed to perform extracellular staining and red blood cell lysis in one step, using dry antibodies. Common markers of white blood cells as well as sepsis biomarkers were tested as a model for modulated antigen expression., Methods: Peripheral blood was stained using the reference and the one-step methods. Recruitment and staining of CD3-, CD4-, CD8-, CD14-, and CD15-positive cells were analyzed. Then, protocol modifications were tested for optimization. Finally, the one-step method was evaluated on subjects in septic conditions, by measuring expressions of CD64 and of HLA-DR., Results: No statistical differences were observed between methods when comparing the proportions of cells. The procedure was optimized by decreasing blood volume from 100 μL to 5 μL, lysis from 1 mL to 500 μL, and time from 30 to 15 min. In the blood samples from septic subjects, an increase of CD64 on neutrophils and a decrease of HLA-DR on monocytes were observed., Conclusions: The one-step method, described here-in, enables an accurate, streamlined flow cytometry sample preparation protocol. The simplified phenotyping procedure reduces training requirements and could help overcome logistic constraints in many flow cytometry applications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Sepsis is the leading cause of mortality for critically ill patients worldwide. Patients develop T lymphocyte dysfunctions leading to T-cell exhaustion associated with increased risk of death. As interleukin-7 (IL-7) is currently tested in clinical trials to reverse these dysfunctions, it is important to evaluate the expression of its specific CD127 receptor on the T-cell surface of patients with septic shock. Moreover, the CD127 low PD-1 high phenotype has been proposed as a T-cell exhaustion marker in chronic viral infections but has never been evaluated in sepsis. The objective of this study was first to evaluate CD127 and CD127 low PD-1 high phenotype in septic shock in parallel with functional T-cell alterations. Second, we aimed to reproduce septic shock-induced T-cell alterations in an ex vivo model., Methods: CD127 expression was followed at the protein and mRNA levels in patients with septic shock and healthy volunteers. CD127 low PD-1 high phenotype was also evaluated in parallel with T-cell functional alterations after ex vivo activation. To reproduce T-cell alterations observed in patients, purified T cells from healthy volunteers were activated ex vivo and their phenotype and function were evaluated., Results: In patients, neither CD127 expression nor its corresponding mRNA transcript level was modified compared with normal values. However, the percentage of CD127 low PD-1 high T cells was increased while T cells also presented functional alterations. CD127 low PD-1 high T cells co-expressed HLA-DR, an activation marker, suggesting a role for T-cell activation in the development of this phenotype. Indeed, T-cell receptor (TCR) activation of normal T lymphocytes ex vivo reproduced the increase of CD127 low PD-1 high T cells and functional alterations following a second stimulation, as observed in patients. Finally, in this model, as observed in patients, IL-7 could improve T-cell proliferation., Conclusions: The proportion of CD127 low PD-1 high T cells in patients was increased compared with healthy volunteers, although no global CD127 regulation was observed. Our results suggest that TCR activation participates in the occurrence of this T-cell population and in the development of T-cell alterations in septic shock. Furthermore, we provide an ex vivo model for the investigation of the pathophysiology of sepsis-induced T-cell immunosuppression and the testing of innovative immunostimulant treatments.

Although human endogenous retroviruses (HERVs) expression is a growing subject of interest, no study focused before on specific endogenous retroviruses loci activation in severely injured patients. Yet, HERV reactivation is observed in immunity compromised settings like some cancers and auto-immune diseases. Our objective was to assess the transcriptional modulation of HERVs in burn, trauma and septic shock patients. We analyzed HERV transcriptome with microarray data from whole blood samples of a burn cohort ( n = 30), a trauma cohort ( n = 105) and 2 septic shock cohorts ( n = 28, n = 51), and healthy volunteers (HV, n = 60). We described expression of the 337 probesets targeting HERV from U133 plus 2.0 microarray in each dataset and then we compared HERVs transcriptional modulation of patients compared to healthy volunteers. Although all 4 cohorts contained critically ill patients, the majority of the 337 HERVs was not expressed (around 74% in mean). Each cohort had differentially expressed probesets in patients compared to HV (from 19 to 46). Strikingly, 5 HERVs were in common in all types of severely injured patients, with 4 being up-modulated in patients. We highlighted co-expressed profiles between HERV and nearby CD55 and CD300LF genes as well as autonomous HERV expression. We suggest an inflammatory-specific HERV transcriptional response, and importantly, we introduce that the HERVs close to immunity-related genes might have a role on its expression.

Sepsis, which is the leading cause of death in intensive care units (ICU), has been acknowledged as a global health priority by the WHO in 2017. Identification of biomarkers allowing early stratification and recognition of patients at higher risk of death is crucial. One promising biomarker candidate is pentraxin-3 (PTX3); initially elevated and persistently increased plasma concentration in septic patients has been associated with increased mortality. PTX3 is an acute phase protein mainly stored in neutrophil granules. These cells are responsible for rapid and prompt release of PTX3 in inflammatory context, but the cellular origin responsible for successive days' elevation in sepsis remains unknown. Upon inflammatory stimulation, PTX3 can also be produced by other cell types, including endothelial and immune cells. As in septic patients immune alterations have been described, we therefore sought to investigate whether such cells participated in the elevation of PTX3 over the first days after septic shock onset. To address this point, PTX3 was measured in plasma from septic shock patients at day 3 after ICU admission as well as in healthy volunteers (HV), and the capacity of whole blood cells to secrete PTX3 after inflammatory stimulation was evaluated ex vivo . A significantly mean higher (100-fold) concentration of plasma PTX3 was found in patients compared to HV, which was likely due to the inflammation-induced initial release of the pre-existing PTX3 reservoir contained in neutrophils. Strikingly, when whole blood was stimulated ex vivo with LPS no significant difference between patients and HV in PTX3 release was found. This was in contrast with TNFα which decreased production was illustrative of the endotoxin tolerance phenomenon occurring in septic patients. Then, the release of PTX3 protein from a HV neutrophil-free PBMC endotoxin tolerance model was investigated. At the transcriptional level, PTX3 seems to be a weakly tolerizable gene similar to TNFα. Conversely, increased protein levels observed in anergy condition reflects a non-tolerizable phenotype, more likely to an anti-inflammatory marker. Hence, altered immune cells still have the ability to produce PTX3 in response to an inflammatory trigger, and therefore circulating white blood cell subset could be responsible of the sustained PTX3 plasma levels over the first days of sepsis setting.

Background: Sepsis is a dysregulated host response to an infection and can result in organ dysfunctions and death. Extracorporeal blood purification techniques aim to improve the prognosis of these patients by modulating the unbalanced immune response. This study reports our experience with the use of the oXiris® membrane for septic shock patients requiring continuous renal replacement therapy (CRRT)., Summary: Thirty-one patients were diagnosed with septic shock and underwent CRRT with the oXiris® membrane between 2014 and 2019. We compared the observed hospital mortality with that predicted by the Simplified Acute Physiology Score II (SAPS II). Change in the Sequential Organ Failure Assessment (SOFA) score and of the main clinical and biological parameters over time were analyzed. Hospital mortality was lower than predicted for the most severe patients (60 vs. 91% for the [74-87] SAPS II quartile and 70 vs. 98% for the [87-163] SAPS II quartile, p < 0.02). There was no significant improvement in the SOFA score from 0h to 48 h. An 88% relative decrease in norepinephrine infusion was observed (median at 0 h was 1.69 [0.52-2.45] µg/kg/min; at 48 h it was 0.20 [0.09-1.14] µg/kg/min, p = 0.002). Lactataemia and pH were significantly improved over time. Patients with intra-abdominal sepsis as well as those with Gram-negative bacilli (GNB) infections seemed to benefit the most from the therapy. Key Messages: CRRT with the oXiris® haemofilter resulted in higher observed survival than predicted by a severity score (SAPS II) for the most severe patients. Haemodynamic status and lactataemia appeared to improve, especially in intra-abdominal sepsis and GNB infections., (© 2019 The Author(s). Published by S. Karger AG, Basel.)

Innovative single cell technologies such as mass cytometry (CyTOF) widen possibilities to deeply improve characterisation of immune alterations mechanisms in human diseases. So far, CyTOF has not been used in sepsis - a condition characterized by complex immune disorders. Here, we evaluated feasibility of CyTOF analysis in patients with septic shock. We designed a mass cytometry panel of 25 extracellular markers to study mononuclear cells from 5 septic shock patients and 5 healthy donors. We explored single-cell data with global and specific unsupervised approaches such as heatmaps, SPADE and viSNE. We first validated relevance of our CyTOF results by highlighting established immune hallmarks of sepsis, such as decreased monocyte HLA-DR expression and increased expressions of PD1 and PD-L1 on CD4 T cells and monocytes. We then showed that CyTOF analysis reveals novel aspects of sepsis-induced immune alterations, e.g. B cell shift towards plasma cell differentiation and uniform response of several monocyte markers defining an immune signature in septic patients. This proof of concept study demonstrates CyTOF suitability to analyse immune features of septic patients. Mass cytometry could thus represent a powerful tool to identify novel pathophysiological mechanisms and therapeutic targets for immunotherapy in septic shock patients.

Objectives: Septic shock is the primary cause of death in ICUs. A better comprehension of its pathophysiology, in particular, the immune alteration mechanisms, opened new therapeutic perspectives such as the recombinant interleukin-7. The use of biomarkers could improve the identification of eligible patients for this therapy. The soluble form of the interleukin-7 appears as a promising candidate in this regard since an association between its high plasmatic level and mortality in critically ill patients has been demonstrated. Because there are no data available on the transcriptional regulation of the interleukin-7 receptor in such patients, this study aimed to explore the expression level of different interleukin-7 receptor transcripts after septic shock and evaluate their association with mortality., Design: Retrospective discovery cohort (30 patients) and validation cohort (177 patients)., Setting: Two French ICUs (discovery study) and six French ICUs (validation study)., Patients: Adult septic shock patients., Interventions: None., Measurements and Main Results: The quantification of several interleukin-7 receptor transcripts using specific reverse transcription quantitative polymerase chain reaction designs allowed for global evaluation of interleukin-7 receptor gene expression in whole blood. In the discovery cohort, all interleukin-7 receptor transcripts studied were expressed at lower levels in septic shock patients than in healthy volunteers. Interleukin-7 receptor gene expression at day 3 after septic shock diagnosis was associated with day 28 mortality. Patients at a lower risk of death showed higher expression levels. These results were confirmed in the independent validation cohort. Interestingly, using a threshold obtained on the discovery cohort, we observed in the validation cohort a high negative predictive value for day 28 mortality for the transcript encoding the membrane form of interleukin-7 receptor (0.86; 95% CI, 0.79-0.93)., Conclusions: Interleukin-7 receptor transcripts appear as biomarkers of impaired adaptive immune response in septic shock patients and as a promising tool for patient stratification in clinical trials evaluating immunoadjuvant therapies.

Septic shock is accompanied by the development of immune dysfunctions whose intensity and duration are associated with increased risk of secondary infections and mortality. Although B lymphocytes play a pivotal role in the immune response to infections, no comprehensive exploration of circulating B cell status has been performed during the immunosuppressive phase of septic shock. Thus, our aim was to extensively characterize the phenotype and function of B cells in septic shock, including IL-10 production. Circulating B lymphocyte phenotype and function were evaluated by flow cytometry on fresh whole blood and after ex vivo stimulation in adult septic shock patients sampled at day 1, 3, and 6 after the onset of shock. The circulating B cell number was reduced in septic shock patients, whereas the B cell proportion among total lymphocytes was increased. The remaining circulating B lymphocytes presented with decreased MHC class II expression and increased CD21 low CD95 high exhausted-like phenotype but showed no change in maturation status. Circulating B cell functions were markedly altered after sepsis with reduced ex vivo activation and proliferation capacities. Finally, B cell response after septic shock was characterized by a clear plasmacytosis and an increased IL-10 production in remaining B cells from patients after ex vivo stimulation. During the sepsis-induced immunosuppression phase, B cell response is altered and is oriented toward an exhausted-like/immunoregulatory profile. Further studies are now needed to confirm the immunoregulatory properties of B lymphocytes and evaluate their role in sepsis-induced immunosuppression., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Functional testing protocols are thought to be the gold standard for the exploration of the immune system. However, in terms of routine analysis, they present numerous drawbacks and consequently their use is mainly limited to research applications. In the clinical context of septic shock, characterized by marked lymphocyte alterations, a new approach for lymphocyte intracellular cytokine measurement in whole blood upon was evaluated in a proof-of-concept study. Following lymphocyte activation, simultaneous intracellular labeling of Interferon-γ (IFN-γ), Tumor Necrosis Factor-α (TNF-α), and Interleukin-2 (IL-2) was performed in CD4 + and CD8 + T cells (identified by surface marking). The analysis was carried out by flow cytometry (6 colors). Results obtained in septic patients (n=22) were compared to those of healthy volunteers (n=8). Independently of lymphopenia, there were significant differences between groups. In particular there was significant decrease in the production of IL-2 and TNF-α in septic patients, while the production of IFN-γ was not significantly altered. Polyfunctional results showed that patients presented with increased percentages of triple negative lymphocytes. In contrast, volunteers had higher proportions of triple positive cells. The approach could be performed in a robust and consistent way, taking 4.5h to complete. Moreover, clear differences could be observed between clinical groups with this modified method. These characteristics illustrate the potential of this novel whole blood protocol for clinical applications. However, further research is required to determine the applicability compared to alternative test and to evaluate clinical performances in larger cohorts of patients., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: A defining pathophysiologic feature of sepsis is profound apoptosis-induced death and depletion of CD4+ and CD8+ T cells. Interleukin-7 (IL-7) is an antiapoptotic common γ-chain cytokine that is essential for lymphocyte proliferation and survival. Clinical trials of IL-7 in over 390 oncologic and lymphopenic patients showed that IL-7 was safe, invariably increased CD4+ and CD8+ lymphocyte counts, and improved immunity., Methods: We conducted a prospective, randomized, double-blind, placebo-controlled trial of recombinant human IL-7 (CYT107) in patients with septic shock and severe lymphopenia. Twenty-seven patients at academic sites in France and the United States received CYT107 or placebo for 4 weeks. Primary aims were to determine the safety of CYT107 in sepsis and its ability to reverse lymphopenia., Results: CYT107 was well tolerated without evidence of inducing cytokine storm or worsening inflammation or organ dysfunction. CYT107 caused a 3- to 4-fold increase in absolute lymphocyte counts and in circulating CD4+ and CD8+ T cells that persisted for weeks after drug administration. CYT107 also increased T cell proliferation and activation., Conclusions: This is the first trial of an immunoadjuvant therapy targeting defects in adaptive immunity in patients with sepsis. CYT107 reversed the marked loss of CD4+ and CD8+ immune effector cells, a hallmark of sepsis and a likely key mechanism in its morbidity and mortality. CYT107 represents a potential new way forward in the treatment of patients with sepsis by restoring adaptive immunity. Such immune-based therapy should be broadly protective against diverse pathogens including multidrug resistant bacteria that preferentially target patients with impaired immunity., Trial Registration: Trials registered at clinicaltrials.gov: NCT02640807 and NCT02797431., Funding: Revimmune, NIH National Institute of General Medical Sciences GM44118.

T lymphocyte alterations are central to sepsis pathophysiology, whereas related mechanisms remain poorly understood. We hypothesized that metabolic alterations could play a role in sepsis-induced T lymphocyte dysfunction. Samples from septic shock patients were obtained at day 3 and compared with those from healthy donors. T cell metabolic status was evaluated in the basal condition and after T cell stimulation. We observed that basal metabolic content measured in lymphocytes by nuclear magnetic resonance spectroscopy was altered in septic patients. Basal ATP concentration, oxidative phosphorylation (OXPHOS), and glycolysis pathways in T cells were decreased as well. After stimulation, T lymphocytes from patients failed to induce glycolysis, OXPHOS, ATP production, GLUT1 expression, glucose entry, and proliferation to similar levels as controls. This was associated with significantly altered mTOR, but not Akt or HIF-1α, activation and only minor AMPKα phosphorylation dysfunction. IL-7 treatment improved mTOR activation, GLUT1 expression, and glucose entry in septic patients' T lymphocytes, leading to their enhanced proliferation. mTOR activation was central to this process, because rapamycin systematically inhibited the beneficial effect of recombinant human IL-7. We demonstrate the central role of immunometabolism and, in particular, mTOR alterations in the pathophysiology of sepsis-induced T cell alterations. Our results support the rationale for targeting metabolism in sepsis with recombinant human IL-7 as a treatment option., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Septic patients develop immune dysfunctions, the intensities and durations of which are associated with deleterious outcomes. LILRB2 (leukocyte immunoglobulin-like receptors subfamily B, member 2), an inhibitory member of the LILR family of receptors, is known for its immunoregulatory properties. In a microarray study, we identified LILRB2 as an upregulated gene in septic shock patients. On monocytes primed with LPS ex vivo, LILRB2 mRNA and protein expressions were dose-dependently downregulated and subsequently highly upregulated versus non-stimulated cells. This is concordant with clinical data, since both LILRB2 mRNA and protein expressions were significantly increased in septic shock patients at day 3. In a cohort of more than 700 patients, only after septic shock were LILRB2 mRNA levels increased compared with non-infected or less severely infected patients. This was preceded by a phase of downregulated mRNA expression during the first hours after septic shock. Interestingly, the intensity of this decrease was associated with increased risk of death after septic shock. LILRB2 protein and mRNA expressions are deregulated on monocytes after septic shock and this can be reproduced ex vivo after LPS challenge. Considering LILRB2 inhibitory properties, we can hypothesize that LILRB2 may participate in the altered immune response after septic shock., (Copyright © 2017. Published by Elsevier Inc.)

Sepsis represents the host's systemic inflammatory response to an infection. In this condition, immune response associates a marked inflammatory response and the delayed development of severe dysfunctions affecting both innate and adaptive responses. As neutrophils are the first line of defense against infection, they are central to the pathophysiology of sepsis in first hours. Nevertheless, their role during immunosuppression phase remains elusive. The main objective of the current work was to perform a transcriptomic study on purified neutrophils from septic shock patients (n=9) so as to identify genes that are differentially expressed during the first week after disease onset both (3-4 and 6-8days) versus healthy donors. Then, 45 septic shock patients were prospectively enrolled to confirm results at the protein level using flow cytometry. Twenty healthy volunteers (HV) were also included for the whole study. By comparing the transcriptome of purified neutrophils, we identified 364 up-regulated and 328 down-regulated genes differentially expressed. Of them, CD177 mRNA, coding for an activation molecule in chemotaxis, had the highest fold change modulation between patients and HV. This increase was then confirmed at the protein level. There was a constant subset of neutrophils that did not express CD177. However, when positive, septic neutrophils presented with significantly increased CD177 expression. Of note, no association between CD177 overexpression and features of immunosuppression has been highlighted. In addition, this up-regulation was negatively correlated with a decreased expression of CD10, a characteristic of immature myeloid cells. In conclusion, in this exploratory work, we shed light on the increased CD177 mRNA and protein expressions in circulating neutrophils after septic shock. Considering the potential dual roles of CD177 neutrophil (i.e., maturation/chemotaxis), negatively correlated in this study, its participation in septic shock pathophysiology deserves further investigation. Furthermore, its potential as a biomarker for sepsis would deserve to be investigated in large cohorts of patients., (Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Septic shock is characterized by lymphocyte alterations associated with increased risk of nosocomial infections and mortality. IL-7, a cytokine required for T cell survival, is thought as a novel therapy for septic patients with severe lymphopenia. We assessed CD4(+) lymphocyte responsiveness to rhIL-7 in septic shock patients ex vivo. Thirteen septic shock patients and 10 controls were included. The MFI of pSTAT5, a key signaling molecule for IL-7, was measured by flow cytometry in CD4(+)FOXP3- (Teffs) and CD4(+)FOXP3(+) (Tregs) lymphocytes after whole-blood incubation with increasing doses of rhIL-7. The basal level of pSTAT5 in nonstimulated T cells was higher in patients. However, the maximal activation level in response to the highest doses of rhIL-7 was similar in both groups. Importantly, low doses of rhIL-7 preferentially activated Teff versus Treg in patients and nonsurvivors tended to present with decreased pSTAT5 expression. This pilot study is the first to highlight, in septic patients, the interest of pSTAT5 measurement in whole blood for the monitoring of rhIL-7 therapy. Such a method could represent an innovative, biologic tool for monitoring leukocyte pharmacological responses to biotherapies in daily clinical practice in other clinical contexts., (© Society for Leukocyte Biology.)

Introduction: The aim of this study was to assess the effect of low-dose corticosteroid therapy in reducing shock duration after severe burn., Methods: A placebo-controlled, double-blind, randomized clinical trial (RCT) was performed on two parallel groups in the burn intensive care unit (ICU). Patients were randomized to receive either low-dose corticosteroid therapy or placebo for seven days. A corticotropin test was performed at the time of randomization, before the administration of the treatment dose. Thirty-two severely burned patients with refractory shock (>0.5 μg/kg/min of norepinephrine) were prospectively included in the study., Results: We included 12 patients in the hydrocortisone-treated group and 15 patients in the placebo group in the final analysis. Among these patients, 21 were nonresponders to the corticotropin test. Median norepinephrine treatment duration (primary objective) was significantly lower in the corticosteroid-treated versus the placebo group (57 hours versus 120 hours, P = 0.035). The number of patients without norepinephrine 72 hours after inclusion was significantly lower in the treated group (P = 0.003, log-rank test analysis). The total quantities of norepinephrine administered to patients were lower in the hydrocortisone-treated versus the placebo group (1,205 μg/kg (1,079 to 2,167) versus 1,971 μg/kg (1,535 to 3,893), P = 0.067). There was no difference in terms of ICU or hospital length of stay, sepsis incidence, cicatrization or mortality., Conclusions: In this placebo-controlled, randomized, double-blind clinical trial, we show for the first time that the administration of low-dose hydrocortisone in burn patients with severe shock reduces vasopressor administration., Trial Registration: Clinicaltrial.gov NCT00149123 . Registered 6 September 2005.

In clinical laboratories, the evaluation of lymphocyte proliferative response (lymphocyte transformation test-LTT) is routinely performed by the measurement of [(3)H]-thymidine uptake after stimulation. In this study we evaluated the performances of a recently developed non-radioactive test based on the detection by flow cytometry of 5-ethynyl-2'deoxyuridine (EdU) incorporation for the measurement of LTT in routine lab conditions. After definition of optimal protocol parameters, EdU incorporation test showed good repeatability and reproducibility. Moreover, this assay was flexible enough to fit important clinical laboratory constraints (delayed stimulation, low number of cells and delayed analysis after staining). Importantly, correlations between results obtained with EdU and [(3)H]-thymidine incorporation assays were excellent both in healthy volunteers and pediatric and septic patients. In particular, the two techniques identified patients presenting with altered LTT. Upon confirmation in a larger cohort of patients, EdU incorporation assay may be a relevant non-radioactive candidate for LLT in clinic., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Coupled plasma filtration adsorption (CPFA) is a blood purification therapy aimed at modulating the host inflammatory response involved in sepsis pathogenesis. One potential drawback of this technique is the unexpected elimination of antibiotics. The aim of this study was to assess the elimination of several antibiotics with CPFA. We performed a retrospective analysis of the serum and ultrafiltrate concentrations of different antibiotics routinely measured during CPFA sessions in five patients experiencing septic shock. The adsorbent extraction ratio (AER) for piperacillin and vancomycin 2 h into the CPFA session were high: 95.4 ± 6.9% and 99.6 ± 0.9%, respectively. These AER decreased significantly by 8 h (at 8 h: 6.3 ± 51.8% and -30.2 ± 25.6%, respectively), suggesting saturation of the resin cartridge. Conversely, the tazobactam AER was low (7.2 ± 15% after 2 h of CPFA). No significant changes in the mean serum concentrations of piperacillin, tazobactam, and vancomycin were observed. Thus, as opposed to tazobactam, we report high adsorption of piperacillin and vancomycin on the CPFA resin but with no reduction in serum concentrations.

Continuous renal replacement therapy (CRRT) with a high cutoff (HCO) membrane is proposed for septic shock as a blood purification technique. The aim of this therapy is to modulate the immune response through the increase of the clearances of the inflammatory mediators as compared to standard CRRT. The use of HCO membranes in daily clinical practice is limited due to the related theoretical albumin loss. Super high-flux (SHF) membranes have an optimized cutoff and, when used in a diffusive mode, may allow for high clearances of middle molecules with limited albumin loss. We report the case of a patient with pneumonia and septic shock treated with continuous hemodialysis with a SHF membrane in order to present the clinical application of this new extracorporeal blood purification technique., (Copyright © 2013 Association Société de néphrologie. Published by Elsevier SAS. All rights reserved.)

High-volume hemofiltration has been suggested as an adjuvant treatment of septic shock (renal support and immunomodulation of the host response via the removal of middle molecular weight molecules such as cytokines). Nevertheless, high-volume hemofiltration presents some important drawbacks, such as the depletion of low molecular weight molecules (nutriments, vitamins, trace elements and antibiotics) due to the high ultrafiltration rate, or the significant financial cost and nursing workload. We describe cascade hemofiltration, a new high-volume hemofiltration system, which has been developed to limit these drawbacks by using a special extracorporeal circuit. Results of the first experimental study using this prototype are also reported. They demonstrate the technical feasibility, security and safety of the cascade system although other experimental and clinical studies are needed to continue evaluating this system.

High-volume hemofiltration (HVHF) has been suggested as an adjuvant treatment of septic shock because of its capacities to remove inflammatory mediators from blood. Nevertheless, HVHF presents some important drawbacks, such as the depletion of low molecular weight molecules (nutriments, vitamins, trace elements and antibiotics) due to the high ultrafiltration rate, or the significant financial cost and nursing workload due to the frequent changes of large amounts of expensive sterile substitution fluids. A new hemofiltration system called "Cascade" has been developed, allowing very high ultrafiltration rates (120 mL/kg/h) limiting these drawbacks by using a special extracorporeal circuit. The objective of this study was to assess the technical feasibility of the Cascade system and to compare its hemodynamic impact to that of the standard HVHF system. Twenty sepsis-induced pigs were randomized in two groups: one group was hemofiltered with the standard HVHF system and the other with the Cascade system during a six-hour session. No technical problems were observed with the Cascade system during the experiment. At the end of the experiment, colloid requirements (989 +/- 355 mL vs. 1913 +/- 538 mL, P = 0.006), epinephrine requirements (0.82 +/- 0.42 mg vs. 3.27 +/- 3.02 mg, P < 0.001), lactic acidosis (pH = 7.33 +/- 0.08 vs. 7.10 +/- 0.07, P < 0.001) and mean pulmonary arterial pressure were less pronounced in the Cascade group. These results suggest that Cascade hemofiltration is technically feasible and safe. Moreover, compared with standard HVHF, it can reduce the severity of porcine septic shock.

Purpose: Septic shock is the leading cause of multiple organ failure and mortality in intensive care units. This condition seems to be related to an overproduction of both pro- and anti-inflammatory mediators, leading to an immunological dysfunction. Therapeutic strategies aimed at reducing blood and tissue concentrations of these mediators have been developed. One of these techniques, based on extra-renal filtration, is called "coupled plasma filtration and adsorption"., Source: English and French language articles published between 1984 and 2008 were identified through a computerized Medline search. Keywords and MeSH terms used were: "coupled plasmafiltration", "coupled plasma filtration", "adsorption", "blood purification", "cytokines", "hemofiltration", "sepsis". Relevant publications were retrieved and scanned for additional sources. Nine publications were found., Principal Findings: Coupled plasma filtration adsorption is an extra-corporeal treatment, based on non-specific cytokine adsorption on a specially designed resin cartridge, coupled with hemofiltration. Experimental studies and the first clinical trials have shown interesting results regarding hemodynamics and respiratory parameters. However, these trials failed to demonstrate any improvement in outcome., Conclusion: Additional evidence is needed to clarify the role of this blood purification technique for the treatment of septic shock.

Background: We tested the hypothesis that sodium nitroprusside (SNP) might improve the impairment of hepatosplanchnic microcirculatory blood flow (MBF) in septic shock., Methods: Fourteen pigs were anaesthetized and their lungs mechanically ventilated. Sepsis was induced with i.v. infusion of live Pseudomonas aeruginosa [1x10(8) colony forming units (CFU) ml(-1) kg(-1)] for 1 h. Sixty minutes later, the animals received in a random succession either SNP or normal saline for 30 min. Mean arterial pressure (MAP), cardiac index (CI), mean pulmonary artery pressure (MPAP), carbon dioxide tension of the ileal mucosa (PCO2; by gas tonometry), ileal mucosal and hepatic MBF by laser Doppler flowmetry, blood gases, and lactates were assessed before, during administration, and 30 min after discontinuing the test drug., Results: Bacterial infusion promoted hypodynamic shock (MAP -18%, CI -33%, ileal MBF -19%, and hepatic MBF -27%), which was converted to normodynamic shock by resuscitation. During SNP infusion, ileal mucosal MBF significantly increased (+19%) compared with control (P = 0.033). Although hepatic MBF increased (+42% from baseline), this did not differ from control. In order to maintain a constant central venous pressure and MAP, fluid loading and norepinephrine (P < 0.01) were increased. Acid-base status was not altered by SNP., Conclusions: In a resuscitated porcine model of the early phase of septic shock, SNP improved ileal mucosal MBF but required a concomitant increase in fluid and norepinephrine supplements to maintain constant systemic haemodynamic parameters.

Coupled plasma filtration adsorption (CPFA) is a new technology proposed for septic shock, removing both pro- and anti-inflammatory mediators. We report the first use of CPFA in France. The patient was 80-years-old and was admitted to the intensive care unit because of septic shock from urinary source. Besides conventional septic shock treatment, four successive CPFA sessions were daily performed and lasted from 6 to 11 hours. At the end of the sessions, a noteworthy hemodynamic and inflammatory improvement was observed. This case confirms the feasibility and the relative simplicity of CPFA. It also illustrates the recent and few scientific data of the medical literature concerning this technology.

Objectives: To develop a standardized bacteraemic porcine model of septic shock with cardiovascular and immunological profiles similar to those observed in human clinical states., Methods: Sepsis was induced by an intravenous challenge of 18 anaesthetized pigs with live Pseudomonas aeruginosa. The pulmonary arterial pressure was monitored and the bacterial infusion was stopped when the systolic pulmonary arterial pressure reached 45 mmHg. Septic shock was treated with fluid resuscitation and epinephrine infusion. The haemodynamic parameters and the rate of different inflammatory cytokines were recorded during 6 h of observation., Results: The mean+/-SD cardiac output increased from 2.4+/-1.2 to 5.7+/-2.1 L/min while the mean+/-SD systemic vascular resistance index decreased from 1957+/-744 to 709+/-221 dyn/s/cm5/m2. The pharmacokinetic profile of the inflammatory cytokines was similar to the one observed in human studies., Conclusions: The control of the systolic pulmonary arterial pressure during a P. aeruginosa infusion leads to a hyperdynamic, reproducible cardiovascular profile similar to the one observed in human septic shock. Since the immunological profile of the inflammatory cytokines is also similar to the human one, this standardized porcine model appears to be appropriate for experimental research concerning sepsis.