Skip to main content

Advertisement

37th International Symposium on Intensive Care and Emergency Medicine (part 3 of 3)

Article metrics

P349 Muscle mitochondrial function and N+/K+ -ATPase activity are unaffected by sepsis in pigs

M Von Seth, L Hillered, A Otterbeck, K Hanslin, A Larsson, J Sjölin, M Lipcsey

Uppsala University, Uppsala, Sweden

Introduction

Imbalance in cellular energetics has been suggested to be an important mechanism for organ failure in sepsis and septic shock. We hypothesized that such energy imbalance would either be caused by metabolic changes leading to decreased energy production or by increased energy consumption. Thus, we set out to investigate if mitochondrial dysfunction or decreased energy consumption alters cellular metabolism in muscle tissue in experimental sepsis.

Methods

We submitted anesthetized piglets to sepsis (n = 12) or placebo (n = 4) and monitored them for 3 hours. Plasma lactate and markers of organ failure were measured hourly, as was muscle metabolism by microdialysis. Energy consumption was intervened locally by infusing ouabain through one microdialysis catheter to block major energy expenditure of the cells, by inhibiting the major energy consuming enzyme, N+/K + -ATPase. Similarly, energy production was blocked infusing sodium cyanide (NaCN), in a different region, to block the cytochrome oxidase in muscle tissue mitochondria.

Results

All animals submitted to sepsis fulfilled sepsis criteria as defined in Sepsis-3, whereas no animals in the placebo group did. Muscle glucose decreased during sepsis independently of N+/K + -ATPase or cytochrome oxidase blockade. Muscle lactate did not increase during sepsis in naïve metabolism. However, during cytochrome oxidase blockade, there was an increase in muscle lactate that was further accentuated during sepsis. Muscle pyruvate did not decrease during sepsis in naïve metabolism. During cytochrome oxidase blockade, there was a decrease in muscle pyruvate, independently of sepsis. Lactate to pyruvate ratio increased during sepsis and was further accentuated during cytochrome oxidase blockade. Muscle glycerol increased during sepsis and decreased slightly without sepsis regardless of N+/K + -ATPase or cytochrome oxidase blocking. There were no significant changes in muscle glutamate or urea during sepsis in absence/presence of N+/K + -ATPase or cytochrome oxidase blockade.

Conclusions

These results indicate increased metabolism of energy substrates in muscle tissue in experimental sepsis. Our results do not indicate presence of energy depletion or mitochondrial dysfunction in muscle and should similar physiologic situation be present in other tissues, other mechanisms of organ failure must be considered.

P350 Pilot study showing reduced bone strength at 96 hours in rodent sepsis

ME Cove1, NS Chew2, LH Vu2, RZ Lim2, Z Puthucheary3

1National University Hospital, Singapore, Singapore; 2Yong Loo Lin School of Medicine, National University Singapore, Singapore, Singapore; 3University College London, London, United Kingdom

Introduction

Bone mineral density (BMD) is reduced in critical care survivors [1], and long-term follow up has shown increased fracture risk [2]. It is unclear if these changes are a consequence of acute critical illness, or reduced activity afterwards. Bone health assessment during critical illness is challenging, and direct bone strength measurement is not possible. We used a rodent sepsis model to test the hypothesis that critical illness causes early reduction in bone strength and changes in bone architecture.

Methods

20 Sprague-Dawley rats (350 ± 15.8g) were anesthetised and randomised to receive cecal ligation and puncture (CLP) (50% cecum length, 18G needle single pass through anterior and posterior walls) or sham surgery (cecum mobilised, no CLP), and then returned to their cages. 10 rodents (5 CLP, 5 sham) were sacrificed at 24 hours, and the remaining 10 at 96 hours. Femur bones were harvested and bone strength testing was conducted using the Instron 5543 (Instron Corp, USA). Trabecular bone strength was measured using a femoral neck break and cortical bone strength tested using a femoral shaft 3-point bending test. Bone architecture was assessed using micro-computerised tomography (microCT) imaging (PerkinElmer, USA), and images analysed with BoneJ [3].

Results

All 20 rats survived to the end of the protocol. The load required to fracture the femoral neck and shaft was not significantly different for CLP and sham groups at 24 hours (97 ± 19N vs 81 ± 10N p = 0.12 and 127 ± 8N vs 119 ± 18N p = 0.35, respectively). However, at 96 hours there was a significant reduction in the fracture force at both the femoral neck and shaft in the CLP group, compared to sham (75 ± 11N vs 97 ± 13N p = 0.02 and 102 ± 20N vs 139.9 ± 28N p = 0.04). In contrast, there were no bone architecture differences, as measured by bone volume/total volume, trabecular thickness/separation, connectivity density, anisotropy and BMD (all p > 0.20) using microCT at 24 or 96 hours.

Conclusions

In this rodent model of sepsis, there is a significant reduction in trabecular and cortical bone strength at 96 hours. In the absence of changes in bone architecture, these findings suggest sepsis may induce early biochemical changes affecting bone strength. We plan further rodent experiments to confirm these results, increase our power, assess nano-mechanics and complete a histological analysis.

References

1 Orford NR et al: Am J Resp Crit Care 2016,193:736–744

2 Rawal J et al: Crit Care 2015,19:165

3 Doube M et al: Bone 2010, 47:1076–1079

P351 Endotoxin clearance by the spleen is unaffected by pre-existing systemic inflammation in porcine septic shock

K Hanslin1, F Wilske2, P Skorup2, E Tano2, J Sjölin2, M Lipcsey1

1Uppsala University, Uppsala, Sweden; 2Uppsala University, Department of Medical Sciences, Uppsala, Sweden

Introduction

As part of the mononuclear phagocytic system, the spleen participates in bacterial and endotoxin clearance. In our previous study we saw decreased endotoxin clearance by the liver in pigs with pre-existing systemic inflammatory response (SIR). We therefore hypothesized that immunosuppression induced by SIR may also lead to decreased trans-splenic endotoxin clearance, and set out to investigate this in a porcine model of sepsis.

Methods

15 anesthetized pigs received an [i]E. coli[/i] infusion intravenously (i.v.) for 3 hours (h). In group Pre-existing SIR (n = 6), SIR was induced by 24 h of i.v. endotoxin infusion prior to the [i]E. coli[/i] infusion. Group Non-Pre-existing SIR (n = 6) received the bacterial infusion without prior endotoxin exposure. To study the effects of 24 h of anesthesia alone, “Controls” (n = 3) received saline instead of endotoxin for 24 h prior to the bacterial infusion (not included in the primary analysis). The kinetic chromogenic LAL-test was used to analyze endotoxin in arterial and splenic venous blood samples.

Results

All animals receiving endotoxin developed SIR prior to the [i]E. coli[/i] infusion. The amounts of [i]E. coli[/i] given to the groups were comparable. Endotoxin levels were similar at 3 h, just before the end of the [i]E. coli[/i] infusion, in the Pre-existing SIR and Non-Pre-existing SIR groups in arterial (Fig. 1) and splenic venous blood (2.40 (1.94-2.60) vs. 2.81 EU/mL (2.73-2.91) median (IQR)). Furthermore, endotoxin levels at 4 h, one hour after completed [i]E. coli[/i] infusion, were lower in Pre-existing SIR vs. Non-Pre-existing SIR group both in arterial (Fig. 1) and splenic venous blood (0.38 (0.31-0.42) vs. 0.51 EU/mL (0.47-0.71); p < 0.05). There was no difference in the ratio of splenic venous to arterial endotoxin levels between Pre-existing SIR and Non-Pre-existing SIR groups.

Conclusions

In our model, the endotoxin clearance by the spleen is not affected by pre-existing inflammatory response in porcine [i]E. coli[/i] septic shock.

Fig. 1 (abstract P351).
figure1

See text for description

P352 The role of autophagy in critical illness-induced organ failure

I Derese, S Thiessen, S Derde, T Dufour, L Pauwels, Y Bekhuis, G Van den Berghe, I Vanhorebeek

University Hospital, Leuven, Belgium

Introduction

Increasing evidence implicates mitochondrial dysfunction and endoplasmic reticulum (ER) stress, which activates the unfolded protein response (UPR), as contributors to critical illness-induced organ failure. Both can be alleviated by autophagy, a cellular defense mechanism. However, a phenotype of insufficiently activated autophagy has been observed during critical illness. We hypothesized that insufficient hepatic autophagy during critical illness aggravates liver damage/failure, hallmarked by mitochondrial dysfunction and ER stress.

Methods

In a centrally catheterized mouse model of critical illness, induced by cecal ligation and puncture, the effect of genetic inactivation of hepatic autophagy (via inducible deletion of autophagy gene 7 in liver) on survival, markers of organ damage, apoptosis, UPR and mitochondrial content and function was evaluated in the acute (30 hrs) and prolonged (3 days) phase. For each time point, 2 groups of critically ill mice and 2 groups of healthy pair-fed mice were included (at least 10 surviving mice per group), where each time autophagy was inactivated in one group but not in the other.

Results

Hepatic autophagy deficiency during critical illness did not affect survival, but increased hepatic damage/dysfunction. In the acute phase, this was illustrated by higher plasma ALT (P = 0.0001), and by elevated markers of apoptosis (P = 0.001) and more mitochondrial dysfunction (Complex V activity, P = 0.02) in liver. In the prolonged phase, hepatic autophagy inactivation increased apoptosis (P = 0.01) and aggravated mitochondrial dysfunction (Complex V activity, P = 0.005) in liver. Autophagy deficiency did not affect mitochondrial DNA content (day 1 P = 0.98, day 3 P = 0.57). Autophagy deficiency time-dependently modulated several branches of the UPR in liver. On day 1, it decreased activation of the IRE1alpha-XBP1s (P = 0.003) and ATF6-CREB3L3 pathway (P = 0.003), coinciding with a diminished inflammatory response as shown by lower C-reactive protein gene expression (P = 0.006), but did not affect the p-eIF2alpha pathway (P = 0.26). At day 3, autophagy deficiency increased the activation of the p-eIF2alpha pathway (P = 0.03), but not the IRE1alpha-XBP1s (P = 0.37) or ATF6-CREB3L3 (P = 0.14) pathway.

Conclusions

Insufficient hepatic autophagy during critical illness aggravates liver damage, coinciding with more mitochondrial dysfunction and a time-dependent modulation of the UPR, hereby likely aggravating liver failure.

P353 Role of leptin and proprotein convertase subtilisin/kexin type 9 in modulating pulmonary inflammation in a murine model of early sepsis

M Khan1, D Dwivedi1, J Zhou1, A Prat2, NG Seidah2, PC Liaw1, AE Fox-Robichaud1

1McMaster University, Hamilton, Canada; 2Montreal Clinical Research Institute, Montreal, Canada

Introduction

Obesity increases the risk of sepsis but how obesity shapes the immune responses to infection is unknown. Similar to patients, we previously demonstrated that Western diet fed obese mice have reduced lung inflammation during early sepsis. In this study we explore the potential mechanisms to explain this finding. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a protein involved in cholesterol homeostasis that is implicated in sepsis survival. Leptin is a hormone produced by adipocytes that regulates energy homeostasis and is increased in obesity and sepsis. We hypothesized that either PCSK9 and/or leptin contributes to the obesity-associated lung protection in sepsis.

Methods

PCSK9 deficient, PCSK9 overexpressing and wild type mice on a C57/Bl6 background were fed either a high fat Western diet (WD) or a normal chow diet (NCD) for 15 weeks (n = 5/group). Sepsis was induced by cecal ligation and puncture (CLP). Tissues were harvested six hours post surgery. For the leptin studies mice were housed in static cages for 10-12 weeks. Mice were injected with recombinant leptin protein (1mg/kg)) one hour prior to CLP, then re-anesthetized and tissues collected at 6 hours. All mice were resuscitated with 2ml of lactated Ringers SQ pre surgery, and 1ml IV post surgery. Lung injury was assessed by myeloperoxidase (MPO in U/mg of tissue) assay of lung tissues and histopathology scores. Data are expressed as mean ± SEM and analyzed using ANOVA or t-test.

Results

Septic PCSK9 over expressing mice fed NCD had greater lung MPO levels (46.5 ± 4.5) compared to PCSK9 deficient mice (31.1 ± 1.7) on NCD (p < 0.01). In mice fed the WD for 15 wks the protection from the loss of PCSK9 was no longer present, however the injury was reduced. Septic PCSK9 deficient (14.4 ± 1.4), wildtype (17.6 ± 0.9) and overexpressing (17.9 ± 1.0) mice on WD had no significant differences in lung MPO levels. This correlated with histopathology scores for PCSK9 deficient (0.7 ± 0.2), wildtype (1.1 ± 0.2) and PCSK9 overexpressing (1.3 ± 0.7) septic mice. We found that leptin treated septic mice had lower lung MPO (32.6 ± 1.6) levels compared to saline treated septic mice (46.6 ± 3.5) (p < 0.001). Sham operated mice had significantly lower MPO levels (12.7 ± 1.7 for leptin and 11.8 ± 1.2 for saline) compared to septic counterparts.

Conclusions

Our data suggests that both lack of PCSK9 and increases in leptin contribute to the lung protection in early sepsis. However, when exposed to a WD the potential benefits of PCSK9 deficiency to further reduce lung injury are no longer evident. These findings have implications for potential therapeutic strategies to reduce sepsis-induced lung injury.

P354 The role of oxygen delivery on plasma lactate and organ failure in experimental septic shock

M Von Seth, P Skorup, L Hillered, A Larsson, J Sjölin, M Lipcsey

Uppsala University, Uppsala, Sweden

Introduction

The concept resuscitation of patients with septic shock, aiming at normalization of oxygen delivery (DO2), to limit tissue dysoxia and organ failure has not been confirmed in recent trials. Elevated plasma lactate in septic shock is considered as a key marker of inadequate DO2. We hypothesized that, apart from severely decreased levels, DO2 is not associated to plasma lactate in a model of septic shock.

Methods

We investigated the effects of circulatory shock and inflammation on plasma lactate in a retrospective analysis of 105 anesthetized endotoxemic (N = 61) or bacteremic (N = 44) piglets in shock. Tumor Necrosis Factor alpha (TNF-α) and Interleukin-6 (IL-6) were measured hourly during 6 hours (h) of shock. Muscle metabolism was monitored by microdialysis. The animals were stratified per degree of shock by DO2. The primary analysis was the breakpoint of insufficient DO2 to yield an elevated plasma lactate. ANOVA and regression models were used.

Results

All animals developed macrocirculatory shock, elevated plasma and muscle lactate levels, elevated levels of cytokines in plasma, as well as renal and pulmonary failure. At 3 h, DO2 was 289 ± 68 mL x min-1 x m-2 (mean ± SD) and plasma lactate levels were 2.7 (2.0-3.6) mmol x L-1 (median(IQR)). Mixed venous saturation (SvO2) decreased and oxygen extraction increased linearly with DO2 (p > 0.001). Oxygen consumption (VO2) was not DO2 dependent.

Plasma lactate increased at DO2 < 250 mL x min-1 x m-2 (p < 0.001). Urinary output decreased at DO2 < 250 mL x min-1 x m-2 (p < 0.01), but static lung compliance was not DO2-dependent. Muscle glucose, lactate and pyruvate, urea and glutamate were not DO2-dependent. Muscle glycerol was DO2-dependent without breakpoint.

Plasma lactate correlated to Mean Arterial Blood Pressure (MAP), DO2 and peak IL-6, but not Systemic Vascular Resistance Index (SVRI) and peak TNF-α.Urinary output correlated to DO2 and MAP. Static lung compliance did not correlate to any parameters above.

Over time, muscle pyruvate increased and muscle glycerol and glucose decreased but no changes in muscle lactate and glutamate were seen. Muscle pyruvate correlated to MAP. Muscle glycerol correlated to MAP and to TNF-α.

Conclusions

In porcine experimental sepsis, elevated plasma lactate was only associated with very low DO2 while oxygen consumption was unaffected by low DO2 despite development of organ failure. Tissue metabolism was associated with both inflammatory and circulatory changes. Our findings suggest that the current concepts of resuscitation focusing on restoration of oxygen delivery must be combined with measures to limit the inflammatory response.

P355 The lung is not a significant source of lactate in a pig model of sepsis

A Otterbeck, K Hanslin, M Lipcsey, A Larsson, M Von Seth

Uppsala Universitet, Uppsala, Sweden

Introduction

We used a porcine sepsis model to investigate pulmonary hypoxia as an explanation for lactate elevation in sepsis. We measured the pulmonary lactate production and shunt fraction during bacteremia. Sepsis is a condition characterized by severe organ failure as a result of a dysregulated response to infection. Central in many pathophysiological theories is the decreased delivery or utilization of oxygen by tissues. Normal physiology dictates that hypoxia leads to lactate production, a prognostic marker in sepsis. Thus, hypoxia has been used as an explanation for both organ dysfunction and elevated plasma (p-)lactate in sepsis. However, new research has implied other mechanisms. Previous studies have reported increased pulmonary lactate production in sepsis [1], the lungs being a generally well-oxygenated organ. However, these studies have not measured pulmonary shunt fraction and hence cannot estimate if the entire lung is ventilated.

Methods

We used 13 anesthetized pigs where 9 were randomized to a sepsis group and 4 were randomized to a sham group. All pigs received a pulmonary artery catheter and an arterial line. Pigs in the sepsis group were infused with live Escherichia coli for 3 hours (h) and in the sham group with NaCl. Blood cultures were used to confirm bacteremia. Blood gases, blood tests and physiological parameters were collected hourly. Lactate production was calculated by the p-lactate gradient from pulmonary artery to systemic artery and cardiac index. Shunt fraction was estimated at 3 h after ventilation with 100% O2 for 5 minutes.

Results

Sepsis occurred in all pigs in the sepsis group, according to the criteria from Sepsis-3, and in no pigs in the sham group (p = 0.03). Global oxygen delivery (DO2) remained equal in both groups. Arterial lactate was higher (p = 0.003) in the sepsis group after 1 hour with a median value of 2.3 mmol/L vs. 0.95 mmol/L. Negative and positive lactate production occurred over the lungs in both groups (Fig. 2). There was no difference in pulmonary lactate production or pulmonary shunt fraction between groups. Neither group had significant shunt formation.

Conclusions

In this study we found a high p-lactate in septic pigs despite a high DO2. In absence of pulmonary shunts, the lung was not a major source, nor a major scavenger, of plasma lactate.

Reference

1. Garcia-Alvarez, M. et al. Sepsis-associated hyperlactatemia. Crit. Care Lond. Engl. 18, 503 (2014).

Fig. 2 (abstract P355).
figure2

See text for description

P356 Association between inflammation, mitochondrial function and lactate clearance

T Correa1, J Pereira1, J Takala2, S Jakob2

1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland

Introduction

Systemic lactate clearance (LaCl) higher than 10% during the first hours of sepsis resuscitation is associated with better outcomes, but the mechanisms are unclear. We aimed to investigate the relationship between lactate clearance, inflammatory response, and mitochondrial respiration.

Methods

Original data from two previously published studies were re-analyzed [1,2]. In cohort 1, pigs were randomized to be resuscitated for 48h starting 6, 12 and 24h, respectively, after fecal peritonitis induction (n = 8, each) [1]. Hemodynamics, inflammatory parameters, and mitochondrial function were analyzed. In cohort 2, 16 pigs with fecal peritonitis were immediately resuscitated for 24h [2]. Regional lactate exchange was measured. Animals of both groups were categorized according to LaCl > =10% or <10% during 6h of resuscitation.

Results

Overall mortality was 20% (4/20) for animals with LaCl > =10% and 60% (12/20) for animals with LaCl > =10% (p = 0.022). In cohort 1, systemic hemodynamics were similar in LaCl > =10% (n = 13) and LaCl < 10% (n = 11) groups. Plasma interleukin-6 levels were lower at study end in LaCl > =10% [45 (37-204) vs. 166 (128-310; median, IQR), p = 0.047]. Complex 1 state 3 [586 (386-688) vs. 353 (242-483), p = 0.026] and state 4 [157 (117-247) vs. 122 (89-151), p = 0.045], and Complex 2 state 3 [1156 (828-1401) vs. 761 (664-932), p = 0.041] and state 4 [376 (281-451) vs. 269 (225-326), p = 0.032] isolated brain but not hepatic, myocardial or skeletal muscle mitochondrial respiration were higher at study end in LaCl > =10% compared to LaCl < 10%. In cohort 2, mesenteric, total hepatic and renal blood flows were higher at study end in LaCl > =10% (n = 7) vs. LaCl < 10% group (n = 9), despite similar cardiac output. Hepatic lactate influx and uptake were approximately 1.5 and 3 times, respectively, higher in LaCl > =10% vs. LaCl < 10% (p = 0.066, both).

Conclusions

Systemic lactate clearance > =10% vs. <10% during early resuscitation after abdominal sepsis was associated with lower plasma interleukin-6, and higher brain mitochondrial respiration. Blood flow redistribution to abdominal organs in animals with high systemic lactate clearance increases the potential to deliver and extract lactate.

References

1. Correa TD et al. Effect of treatment delay on disease severity and need for resuscitation in porcine fecal peritonitis. Crit Care Med 40:2841–9, 2012.

2. Brandt S et al. Effect of fluid resuscitation on mortality and organ function in experimental sepsis models. Crit Care 13:R186, 2009.

P357 Dynamics of endotoxin, interleukin-6 and organ dysfunction after treatment with antibiotics in an E.coli porcine intensive care sepsis model

P Skorup1, L Maudsdotter2, E Tano1, M Lipcsey3, M Castegren1, A Larsson1, J Sjölin1

1Dept. of Medical Sciences, Uppsala, Sweden; 2Dept. of Molecular Biosciences, Stockholm, Sweden; 3Dept. of Surgical Sciences, Uppsala, Sweden

Introduction

Endotoxin released during Gram-negative bacterial infections induces the production of pro-inflammatory cytokines and may accentuate the development of septic shock [1]. Gram-negative bacteria exposed to â-lactam antibiotics in vitro release endotoxin but in a minor extent when the â-lactam antibiotic is combined with an aminoglycoside [2]. The primary purpose of the study was to investigate the dynamics in endotoxin and interleukin-6 (IL-6) concentration as well as leukocyte activation and subsequent organ dysfunction in a large animal intensive care sepsis model in order to explore the relevance of antibiotic-induced endotoxin liberation and inflammatory response in vivo. Whether the addition of an aminoglycoside to a â-lactam antibiotic results in a reduced endotoxin release and systemic inflammation constituted a secondary aim.

Methods

A prospective placebo-controlled study was conducted on anesthetized pigs in an intensive care setting. All pigs were administered Escherichia coli as a 3h intravenous infusion. At 2h the animals were subjected to antibiotic treatment (n = 18) receiving either cefuroxime alone (n = 9) or the combination of cefuroxime and tobramycin (n = 9), whereas controls received saline (n = 18). During 4h after administration of antibiotics/saline, plasma endotoxin, IL-6, leukocytes and organ dysfunction variables were recorded hourly and differences to the values before treatment were calculated.

Results

All animals developed sepsis. Antibiotic-treated animals demonstrated a higher IL-6 response (p < 0.001), stronger leukocyte activation (p < 0.001) and more pronounced deterioration in pulmonary static compliance (p < 0.01) over time in comparison with controls. Animals treated with the combination demonstrated only a trend towards less inflammation in comparison with animals treated with cefuroxime alone. In plasma no differences in endotoxin concentration were observed between the groups.

Conclusions

Treatment with antibiotics elicits an inflammatory IL-6 response which is associated with leukocyte activation and pulmonary organ dysfunction, whereas no observable differences were seen in plasma endotoxin concentration. The reduction in cefuroxime-induced endotoxin release after the addition of an aminoglycoside in vitro could not be reproduced in vivo.

References

1. Bozza FA et al.: Crit Care 2007; 11: 1.

2. Goscinsky G et al.: Scand J Infect Dis 2003; 35: 40–6

P358 Persisitent shift of th1 to th2 in one week after diagnosis of community-acquired severe sepsis predicts mortality

M Xue, JY Xu, L Liu, YZ Huang, FM Guo, Y Yang, HB Qiu

Southeast University, Nanjing, China

Introduction

Recent studies have revealed that inflammation mediated by CD4+ T cells may contribute to the pathogenesis of sepsis. The role of the Th(T helper)1/Th2 balance in sepsis remains largely unknown. The aim of this study was to investigate the th2/th1 pattern and its impact on disease severity and outcomes in patients with new onset community-acquired severe sepsis.

Methods

This was a prospective observational study. Patients with community-acquired severe sepsis admitted to ICU within 24 hours were included. Blood sample was collected on day of admission(Day0, D0), 3rd Day (D3) and 7th Day (D7) after admission. Th2 and Th1 in lymphocyte were tested by flow cytometry. The increase of th2/th1 (>0.22) indicated immunosuppression. According to the change of th2/th1, patients were divided into 3 groups: immunosuppression recovered in early stage (th2/th1 began to decrease on D3, Group 1), immunosuppression recovered in late stage (th2/th1 began to decrease on D7, Group 2), immunosuppression worsened (th2/th1 kept increasing in a week, Group 3). The organ dysfunction, hospital-acquired infection(HAI) and 28-day prognosis was recorded. All patients or their legal representatives provided written informed consent. The study is registered with ClinicalTrials.gov, NCT 02883218.

Results

Seventy-four patients were eligible for study during Sept 18, 2014 to Sept 30, 2016. There were 34 cases in Group 1, 19 cases in Group 2, and 21 cases in Group 3. Baseline characteristics(age, sex, source of bacteremia, presence of comorbidities) of the population in different groups were similar.

(1) Compared with Group1(11.8%), Group3(52.4%) showed a significantly higher 28-day mortality(P = 0.001), while group 2 showed a higher 28-day mortality of 31.6% with no significant difference (Fig. 3).

(2) There were no significant differences in terms of incidence of HAI and organ dysfunction among groups.

(3) The areas under the receiver operating characteristic (AUC) curves of value of th2/th1 on D7 was of great value(0.875)(Fig. 4). Using a th2/th1 on day7 cutoff value of >2.74 to determine 28-day mortality, the sensitivity was 76.2% with 96.1% specificity.

Conclusions

Persisitent shift of th1 to th2 in one week after diagnosis of community-acquired severe sepsis may be a predictor for immunosuppression. Patients with persistently increasing th2/th1 have poor outcome.

References

[1] Leentjens J, Kox M, Rebecca M, et al. Reversal of immunoparalysis in humans in vivo. Am J Respir Crit Care Med. 2012, 186: 838–845.

[2] Monneret G, Venet F, Pachot A et al. Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony. Mol Med. 2008, 14: 64–78.

[3] Inoue S, Suzuki-Utsunomiya K, Okada Y, et al. Reduction of immunocompetent T cells followed by prolonged lymphopenia in severe sepsis in the elderly. Crit Care Med. 2013, 41:810–819.

Fig. 3 (abstract P358).
figure3

28-day mortality in different groups

Fig. 4 (abstract P358).
figure4

Receiver operating characteristics (ROC) curves comparing the ability of value of th2/th1 on Day0, Day3, Day7 as well as change of th2/th1 in one week. D3-0, change of th2/th1 between Day3 and Day0; D7-0, change of th2/th1 between Day7 and Day0; D7-3, change of th2/th1 between Day7 and Day3. Legend 2 : Figure 4. Receiver operating characteristic (ROC) curves of value of th2/th1

P359 Genetic variants of intron region of aquaporin AQP5 gene and development of pulmonary edema in lung infection complicated by septic shock

A Kuzovlev, V Moroz, A Goloubev, A Myazin, A Chumachenko, V Pisarev

V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia

Introduction

Product of AQP5 gene belongs to a family of aquaporins (AQPs), membrane proteins, responsible for the selective transmembrane transport of water. However, the value of polymorphic variants AQP5 in the development and progression of pul_monary edema in severe lung infection was studied so far. The aim of the investigation was to determine the value of genetic variants of a single nucleotide polymorphic site rs3736309 of intron 3 of aquaporin_5 (AQP5) gene in the course of critical illness in patients with documented pulmonary infection.

Methods

Patients with critical illness admitted to the intensive care units were examined during the course of treatment (n = 86, age 27 to 82 years, mean age 53.20 ± 14.34 years). Main diagnosis included malignancies (15%), peritonitis (16%) and necrotizing pancreatitis (37%). Patients developed nosocomi_al pneumonia (55%), acute respiratory distress syndrome (ARDS) (54%), septic shock (48%), ARDS combined with septic shock (33%). DNA genotyping was carried out using tetra_primer polymerase chain reaction (PCR). Statistical processing was performed using GraphPad InStat program (GraphPad, USA).

Results

The distribution of frequencies of genotypes AA, GA and GG (AQP5, rs3736309) in cohort of patients corresponded to Hardy_Weinberg equilibrium (P = 0.923) and was similar to frequencies of the alleles determined in healthy Caucasian individuals (literature data) (P > 0.05). In a subgroup of patients with septic shock and AQP5 AA (rs3736309) genotype the lower EVLWI values were found compared to patients with geno_types GG and GA with septic shock in spite of the same approach to treatment. Genetic variant AQP5 G+ (rs3736309) contributed to the development of pulmonary edema resistant to treatment (odds ratio, OR = 6,75; P = 0.032). Only the subgroup of patients with septic shock and geno_type G + (but not all patients or the subgroup of patients without septic shock of the same genotype) were char_acterized by significantly elevated levels of surfactant protein SP_D in plasma compared to patients of genotype AQP5 AA with septic shock (P < 0.05).

Conclusions

In septic shock, the presence of homozygous variant allele A (AA) of AQP5 rs3736309 is a favor_able factor for patients developing the pulmonary edema. The presence of allele AQP5 G (rs3736309) is a risk fac_tor for developing severe pulmonary edema and unfavorable prognosis in spite of treatment.

P360 Withdrawn

P361 Altered T cell repertoire diversity and increased PD-1 expression predict mortality in patients with septic shock

N Takeyama, M Tsuda, H Kanou, R Aoki, Y Kajita, M Hashiba, T Terashima, A Tomino

Aichi Medical University, Aichi, Japan

Introduction

Sepsis causes impairment of innate and adaptive immunity by multiple mechanisms, including depletion of immune effector cells and T cell exhaustion. Although lymphocyte dysfunction is associated with increased mortality and potential reactivation of latent viral infection in patients with septic shock, the relation between viral reactivation and lymphocyte dysfunction is obscure. The objectives of this study were 1) to determine the relation of lymphocyte dysfunction to viral reactivation and mortality, and 2) to evaluate recovery of lymphocyte function during septic shock, including T cell receptor (TCR) diversity and the expression of programmed death 1 (PD-1).

Methods

In 18 patients with septic shock and latent cytomegalovirus infection, serial blood samples were obtained on days 1, 3, and 7 after the onset of shock, and immune cell subsets and receptor expression were characterized by flow cytometry. TCR diversity of peripheral blood mononuclear cells was analyzed by Multi-N-plex PCR, and cytomegalovirus DNA was quantified using a real-time PCR.

Results

Monocytes showed a decrease of TCR diversity and HLA-DR expression in the early stage of septic shock, while CD4+ T cells displayed an increase of PD-1 expression. Normalization of TCR diversity and PD-1 expression was observed by day 7, except in patients who died. cytomegalovirus reactivation was detected in 3 of the 18 patients during the first week of their ICU stay and all 3 patients died.

Conclusions

These changes are consistent with the early stage of immune cell exhaustion and indicate the importance of normal lymphocyte function for recovery from septic shock. Ongoing lymphocyte dysfunction is associated with cytomegalovirus reactivation and dissemination, as well as with unfavorable outcomes.

P362 Vasopressin alone and with noradrenaline attenuates TNF-α production in an in-vitro model of monocyte priming and deactivation

R Davies, KP O´Dea, S Soni, JK Ward, DJ O´Callaghan, M Takata, AC Gordon

Imperial College, London, United Kingdom

Introduction

Vasopressin is a safe and effective ‘catecholamine-sparing’ vasopressor in septic shock [1]. It also has anti-inflammatory properties, including inhibition of endotoxin-induced inflammatory cytokine release from macrophages in-vitro [2]. To further assess vasopressin’s anti-inflammatory effects in sepsis, we developed an in-vitro assay of monocyte priming and deactivation to model the pro- and anti-inflammatory responses, respectively.

Methods

Healthy volunteer CD14+ monocytes were cultured at 37°C for 20hrs with lipopolysaccharide (LPS, 100pg/ml) or IL-10 (10ng/ml), in combination with noradrenaline (5000pg/ml) and ‘high’ (300pmol/L) or ‘low’ (100pmol/L) dose vasopressin. Monocytes were analysed for HLA-DR and CD86 (T-cell co-stimulatory ligand) expression by flow cytometry, or stimulated with a ‘second-hit’ of LPS (10ng/ml) and TNF release measured by ELISA.

Results

Pre-treatment of monocytes with LPS resulted in a primed phenotype of higher HLA-DR expression and TNF release on stimulation, whereas IL-10 reduced HLA-DR, CD86, and TNF release indicating a deactivated phenotype. Under normal and priming conditions, co-incubation with vasopressin alone or with noradrenaline significantly reduced TNF release (Fig. 5), but not HLA-DR/CD86 expression. In contrast, neither vasopressin nor noradrenaline affected the IL10-induced deactivated phenotype.

Conclusions

The vasopressin-mediated suppression of TNF release in normal or primed monocytes, but not in deactivated monocytes, suggests a selective immune-modulatory activity that may be beneficial in septic patients and warrants further investigation.

References

1. Gordon AC et al. JAMA.316:509–18,2016

2. Peng T-C et al. Tzu Chi Medical Journal.25:150–4,2013

Funding: Intensive Care Foundation

Fig. 5 (abstract P362).
figure5

Effect of vasoprissin & noradrenaline on second-hit LPS induced monocyte TNF release. n = 4, **p < 0.01; ***p < 0.001

P363 Relationship between lymphocyte subset expression and serum concentrations of pd-1/pd-l1 in sepsis – pilot study

J Wilson1, Y Zhao1, M Singer2, J Spencer1, M Shankar-Hari1

1King’s College London, London, United Kingdom; 2Bloomsbury Institute of Intensive Care Medicine, London, United Kingdom

Introduction

Programmed death antigen (PD-1) and ligand (PD-L1) are inducible negative regulators on leukocyte surface. The PD-1/PD-L1 pathway contributes to lymphocyte exhaustion and immunosuppression in sepsis [1]. A clinical trial is currently evaluating the safety of an anti-PD-L1 antibody in sepsis patients [2]. However, serum levels and differences in PD-1/PD-L1 expression by B and T cell subsets are unknown and are likely to influence the efficacy of this intervention. We tested the hypothesis that surface PD-1/PD-L1 expression will differ in B and T cell subsets, and that serum PD-1/PD-L1 levels will be high in sepsis.

Methods

A prospective observational cohort study in 22 critically ill adult sepsis patients, excluding those with immune deficiency states, was done with ethics approval and informed consent. Blood was taken on ICU admission day and PBMCs isolated. Patients (11 survivors & 11 non-survivors) were compared with contemporaneously collected and analysed samples from 11 healthy controls. Cell surface staining was performed with antibodies to CD3, CD19 [BD Biosciences], CD4, CD27, PD-1, PD-L1 and PD-L2 [Biolegend], and live dead stain Amcyan [Invitrogen]. FACS analyses were performed on a FACScalibur flow cytometer [BD Biosciences] and using Tree Star FlowJo software. Serum PD-1 and PD-L1 in the same cohort were measured by ELISA [Proteintech]. Data was analysed using PRISM.

Results

The percentages of B and CD4+ T cells expressing PD-1 and PD-L1 were significantly higher in survivors and non-survivors of sepsis compared to healthy controls. There were no significant differences betwe