Christelle Rouget

Thibaut Girardot

Julien Textoris

Guillaume Monneret

Thomas Rimmelé

Fabienne Venet
email: fabienne.venet@chu-lyon.fr

Biological markers of injury-induced immunosuppression

Keywords:

 When persistent or intense, immune paralysis is associated with the development of secondary infections and pejorative outcomes.

(granulocyte-macrophage colony-stimulating factor) therapy enables to shorten the time of mechanical ventilation and hospital/intensive care unit stay. [START_REF] Meisel | granulocyte-macrophage colony-stimulating factor to reverse sepsis-associated immunosuppression: a double-blind, randomized, placebo-controlled multicenter trial[END_REF] Because of the aforementioned heterogeneity, critically ill patients may not all benefit from immunostimulatory drugs. therefore, clinicians need to know the particular immune status of each patient in real time to propose the best personalized care. Finally, markers of patient immune status could be used to monitor treatment efficacy or side effects.

Biomarkers of injury-induced immunosuppression

Innate immunity

Neutrophils aNd immature graNulocytes

Neutrophils represent the first line of innate immune defense against infection and are the most abundant leukocyte subset. Paradoxically, data about injury-induced neutrophil alterations are scarce. sepsis leads to the massive recruitment of immature neutrophils. the immature granulocyte fraction can be assessed by a blood cell analyzer, expressing the result in Delta neutrophil index (Dn), which independently correlates with 28-day mortality in patients with sepsis. [START_REF] Seok | Delta neutrophil index: a promising diagnostic and prognostic marker for sepsis[END_REF] consistently, Mare et al. found a significantly lower immature granulocyte count in the >4-week survival group compared with the patients who died within the first week of sampling. [START_REF] Shankar-Hari | the diagnostic and prognostic significance of monitoring blood levels of immature neutrophils in patients with systemic inflammation[END_REF] We recently studied functional neutrophil alterations during sepsis-induced immunosuppression (day 3-4 and day 6-8), on 43 septic shock patients and 23 healthy volunteers. [START_REF] Demaret | Marked alterations of neutrophil functions during sepsis-induced immunosuppression[END_REF] a high immature granulocyte count was associated with mortality. in addition, we performed a global evaluation of the neutrophil alterations in septic shock, and highlighted a markedly altered neutrophil chemotaxis (reduced integrin, selectin, and chemokine receptor expression with altered ex vivo migration in response to different chemo attractants) and oxidative burst, also associated with mortality. conversely, and in accordance with the work of Drifte et al., [START_REF] Drifte G, Dunn-Siegrist I, Tissières | innate immune functions of immature neutrophils in patients with sepsis and severe systemic inflammatory response syndrome[END_REF] phagocytose and activation capacities were Need for biomarkers even considering the same diagnosis, there is a huge clinical and biological diversity resulting from the complex and highly dynamic interaction between host and injury. the panel of disease severity and patients' comorbidities increases even more this heterogeneity. this has been suggested to participate in the failure of clinical trials in septic shock. in line, icU patients' immune status is heterogeneous but cannot be determined based on clinical signs. Biomarkers of patients' immune status could help patient stratification such as proposed in the Piro (predisposition, insult, response, and organ dysfunction) score. [START_REF] Jc | the Piro (predisposition, insult, response, organ dysfunction) model[END_REF] reversing and/or shortening the immunoparalysis phase might be of crucial interest to improve patients' outcomes. [START_REF] Delano | sepsis-induced immune dysfunction: can immune therapies reduce mortality?[END_REF] For instance, one of these promising immunoadjuvant therapies is interferon-gamma (IFNγ). It has been shown to restore the expression of transcripts associated with the development of immunosuppression in an ex vivo monocyte endotoxin tolerance model. [START_REF] Allantaz-Frager | Identification of Biomarkers of response to iFng during endotoxin tolerance: application to septic shock[END_REF] IFNγ also partially reversed sepsis-induced immunoparalysis in a human in vivo model of gram negative sepsis. [START_REF] Leentjens | reversal of immunoparalysis in humans in vivo: a double-blind, placebo-controlled, randomized pilot study[END_REF] We can also refer to the work of Meisel et al., who have shown that mHla-Dr guided gM-csF Figure 1.-Simplified description of immune response to injury: a complex balance. After injury, inflammatory and immunosuppressive processes coexist (orange and red dotted lines), with an usual initial proinflammatory and lately anti-inflammatory resultant (green continuous line). severe injuries lead to a biphasic situation, with a long lasting immunoparalysis following the initial inflammatory burst. Both extreme pro-and anti-inflammatory states lead to unfavorable outcome (dashed lines). overall, mHla-Dr is a robust prognostic marker of mortality and secondary infections in a broad variety of injury contexts. Multicenter clinical trials are still needed to define precise cut-off values, as they may depend on the type of injury.

IL10 and IL10/TNF ratio interleukin 10 (il10) is a powerful anti-inflammatory mediator, which namely suppresses dendritic cell and macrophage functions. Many different cell populations may be involved in its synthesis, such as monocytes, and its regulation is complex. [START_REF] Couper Kn | il-10: the Master regulator of immunity to infection[END_REF] in a septic shock population, Monneret et al. showed that il10 correlates with Hla-Dr expression and that values were significantly higher among nonsurvivors. [START_REF] Monneret G | The anti-inflammatory response dominates after septic shock: association of low monocyte Hla-Dr expression and high interleukin-10 concentration[END_REF] interestingly, il10 elevation was already significantly associated to mortality at time of admission and il10 remained higher during 15 days in the non-survivor group. these results were consistent with previous literature regarding il10 dosage [START_REF] Van Dissel | Anti-inflammatory cytokine profile and mortality in febrile patients[END_REF][START_REF] Bassaris | skoutelis a. Proversus anti-inflammatory cytokine profile in patients with severe sepsis: a marker for prognosis and future therapeutic options[END_REF][31] or il10 mrna measurement. [START_REF] Karakantza | cytokine production and monocyte Hla-Dr expression as predictors of outcome for patients with community-acquired severe infections[END_REF][START_REF] Bohé | longitudinal study of cytokine and immune transcription factor mrna expression in septic shock[END_REF][START_REF] Hirasawa | Up-regulation of interleukin-10 mrna expression in Peripheral leukocytes Predicts Poor outcome and Diminished Human leukocyte antigen-Dr expression on Monocytes in septic Patients[END_REF] the association between il10 and septic complication or mortality was also described in burned patients [START_REF] Pileri | amelio l, D'arpa n, amato g, Masellis a, et al. concentrations of cytokines il-6 and il-10 in plasma of burn patients: their relationship to sepsis and outcome[END_REF][START_REF] Foldi V, Pálinkas L | Time course of pro-and anti-inflammatory cytokine levels in patients with burns--prognostic value of interleukin-10[END_REF] and in trauma patients. [START_REF]rasmussen ls. the early il-6 and il-10 response in trauma is correlated with injury severity and mortality[END_REF] il10 may therefore constitute an early marker of forthcoming immunoparalysis. some authors also evaluated IL10/TNF ratio, which reflects the pro/antiinflammatory balance and also correlates with unfavorable outcome. [START_REF] Monneret G | The anti-inflammatory response dominates after septic shock: association of low monocyte Hla-Dr expression and high interleukin-10 concentration[END_REF][START_REF] Van Dissel | Anti-inflammatory cytokine profile and mortality in febrile patients[END_REF][START_REF] Bassaris | skoutelis a. Proversus anti-inflammatory cytokine profile in patients with severe sepsis: a marker for prognosis and future therapeutic options[END_REF] Other monocyte-related markers During sepsis, monocytes show not only impaired antigen presentation, but also decreased pro-inflammatory response, as assessed by the down-regulation of surface markers such as cD10, cD86, cD14, gM csF receptor, or cX3cr1. [START_REF] Preiser | Monocyte cD40 expression in severe sepsis[END_REF][START_REF] Wolk | reduced monocyte cD86 expression in postinflammatory immunodeficiency[END_REF][START_REF] Heinzelmann | cD14 expression in injured patients correlates with outcome[END_REF][START_REF] Kautiainen | Monocyte cD14 and soluble cD14 in predicting mortality of patients with severe community acquired infection[END_REF][START_REF] Pangault | Down-modulation of granulocyte macrophage-colony stimulating factor receptor on monocytes during human septic shock[END_REF][START_REF] Pachot | Decreased expression of the fractalkine receptor cX3cr1 on circulating monocytes as new feature of sepsis-induced immunosuppression[END_REF] For all these markers, a reduced expression was associated with mortality. to our knowledge, none of them has been associated with poor prognosis in surgical, burn or trauma context.

another interesting monocyte surface protein is PD-l1. this marker will be discussed preserved, indicating that neutrophil function is reconfigured rather than globally suppressed. impaired neutrophil chemotaxis, altered phagocytosis and bacterial killing were also described in burn [START_REF] Fikrig Sm | neutrophil chemotaxis in patients with burns[END_REF][START_REF] Brom | Decreased expression of leukotriene B4 receptor sites on polymorphonuclear granulocytes of severely burned patients[END_REF][START_REF] Brom | gtP-binding proteins in polymorphonuclear granulocytes of severely burned patients[END_REF][START_REF]arturson g. neutrophil granulocyte functions in severely burned patients[END_REF] and trauma patients. [START_REF] Hazeldine | the impact of trauma on neutrophil function[END_REF] However, the possible association between neutrophils and mortality/morbidity has only been investigated in sepsis. [START_REF] Shankar-Hari | the diagnostic and prognostic significance of monitoring blood levels of immature neutrophils in patients with systemic inflammation[END_REF][START_REF] Demaret | Marked alterations of neutrophil functions during sepsis-induced immunosuppression[END_REF] moNocytes Monocytes are myeloid cells which play a central role in innate immunity. these circulating cells are also able to release pro and antiinflammatory molecules. After injury, many aspects of monocyte physiology have been studied.

HLA-DR

one of the most promising biomarkers of injury-induced immunoparalysis is monocyte human leukocyte antigen Dr (mHla-Dr). as developed elsewhere, monocytes from septic patients display an altered antigen presentation, related to lower expression of major histocompatibility (MHc) class ii system. this decrease in cell-surface expression of mHla-Dr is a marker of monocyte dysfunction, objectified by lower synthesis of pro-inflammatory cytokines in response to bacterial challenges, lower proliferation rate, and interestingly recovery of monocyte functions after restoration of normal mHla-Dr levels after treatment ex vivo and in vivo. [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF] low mHla-Dr expression has been associated to both mortality and secondary infections in various clinical backgrounds: septic shock, severe burn, trauma, pancreatitis, cirrhosis etc. For instance, gouel-chéron et al. observed that after severe trauma, a ratio of mHla-Dr expression between day 3 and day 2 below 1.2 is an independent predictor of septic complication with an odd ratio of 5 (95% ci [1.6; 19], P=0.008) after multivariate analysis. [START_REF] Gouel-Chéron | g. early daily mHla-Dr monitoring predicts forthcoming sepsis in severe trauma patients[END_REF] similarly, venet et al. reported an association between mHla-Dr decrease and severity as well as secondary septic shock after severe burn injury. [START_REF] Debard A-L | Decreased monocyte human leukocyte antigen-Dr expression after severe burn injury: correlation with severity and secondary septic shock[END_REF] Biological MarKers oF inJUrY-inDUceD iMMUnosUPPression roUget vol. 83 -no. 3 Minerva anestesiologica 305 that was chosen (day 0). Further multicentric clinical trials are warranted to confirm the prognostic value of this functional approach in different injury contexts, and to determine its optimal timing.

deNdritic cells

Dendritic cells (Dcs) are professional antigen-presenting cells (aPcs), playing a key role in linking innate and adaptive immune systems. Dcs capture and process antigens, before migrating to lymph nodes, where they present antigens to lymphocytes. literature provides strong evidences for both quantitative and qualitative Dc alterations after injury. Hotchkiss et al. showed a dramatic reduction in the number of follicular Dcs in septic patients compared with controls. [START_REF] Hotchkiss | Depletion of dendritic cells, but not macrophages, in patients with sepsis[END_REF] MHc class ii expression by Dcs was also lower in septic patients. this is consistent with the observation that circulating Dc count is lower in septic shock patients than in controls (shocks other than septic) as early as day 1, and persistent low circulating myeloid Dc count is associated with nosocomial infection. [START_REF] Pène | Profound and persistent decrease of circulating dendritic cells is associated with icU-acquired infection in patients with septic shock[END_REF] Furthermore, Dc loss in septic patients is higher in non-survivor septic patients than in survivors and is inversely correlated with severity scores. [START_REF] Dilhuydy | Decrease in circulating dendritic cells predicts fatal outcome in septic shock[END_REF] after surgery, Dc count increases acutely, and then drops below preoperative levels on days 2-3. [START_REF] Ho | surgical and physical stress increases circulating blood dendritic cell counts independently of monocyte counts[END_REF] surgery-induced Dc depletion is associated with IL12 -a pro-inflammatory cytokine -deficiency. [START_REF] Yadavalli Gk, Chien | interleukin 12 and interferongamma synthetic deficiency is associated with dendritic cell cytopenia after cardiac surgery[END_REF] Dc functional impairment has also been observed in trauma patients with an in vitro reduced differentiation into Dcs, a lower il12 production, and an impaired t cell proliferation. [START_REF] De | Failure of monocytes of trauma patients to convert to immature dendritic cells is related to preferential macrophagecolony-stimulating factor-driven macrophage differentiation[END_REF] Furthermore, life span and signaling function of Dcs may be altered by trauma. [START_REF] Maier | altered gene expression patterns in dendritic cells after severe trauma: implications for systemic inflammation and organ injury[END_REF] consistently, severe burn patients displayed early low Dc count. [START_REF] Pileri | Decrease of circulating Dendritic cells in Burn Patients[END_REF] in addition Dcs from burn patients had impaired reactivity, an anti-inflammatory phenotype, and dysfunctional t cell-priming ability. [START_REF] Shen | Burn injury triggered Dysfunction in Dendritic cell re-g, et al. evaluation of serum c-reactive protein, procalcitonin, tumor necrosis factor alpha, and interleukin-10 levels as diagnostic and prognostic parameters in patients with community-acquired sepsis, severe sepsis, and septic shock[END_REF] to sum up, functional and quantitative impairments of Dcs have been observed in many in a following subsection, together with the protein it binds.

cD163 is a scavenger receptor expressed on monocytes, macrophages, and dendritic cells. it is also known as haptoglobin-hemoglobin receptor, and also exists as a soluble form, scD163, which is shed into plasma after stimulation by mediators of inflammation. [START_REF] Droste | shedding of cD163, a novel regulatory mechanism for a member of the scavenger receptor cysteine-rich family[END_REF] three clinical studies in septic populations showed that monocyte expression of cD163, or scD163 level, were higher in non-survivors, [START_REF] Feng L | clinical Significance of Soluble Hemoglobin Scavenger Receptor cD163 (scD163) in sepsis, a Prospective study[END_REF][START_REF] Su L, Feng L, Song | Diagnostic value of Dynamics serum scD163, streM-1, Pct, and crP in Differentiating sepsis, severity assessment, and Prognostic Prediction[END_REF][START_REF] Kjaergaard Ag, Rødgaard-Hansen S, Dige A | Monocyte expression and soluble levels of the Haemoglobin receptor (cD163/ scD163) and the Mannose receptor (Mr/sMr) in septic and critically ill non-septic icU Patients[END_REF] even at early time points. nevertheless, cD163 has not been evaluated in severe burn, surgery or trauma.

Functional testing: endotoxin tolerance

Functional approaches may be interesting, for they give an insight into in vivo cell response to an immune challenge. Monocytes from septic patients have been particularly studied; they display a predominantly anti-inflammatory phenotype, this observation leads to the concept of leukocyte reprogramming. [START_REF]Bench-to-bedside review: endotoxin tolerance as a model of leukocyte reprogramming in sepsis[END_REF] Indeed, these cells release less pro-inflammatory mediators (such as tnF, il6 or il12) and as much or even slightly more anti-inflammatory molecules (il10, il1ra). [START_REF]Bench-to-bedside review: endotoxin tolerance as a model of leukocyte reprogramming in sepsis[END_REF] this reduced responsiveness after lPs stimulation has been described as Endotoxin Tolerance. Hence, this method may reveal immune paralysis. endotoxin tolerance has also been described in severe trauma [START_REF] Keel | endotoxin tolerance after severe injury and its regulatory mechanisms[END_REF][START_REF] West Sd | Monocyte deactivation correlates with injury severity score, but not with heme oxygenase-1 levels in trauma patients[END_REF] as well as in major surgery. [START_REF] Kohriyama | role of interleukin-10 on hyporesponsiveness of endotoxin during surgery[END_REF][START_REF] Flohé | Influence of surgical intervention in the immune response of severely injured patients[END_REF] clinical impact of this reprogramming has been recently studied. in a heterogeneous pediatric population with multiple organ failure syndrome, Hall et al. found an association between a reduced ex vivo lPs-induced tnF response (in whole blood, on day 7 after admission) and increased nosocomial infection (rr 3.3 [1.8-6.0]) and mortality (rr 5.8 [2. 1-16]). [START_REF] Hall | immunoparalysis and nosocomial infection in children with multiple organ dysfunction syndrome[END_REF] conversely, van vught et al. observed an endotoxin tolerant phenotype on septic patient monocytes, but it was not associated with the development of nosocomial infections. [START_REF] Van Vught | reduced responsiveness of Blood leukocytes to lipopolysaccharide does not Predict nosocomial infections in critically ill Patients[END_REF] as suggested by the authors, this negative result may be related to the early time point Neutrophil-to-lymphocyte ratio.-another way to take into consideration lymphopenia is to use the neutrophil-to-lymphocyte ratio (ntl). [START_REF] Zahorec R | ratio of neutrophil to lymphocyte counts--rapid and simple parameter of systemic inflammation and stress in critically ill[END_REF] From a pathophysiological perspective, NTL reflects the complex balance between inflammatory insult and immune suppression. In clinical setting, NTL was first investigated in abdominal surgery. increased nlr after digestive surgery was associated with an increased risk of complications or death. [START_REF] Walsh Sr | Post-operative neutrophil-lymphocyte ratio predicts complications following colorectal surgery[END_REF][START_REF] Forget | is the neutrophil-tolymphocyte ratio more correlated than c-reactive protein with postoperative complications after major abdominal surgery?[END_REF][START_REF] Ky | neutrophil to lymphocyte ratio as predictor of surgical mortality and survival in complex surgery of the upper gastrointestinal tract[END_REF] Moreover, time points and cut-offs were very heterogeneous among these studies, and cancer in itself may interfere with ntl.

three clinical studies recently assessed ntl in the icU. in cirrhotic patients with acute complication, high nlr at day 1 was associated with mortality in a multivariate logistic regression. [START_REF] Moreau N | neutrophil-to-lymphocyte ratio is associated with mortality in critically-ill cirrhotic patients[END_REF] a retrospective study suggested that ntl at admission is associated with 28-day mortality in unselected critically ill patients, however this association was not observed in the septic patient subgroup. [START_REF] Salciccioli | the association between the neutrophil-to-lymphocyte ratio and mortality in critical illness: an observational cohort study[END_REF] riché et al. refined this result in a septic shock cohort. they highlighted a reversed ntl evolution according to the timing of death: [START_REF] Barthélémy R, Le Dorze | reversal of neutrophil-to-lymphocyte count ratio in early versus late death from septic shock[END_REF] early death was associated with low ntl at admission, whereas late death (≥day 5) was associated with high ntl between day 1 and day 5.

all in all, ntl is an easy and affordable biomarker which may be of interest to detect immune failure, yet the best timing and cutoffs are still to be determined, and, like mHla-Dr, these parameters may depend on the clinical situation.

regulatory t cells

Regulatory T cells (Tregs) are a very specific subset of t cells, crucial for immune tolerance and homeostasis. indeed, they inhibit the activation and proliferation of most of the other immune cells, by directly killing cytotoxic cells, inhibiting their cytotoxic production, and secreting immunomodulatory cytokines (TGFβ, il10). [START_REF] Kessel A, Bamberger E | the role of t regulatory cells in human sepsis[END_REF] as presented recently, the percentage of tregs increases after the onset of septic shock, [START_REF] Venet | increased circulating regulatory t cells (CD4+CD25+CD127-) contribute to lymphocyte roUget Biological MarKers oF inJUrY-inDUceD iMMUnosUPPression 314 Minerva anestesiologica[END_REF] burn and trauma. the enhancement of their suppressive functions and the relative and different injury situations, and may be key players of injury-induced immune paralysis. As murine administration of purified DCs [START_REF] Benjamim | reversal of long-term sepsis-induced immunosuppression by dendritic cells[END_REF][START_REF] Wang | adoptive transfer of bone marrow-derived dendritic cells decreases inhibitory and regulatory t-cell differentiation and improves survival in murine polymicrobial sepsis[END_REF] and of Dc growth factor (Fms-related tyrosine kinase 3 ligand, Flt3-l) [START_REF] Toliver-Kinsky Te | enhancement of dendritic cell production by fms-like tyrosine kinase-3 ligand increases the resistance of mice to a burn wound infection[END_REF][START_REF] Wysocka | Flt3 ligand treatment reverses endotoxin tolerance-related immunoparalysis[END_REF] reversed mortality and secondary sepsis, monitoring and targeting Dcs represent a promising therapeutic strategy.

Adaptive immunity: T and B cells

lymphocytes are central actors in immunity, acting as both effectors and regulators. not only are they quantitatively altered during injury, but they also display functional anomalies.

cell couNts

Lymphopenia.-apoptosis-related loss of immune cells is an important feature of injuryinduced immune paralysis. this cell loss affects both t cells (except for treg cells) and B cells. lymphopenia is a hallmark of sirs and septic syndrome, and has also been described in trauma, burn and surgical patients. at day 4 of sepsis, lymphopenia was independently associated with 28-day and 1-year survival and severe lymphopenia was associated with increased development of secondary infections. [START_REF] Fuller | Persistent lymphopenia after diagnosis of sepsis predicts mortality[END_REF] in this study, lymphopenia at admission was not associated with mortality, contrary to the results presented by chung et al. [START_REF] Chung | severe lymphopenia is associated with elevated plasma interleukin-15 levels and increased mortality during severe sepsis[END_REF] consistently, in emergency general surgical patients, lymphopenia was independently associated with increased in-hospital mortality (or 3.5 [1.7-7.3]). [START_REF] Vulliamy Pe | Persistent lymphopenia is an independent predictor of mortality in critically ill emergency general surgical patients[END_REF] in trauma as well, failure to normalize lymphopenia in severely injured patients is associated with significantly higher mortality, [START_REF] Ds | Failure to normalize lymphopenia following trauma is associated with increased mortality, independent of the leukocytosis pattern[END_REF] and with higher rate of nosocomial infections after multivariate analysis (personal data). Moreover, lymphocyte count is cheap and included in routine biology. However, most of the severely injured patients display lymphopenia, which may limit its discriminating power. Effective stratification strategies including lymphopenia are still to be determined. cD4 + t cells during the early stage of sepsis was associated with severity and 28-day mortality. [START_REF] Fang | low B and t lymphocyte attenuator expression on cD4(+) t cells in the early stage of sepsis is associated with the severity and mortality of septic patients: a prospective cohort study[END_REF] ctla-4 and Btla might constitute interesting biomarkers for injury-induced immune paralysis, but data are still scarce. to address this question, more clinical studies are needed, including the monitoring of nosocomial infections, and exploring a wider range of injuries. other negative immune regulators have been proposed, such as lymphocyte-activation gene 3 (lag-3) or t-cell immunoglobulin and mucin domain 3 (Havcr2, also known as tiM3), as their expression is increased in septic patients presenting an exhausted t cell phenotype. [START_REF] Js | a prospective analysis of lymphocyte phenotype and function over the course of acute sepsis[END_REF] PD-1/PD-L1.-another promising biomarker is PD-1 (for Programmed Death one, official symbol PDCD1). The protein is expressed on the cell membrane of lymphocytes, myeloid and dendritic cells. it binds two ligands: PD-l1 (cD274, also known as B7-H1), PD-l2 (PDcD1lg2, also known as B7-Dc or cD273). Both belong to the B7:cD28 family, [START_REF] Me | PD-1 and its ligands in tolerance and immunity[END_REF] and are expressed by epithelial, endothelial and antigen presenting cells. this complex pathway is involved in co-ligation with t cell receptor and leads to lower cytokine production and to an inhibition of cellular proliferation, negatively controlling the immune response.

a murine PD-1 knock-out model exposed to sepsis showed lower mortality and decreased bacterial burden and inflammatory response, in link with a macrophage dysfunction. [START_REF] Huang | PD-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis[END_REF] in clinical setting, Zhang et al. provided evidence for an up-regulation of PD-1 on t cells and PD-l1 on monocytes of 19 septic shock patients. interestingly, PD-l1 blockade leads to a decreased apoptosis of t cells after tnF stimulation or t cell receptor ligation. [START_REF] Zhang | Upregulation of programmed death-1 on t cells and programmed death ligand-1 on monocytes in septic shock patients[END_REF] guignant et al. found consistent results, and highlighted a correlation between increased PD-1, mortality and nosocomial infections after septic shock. [START_REF] Huang | Programmed death-1 levels correlate sponse to tlr9 activation and resulted in skewed t cell Functions[END_REF] in this study, trauma patients did not express increased PD1 or PD-l1, contrary to the results presented elsewhere. [START_REF] Bandyopadhyay G, De A, Laudanski | negative signaling contributes to t-cell anergy in trauma patients[END_REF] absolute elevation in their count were associated with mortality in burn. [START_REF] Huang Lf | association between regulatory t cell activity and sepsis and outcome of severely burned patients: a prospective, observational study[END_REF] chen et al. also described a significant association between Treg count increase at day 7 and mortality in icU patients with sepsis. 82 cD39 + treg cells form a specific subset displaying increased suppressive skills. in septic patients, increased expression of cD39 + tregs was associated with mortality and severity of sepsis. [START_REF] Huang | High circulating cD39(+) regulatory t cells predict poor survival for sepsis patients[END_REF] conversely, after traumatic brain injury, the level of circulating tregs was positively associated with neurologic recovery and lower hospital mortality. [START_REF] Li | role of regulatory t cell in clinical outcome of traumatic Brain injury[END_REF] indeed, treg action is thought to be neuroprotective because, in this context, a down-regulation of inflammation may promote cell survival.

as a conclusion, tregs may play a role in injury-induced immunosuppression, given their important regulatory properties.

iNcreased co-iNhibitory receptors CTLA-4, BTLA.-after antigen recognition, t cell full activation depends on the balance between co-stimulatory and co-inhibitory signals. ctla4 -cytotoxic t lymphocyte-associated antigen 4 -is expressed in t cells and delivers a negative signal to the primed lymphocyte, [START_REF] Walker | confusing signals: recent progress in ctla-4 biology[END_REF] directly antagonizing the costimulatory receptor cD28, and participating in t cell tolerance. ctla-4 expression is increased after burn injury, [START_REF] Fazal N | Burn-injury affects gut-associated lymphoid tissues derived cD4+ t cells[END_REF] after trauma-hemorrhage, [START_REF] Bandyopadhyay G, De A, Laudanski | negative signaling contributes to t-cell anergy in trauma patients[END_REF][START_REF] Hsieh Ch | suppression of activation and costimulatory signaling in splenic cD4+ t cells after trauma-Hemorrhage reduces t-cell Function[END_REF] and in sepsis, and inhibits immune cell functions. [START_REF] Chang Kc | Blockade of the negative co-stimulatory molecules PD-1 and ctla-4 improves survival in primary and secondary fungal sepsis[END_REF] inoue et al. showed a dosedependent effect of anti-ctla-4 on survival in a rodent model of sepsis, [START_REF] Bian | Dose-Dependent effect of anti-ctla-4 on survival in sepsis[END_REF] with decreased cD8 + and cD4 + lymphocyte apoptosis. consistently, blockade of ctla-4 improved survival in rodent fungal sepsis. [START_REF] Chang Kc | Blockade of the negative co-stimulatory molecules PD-1 and ctla-4 improves survival in primary and secondary fungal sepsis[END_REF] to date, no data is available in human clinical setting regarding its prognostic value.

B and t lymphocyte attenuator (Btla) is also a lymphocyte inhibitory receptor, expressed in a wide range of cells. Btla decreases cytokine production and inhibits survival signaling in cD4+ lymphocytes. [START_REF] Ma | B and t lymphocyte attenuator exhibits structural and expression Polymorphisms and is Highly induced in anergic cD4+ t cells[END_REF] consistently with previous literature, [START_REF] Monaghan Sf | B and t lymphocyte attenuator expression on cD4+ t-cells associates with sepsis and subsequent infections in icU patients[END_REF] STAT5 phosphorylation.-stat5 (signal transducer and activator of transcription 5) is a key molecule for the response to il7. its phosphorylation occurs in response to the recruitment of IL7R and reflects the activated status of the cell. stat5 phosphorylation (pstat5) is enhanced by ex vivo rhil7 (recombinant human il7) in septic shock patients. 100 low doses of rhil7 preferentially sustain effector t cells activation, whereas rhil2 activates regulatory t cells. 104 a pilot study from our team suggested that pstat5 in septic shock patients was higher in survivors. non-survivors failed to phosphorylate stat5 in effector t cells in response to rhil7. 105 interestingly, pSTAT5 can be assessed by flow cytometry on whole blood samples, making it suitable for daily clinical practice. b cells regarding B cells, human data are limited. B lymphocytes are a heterogeneous cell population; they can not only differentiate into immunoglobulin secreting plasma cells, but they also produce cytokines and present antigens. However, their role in the pathogenesis of injury-induced immunosuppression has not been firmly established. Sepsis-induced cell depletion also deeply affects B cells, but this has not been specifically associated with mortality. 106 interestingly, patterns in B subset distribution and activation were different between survivors and non survivors. at time of admission, a high percentage of cD23 + (a marker of activation and regulation) B cells appeared to be associated with good outcome, whereas cD80 + (a t cell co-stimulation marker) and cD95 + (a PD-1/PD-l1 expression on immune cells was also increased after surgery, and correlates with the severity of surgical trauma. [START_REF] Xu | surgical trauma induces postoperative t-cell dysfunction in lung cancer patients through the programmed death-1 pathway[END_REF] similarly, t cell apoptosis was partially reversed by anti-PD-1 antibody. overall, these data indicate that PD-1 might be an attracting biomarker for injury-induced immunosuppression. cd127 interleukin 7 (il7) is an essential cytokine involved in survival, development and maturation of t and B cells. it binds to a heterodimeric receptor, consisting of an alpha chain (IL7Rα, also known as CD127) and a gamma chain (cD132), the latter is common to several interleukin receptors. administration of il7 restored lymphocyte functions in septic patients. 100 il7 level and cD127 were not associated with mortality in this study. interestingly, a high level of soluble fraction of CD127 (sCD127) was significantly associated with the development of nosocomial infection. recently, in a larger cohort of septic shock patients, Demaret et al. found a significant association between increased plasmatic scD127 (at day 1 and day 3) and mortality. 101 However, the function and the regulation of scD127 and of cD127 are still widely unknown. soluble cD127 could be an interesting biomarker for identification of a group of patients presenting with higher risk of secondary infections or mortality, but it is unclear whether it is involved in immune paralysis pathophysiology.

FuNctioNal testiNg

T cell proliferation.-another hallmark of sepsis is the decreased lymphocyte proliferation in response to stimulation. Yet, lymphocyte proliferation is a fundamental part of immune response. its decrease has been described in sepsis, trauma, severe burn, and after major surgery. it has been associated with nosocomial infections, poor outcome and multiorgan failure syndrome. [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF] recent data suggested that reduced lymphocyte proliferation may be Biological MarKers oF inJUrY-inDUceD iMMUnosUPPression roUget vol. 83 -no. 3 Minerva anestesiologica 309 nomics, transcriptomics and proteomics have expanded rapidly within the past years. transcriptomic approach evaluates the transcripts (messenger rna, also known as mrna) for many genes. assuming that certain genes may be over-or under-expressed in reaction to injury, researchers expect to find or confirm diagnostic, prognostic and follow-up markers, and to progress in pathophysiologic understanding. Being able to guide therapeutic strategies disclosing patient immune profile is a seducing concept, however actual scientific knowledge is far from achieving this goal. 108 still, literature dealing with injury-induced immunoparalysis and transcriptome is abundant (table i).

General clinical data

as reported by Xiao et al., leukocytes of injured patients (in this study, severe trauma and burns) responded to stress by a global repri-marker of apoptosis susceptibility) B cell percentages were associated with increased 28day mortality. the reason of this observation remains to be elucidated. recently, only B cell and cD16 -monocyte counts were associated with increased mortality. 107, 108 consequently we may hypothesize that, similarly to other immune cells, B cell functional and quantitative alterations may play a role in injury-induced immune dysfunction. thus, it might be interesting to shed light on B cell involvement and to develop B cell markers.

Both innate and adaptive immunity:

transcriptomic approach the host response to injury involves numerous intricate pathways (e.g. immune, neurologic and endocrine systems). [START_REF] Lord | the systemic immune response to trauma: an overview of pathophysiology and treatment[END_REF] given this complexity, rather than focusing on very specific markers, some researchers have recently aimed for a systemic perspective. ge- cyte subpopulations. it is involved in adhesion and migration of leukocytes and is thought to amplify pro-inflammatory immune response. in a microarray study, we observed an 8-fold increase of cX3cr1 in survivors compared with non survivors, which corresponded to the highest factor of change. 114 We later reported that cX3cr1 (mrna and protein) was strongly down-regulated in monocytes. [START_REF] Pachot | Decreased expression of the fractalkine receptor cX3cr1 on circulating monocytes as new feature of sepsis-induced immunosuppression[END_REF] consistently, this down-regulation was associated with mortality. although these data are preliminary, both examples suggest that transcriptomic approach may allow to discover and to evaluate new biomarkers of immunoparalysis.

Conclusions

In parallel with explosive inflammatory processes, injury causes major immune dysfunctions leading to a significant morbi-mortality. innate and adaptive immunity may both be severely compromised, in different extents depending on the patient immune profile. Being able to detect and follow immunoparalyzed patients and dysfunction subtypes is an important issue, for a targeted immunostimulating treatment may improve greatly their outcome. to date, available markers have a prognostic value, but researchers now aim to develop diagnostic tools. such biomarkers could help targeting diagnosis procedures, allowing the clinician to define patients at risk to develop opportunistic infections. the most promising markers -i.e. markers both associated with clinical outcome in several types of injury and performable on routine basis -are decreased mHla-Dr and lymphopenia. in the light of the complexity of injury-induced immune response, combinations of biomarkers may represent an interesting line of investigation.

this review outlines current knowledge on biomarkers of injury-induced immunosuppression. it was not intended to be an in-depth survey of the whole subject and does not represent an exhaustive listing of studies in the field. We sincerely apologize for works not cited in this manuscript. oritization affecting >80% of the cellular functions and pathways. 109 authors refer to this phenomenon as "genomic storm". researchers observed simultaneous increase in expression of innate immune genes and suppression of adaptive immunity, in both injury situations. this supports the hypothesis of a common behavior, which would not rely on the stress type. this novel paradigm has also been described after severe blunt trauma 110 and in sepsis. 111 Xiao et al. studied differentially expressed genes in complicated (recovery >14 days, no recovery, or death) versus uncomplicated clinical recovery (recovery in <5 days). interestingly, authors highlighted an up-regulation of pro-inflammatory pathways in the complicated group, associated with down-regulated antigen presentation and t cell regulation.

to date, the literature gives many other examples of the ability of the transcriptomic approach to analyze the patient's immune status in a global way. as proposed by rittirsch et al. in trauma patients, the combination of clinical and transcriptomic markers may represent an even more interesting tool. 112 Further studies are needed to develop predefined set of markers and to evaluate their predictive value regarding the diagnosis and prognosis of immune dysfunction.

Examples of transcriptomic-discovered markers: CD74, CX3CR1

cazalis et al. used transcriptomic approach in a more targeted manner. in a prospective septic shock cohort, they investigated the link between MHc class ii-related gene expression (by qrt-Pcr) and mortality at day 28. 113 among these, low cD74 at day 3 after the onset of shock was associated with 28-day mortality after multivariate logistic regression analysis (or 3.4 [1.2 to 9.8], P=0.026). cD74, also called Hla-Dr antigen-associated invariant chain, is a protein involved in MHc ii heterodimer synthesis and export towards the cell surface. cD74 still needs validation in a larger multicenter study.

cX3cr1 is a chemokine receptor expressed on monocytes, nK cells, and some lympho-

  shao et al. showed that lower percentage of Btla + /

	roUget	Biological MarKers oF inJUrY-inDUceD iMMUnosUPPression
		driven by an indirect mechanism: in an ex vivo
		human model derived from endotoxin toler-
		ance, Poujol et al. suggested that lPs priming
		indirectly impairs lymphocyte functions by re-
		ducing aPcs capacity to activate t cells. 102, 103
		nevertheless, such functional approaches are
		time-consuming, as proliferation requires sev-
		eral days of incubation. this precludes most
		applications in clinical settings.
	308	Minerva anestesiologica	March 2017

table i .

 i -Biomarkers: feasibility and clinical evidence.

	roUget			Biological MarKers oF inJUrY-inDUceD iMMUnosUPPression
			Biomarker	associated clinical outcome	laboratory technique	approximate minimal turn-around time	Possible in routine?
	innate	Pnn	↑ immature forms	Death	Fc, hematological	1h30	Yes
	immunity				analyzers
		Monocytes	↓ mHLA-DR	Death, Hai	Fc, iHc, Pcr	1h30	Yes
			scD127	Few data. Death?	elisa	5h	no
			endotoxin tolerance	Conflicting data	cell culture + elisa	3 days	no
			↑ PD-L1	Hai	Fc, iHc	1h30	not yet
			il10 ad il10/tnF	Death	elisa or	5h	no
					comparable method
		Dc	↓ DC count	Death, Hai	Fc	1h30	not yet
			Function :	no	cell culture, elisa	3 days	no
			il-12 synthesis,
			proliferation
	adaptive	all lymphocytes	lymphopenia	Death	Fc, hematological	30 min	Yes
	immunity				analyzers
			ntl	Death	Fc, hematological	30 min	Yes
					analyzers
			↑ CTLA4, BTLA	Few data	Fc, iHc	1h30	not yet
			↑ PD-1	Death	Fc, iHc	1h30	not yet
			cD127	Death, Hai	Fc, iHc	1h30	not yet
		t cells	Proliferation	Death, Hai, MoF	cell culture + Fc	3 days	no
		tregs	↑ Treg percentage	Death	Fc	1h30	not yet
	Both	transcriptomic	cD74, cX3cr1	Few data. Death?	qrt Pcr	3h*	Possibly
					microarray	3 days	no
	310		Minerva anestesiologica		March 2017

Hai: healthcare-associated infections; MoF: multi organ failure; Pnn: polynuclear neutrophils; Dc: dendritic cells; tregs: regulator t cells; mHla-Dr: monocyte human leukocyte antigen Dr; PD-1: programmed death one; PD-l1: programmed death ligand one; il: interleukin; TNFα: tumor necrosis factor alpha; NLT: neutrophil to lymphocyte ratio; CTLA4: cytotoxic T lymphocyte-associated antigen 4; BTLA: B and T lymphocyte attenuator; FC: flow cytometry; IHC: immunohistochemistry; ELISA: Enzyme-Linked ImmunoSorbent Assay. * if a unitized industrial method were developed.

Key messages

-injury-induced immunosuppression is a healthcare burden; patients suffering from it may benefit from immunostimulating treatments.

-Biomarkers of injury-induced immunosuppression are needed to specifically identify those patients that may benefit from immunoadjuvant therapies.

-For now, the most promising biomarkers are low mHla-Dr, lymphopenia and increased PD-1/PD-l1 pathway. This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies (either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo, or other proprietary information of the Publisher.


Léonard Serrano
email: leonard.serrano@irt-saintexupery.com

Philippe Olivier
email: philippe.olivier@iut-tlse3.frc

Jacques Cinquin
email: jacques.cinquin@airbus.com

Compaction Behavior Of Out-of-Autoclave Prepreg Materials

The main challenges with composite parts manufacturing are related to the curing means, mainly autoclaves, the length of their cycles and their operating costs. In order to decrease this dependency, out of autoclave materials have been considered as a solution for high production rate parts such as spars, flaps, etc… However, most out-of-autoclave process do not possess the same maturity as their counterpart, especially concerning part quality 1 . Some pre-cure processes such as compaction and ply lay-up are usually less of a concern for autoclave manufacturing: the pressure applied during the cycle participates to reduce the potential defects (porosity caused by a poor quality lay-up, bad compaction, entrapped air or humidity…). For out-of-autoclave parts, those are crucial steps which may have many consequences on the final quality of the laminate 2 . In order to avoid this quality loss, those steps must be well understood.

INTRODUCTION

Composite parts for aerospace applications are mainly produced by lay-up using automated methods (ATL, AFP, FLU...), followed by compaction and cure in a pressure vessel called an autoclave. Autoclave manufacturing, however, is not completely defect-free and expensive. Drawbacks to autoclaves include the high capital investment required to obtain the equipment, the high cost of the nitrogen gas used to pressurize the vessel, poor energy efficiency, and long cycle times. Autoclaves are also size-limiting, restricting the dimensions of the part that can be produced to the inner diameter of the vessel. As it will be impossible to meet the growing demand for composite aircraft structures with autoclave cure methods, the industry is looking to out-of-autoclave (OOA) processing. However, while traditional autoclave processing involves high pressures that suppress formation of porosity, OOA methods supply a maximum pressure differential of 1 atm. Thus, implementation of low pressure processing methods requires a clear scientific understanding of the effects of process parameters on porosity and defects.

One of the most critical factors restricting composite part acceptance for aerospace applications is void content. Voids, or porosity, are open spaces within a composite part, which act as strength-limiting defects. A level of porosity below 2% is required for aerospace structures, rejecting parts with void content exceeding this value.

There are many studies examining the influence of porosity on part quality for autoclave processed composite parts [START_REF] Liu | Effects of cure cycles on void content and mechanical properties of composite laminates[END_REF][START_REF] Liu | Effects of cure pressure induced voids on the mechanical strength of carbon/epoxy laminates[END_REF] . Mechanical properties such as the interlaminar shear strength, material toughness and moisture uptake are greatly affected by void content. Olivier et al. investigated the influence of autoclave cure pressure on composite void content [START_REF] Olivier | Effects of cure cycle pressure and voids on some mechanical properties of carbon/epoxy laminates[END_REF] (Figure 1). These results indicate that autoclave cure pressures exceeding 0.8 MPa will result in void free In order to suppress voids and facilitate compaction, composite parts manufacturers have relied upon high autoclave pressures (up to 7 Bars or 0.7 MPa) applied during the cure cycles. With Out-of-Autoclave processing, however, the safeguards to void formation supplied by high cure pressures are removed. Thus, an understanding of the mechanisms of void formation is critical to successful defect reduction in low-pressure processed parts. The importance of pre-cure compaction process will also be studied in this document.

MATERIALS AND METHODS

The influence of pre-cure Compaction

Vacuum Bag Only process (or VBO) was selected as our Out-of-Autoclave processing way. The material used for this study was a UD prepreg made of a thermosetting matrix reinforced by IM7-12K carbon fibers. The curing temperature recommended by the material manufacturer was of 180°C. 4 plies of the prepreg were laid at 0° on a mold following the scheme on Figure 3. In order to study the pre-cure compaction, 5 samples were prepared with different compaction time under a constant vacuum of -0.08 MPa.

Those uncured samples are analyzed by X-Ray Tomography, under a resolution of 12.57 µm per voxel, for a total scanned volume of 10x20x0.5 mm per sample. The porosity was measured by threshold on grayscale images having 256 level of light (0 to 255), following the Y Slices.

This porosity level calculation was made in python, computing the convex hull of the set of pixels in order to separate the pixels of the background from the pixels of the sample, binarizing the images with the threshold and calculating the proportion of pixels corresponding to voids in the sample. Those operations were repeated in the whole length of the sample in order to obtain an average value of porosity level (see Table 1)

FIGURE 3.
Laminating configuration and ancillary products used for manufacturing samples

Porosity level during the cure cycle

We choose to follow the cure changes through rigidity evolution during the cure cycle, with the use of the Vanhograph machine 7 .

The specimen of 50 x 75 mm is made of 7 plies oriented in this way [0° 90° 0° 90° 0° 90° 0°], the 0° are in the direction of the jaws in order to resist the holding of the specimen between the jaws, and making the cross lay-up in order to resist the holding between the jaws and the shear stress produced by the motor.

The resulting curve with the applied cure cycle is shown in figure 5, which can be separated into 4 domains:  Domain 1: Classical evolution of viscosity with temperature increase. Objective is to have a compromise between minimum viscosity and maximum duration at minimum viscosity  Domain 2 : Objective is to reduce porosity of the prepreg as the resin is very fluid, the dry fibers get easily soaked by the resin  Domain 3: The objective is to produce the gelation of the resin, the transition between the liquid and solid state of the resin, when cross-linking exceeds a critical value (which depends only on the compounds of the resin)  Domain 4: The objective is to completely polymerize the resin, which is the longest part because after gelation, the polymerization is controlled by diffusion of the molecules. Five specimens were manufactured according to the configuration of Figure 3 with a lamination [0°]4 each sample was compacted during 30 minutes before curing, and the cycle was stopped by thermal quenching, according to the points on Figure 4. The samples were analyzed by the same method (XR Tomography, 7 µm/Voxel, 10x20mm) as the previous test campaign. Porosity levels were obtained by following the same method as previously (see Table 2). As we can see in Figure 5, the porosities from entrapped air between layers (black), and porosities from dry fibers (darker grey) can be easily observed. The Convex Hull is accurate enough to allow us to make the calculation of the blue pixels in the bottom image (corresponding to the voids in the top image), without the pixels from the background. The grayscale contrast does not allow to separate the fiber and the matrix, a wider scale of gray is needed and longer acquisition times are required for this experiment. As expected, the porosity decreases with the compaction time (Figure 6), as air gets sucked away from the laminate, although, we can expect it to reach a minimum even after 24 hours, as the dry fibers will not get impregnated by the resin at room temperature, as a consequence, there will still be void inside the laminate before curing despite of the time spent for pre-cure compaction. In a further study, it would be interesting to observe the influence of the vacuum level and the thickness of the specimen on the porosity level, as in industrial production, composite parts are usually quite thick and the vacuum is distributed by inlets distant from each other.

RESULTS AND DISCUSSIONS

FIGURE 7. Porosity level as a function of the Cure cycle

As it was expected, the porosity decreases faster when applying heat to the laminate, as the viscosity decreases, the resin can move through the laminate, filling the voids and impregnating the dry fibers. The important decrease in void content occurs before the first dwelling step. It could be interesting to change the heat rate or the dwelling temperature and to observe its effect on the porosity level of laminates with the same configuration.

In spite of using the recommended cure cycle, we could only reach a porosity level of approximately 3%, which is constant after gelation as there is no movement of the matrix inside the laminate, when the degree of cure reaches a certain value, depending on the stoichiometry and functionalities of the reactants [START_REF] Flory | Constitution of Three-dimensional polymers and the theory of Gelation[END_REF] . A complementary work will be based on the optimization of the cure cycle according to the evolution of rigidity and porosity levels that can be reached in order to comply with aeronautical exigencies.

SUMMARY

The influence of the pre-cure compaction time and cure cycle on porosity level were investigated. Void contributions were easily observed through the whole length of the specimen, although, because of the low difference in density between the fibers and the resin, we were not able to accurately separate one from another. We demonstrated that the pre-cure compaction plays an important part in the manufacturing of "void-free" laminates, whichwe hypothesizecan even become crucial for thick laminates.

FIGURE 1 .

 1 FIGURE 1.Void content as a function of pressure applied during process[START_REF] Professor | Dynamic Thermomechanical Analysis of a Prepreg-Applications to Industrial Curing[END_REF]

FIGURE 2 .

 2 FIGURE 2. Interlaminar Shear Strength (ILSS) as a function of void content 6

FIGURE 4 .

 4 FIGURE 4. Vanhograph rigidity curve as a function of the cure cycle (Temperature, Time)

FIGURE 5 .

 5 FIGURE 5. Tomography image of sample A (top); Convex Hull obtained with Python (middle) and binary image obtained by threshold (bottom).

FIGURE 6 .

 6 FIGURE 6. Porosity level as a function of the Compaction time and void representative pictures (Blue)

TABLE 1 .

 1 Sample manufacturing conditions and configuration

Sample Name Pre-cure Compaction Time (min) Porosity Level (%)

	C01	0	37.45
	C02	30	25.02
	C03	60	21.46
	C04	120	15.42
	C05	1440 (24h)	6.38

TABLE 2 .

 2 Sample manufacturing conditions and porosity level

	Sample Name	Sample width/length (mm)	Porosity Level (%)
	C02	10/50	25.02
	A	10/50	7.73
	B	10/50	3.67
	C	10/50	3.27
	D	10/50	3.19
	E	10/50	2.98