Sepsis 2016 Agra, India

Table of contents P1 D-Dimer in adult patients with presumed sepsis and their clinical outcomes Surinder Kumar Sharma, Anurag Rohatgi, Mansi Bajaj P2 Diagnosis of infection utilizing Acellix CD64 Charles L. Sprung, Ricardo Calderon Morales, Harvey Kasdan, Allon Reiter, Tobias Volker, Julien Meissonnier P3 High levels of phenylcarboxylic acids reflect the severity in ICU patients and affect phagocytic activity of neutrophils Natalia Beloborodova, Viktor Moroz, Aleksandra Bedova, Yulia Sarshor, Artem Osipov, Katerina Chernevskaya P4 The role of bacterial phenolic metabolites in mitochondrial dysfunction Nadezhda Fedotcheva, Ekaterina Chernevskaya, Natalia Beloborodova P5 The early diagnosis of severe sepsis and judgment of rapid transport to critical care center: better prognostic factor Hisashi Imahase, Kosuke C Yamada, Yuichiro Sakamoto, Miho Ohta, Ryota Sakurai, Mayuko Yahata, Mitsuru Umeka, Toru Miike, Hiroyuki Koami, Futoshi Nagashima, Takashi Iwamura, Satoshi Inoue P6 Translational neuromodulation of the immune system Zhifeng Li, Dennis Grech, Patrick Morcillo, Alex Bekker, Luis Ulloa P7 Pathway-level meta-analysis reveals transcriptional signature of septic shock Samanwoy Mukhopadhyay, Abhay D Pandey, Samsiddhi Bhattacharjee, Saroj K Mohapatra P8 Antibiotic dosing in septic patients on the critical care unit - a literature review Julie K Wilson P9 Pandemic of Escherichia coli clone O25: H4-ST131 producing CTX-M-15 extended spectrum- β- lactamase- as serious cause of multidrug resistance extraintestinal pathogenic E. coli infections in India Savita Jadhav, Rabindra Nath Misra, Nageswari Gandham, Kalpana Angadi, Chanda Vywahare, Neetu Gupta, Deepali Desai P10 Detection and characterization of meningitis using a DDA-based mass spectrometry approach Anahita Bakochi, Tirthankar Mohanty, Adam Linder, Johan Malmström P11 Diagnostic usefulness of lipid profile and procalcitonin in sepsis and trauma patients Dimple Anand, Seema Bhargava, Lalit Mohan Srivastava, Sumit Ray P12 Heparin – a novel therapeutic in sepsis? Jane Fisher, Peter Bentzer, Adam Linder P13 Hypothalamic impairment is associated with vasopressin deficiency during sepsis Luis Henrique Angenendt da Costa, Nilton Nascimentos dos Santos Júnior Carlos Henrique Rocha Catalão, Maria José Alves da Rocha P14 Presepsin (soluble CD14 subtype) is a dependable prognostic marker in critical septic patients Alfredo Focà, Cinzia Peronace, Giovanni Matera, Aida Giancotti, Giorgio Settimo Barreca, Angela Quirino, Maria Teresa Loria, Pio Settembre, Maria Carla Liberto, Bruno Amantea P15 Safety and efficacy of gelatin-containing solutions versus crystalloids and albumin - a systematic review with quantitative and qualitative summaries Christiane Hartog, Christiane Hartog, Claudia Moeller, Carolin Fleischmann, Daniel Thomas-Rueddel, Vlasislav Vlasakov, Bram Rochwerg, Philip Theurer, Konrad Reinhart P16 Immunomodulatory properties of peripheral blood mesenchymal stem cells following endotoxin stimulation in an equine model Anna E. Smith, Sandra D. Taylor P17 Frequency and outcome of early sepsis-associated coagulopathy Christopher Da Costa, Amanda Radford, Terry Lee, Joel Singer, John Boyd, David Fineberg, Mark Williams, James A Russell

The higher the D-dimer levels on day 0 and day 2, the higher the percentage of patients who require ICU admission during 30 day period (Figs. 3 and 4). The higher the D-dimer on day 0 and day 2, the higher the percentage of patients who succumbed to death during 30-day period (Figs. 5 and 6). In patients with presumed sepsis, 66.7 % patients had positive D-dimer levels on day 0 which was found to have a statistically significant association with organ dysfunction at presentation (p value <0.001) and ICU admission (p value <0.01), respectively. However, the association of Ddimer levels on day 0 with mortality was not statistically significant (p value ≥ 0.05). On day 2, positive D-dimer levels were demonstrated to have a statistically significant association with organ dysfunction on day 2 (p value <0.001) and ICU admission (p value <0.01), respectively. However, the association of D-dimer levels on day 2 with mortality was not statistically significant (p value ≥ 0.05). A positive strong correlation was demonstrated between D-dimer levels and SOFA Scores both on Day 0 (Pearson's correlation coefficient r = .850, p < .001) and Day 2 (Pearson's correlation coefficient r = .870, p < .001) (Figs. 7 and 8).
Conclusions: It can be concluded that fibrin D-dimer is a promising biomarker that may identify, in a simple and rapid way, infected patients who are at increased risk for organ dysfunction, ICU admission and death; thus helping in triage. These patients, consequently, could require special attention upon admission to the emergency service. Background: Differentiating patients who are infected or not in the intensive care unit (ICU) can be very difficult. Present diagnostic tests remain inadequate. CD64 has been found to be a potentially useful marker to identify infected patients. Unfortunately, CD64 measured by standard flow cytometers in a laboratory takes hours to perform. The Accellix table top flow cytometer automates the process: sample preparation and reading are performed in a dedicated disposable cartridge, and analytical data processing utilizing proprietary algorithms provides answers directly to the user. The purpose of this study was to evaluate the Accellix CD64 instrument, which provides results in 20 minutes in ICU patients with and without infections. Materials and methods: The Accellix disposable cartridge-based platform implements sample preparations using three reagent blisters. The three Accellix CD64 cartridge blisters contain staining cocktail of conjugated monoclonal antibodies, lysis buffer and reference beads respectively. Once sample processing is complete, the sample flows through a dedicated reading channel where data is acquired. Infected (ICUi) and non-infected ICU patients (ICU Control-ICUc) and normal volunteers (C) had CD64 levels measured by the Accellix CD64 instrument. Measurements were calculated as 'CD64 index' , i.e. the ratio between the fluorescence of the PMN population and the fluorescence of control beads. ICU infection, ICU control and normal control patients' results can be seen in Fig. 9. Results: 72 subjects were studied (ICUi-27, ICUc-22 and C-23). CD64 Index levels were higher (mean ± SEM) in ICU infection patients then ICU control and normal control patients (2.62 ± 0.39 vs. 1.31 ± 0.24 vs. 0.46 ± 0.04, p < 0.01 for ICUi vs. ICUc, p < 0.001 for ICUi vs. C). Conclusions: CD64 Index levels are higher in infected than noninfected ICU patients. Accellix CD64 is a promising instrument to differentiate infected from non-infected ICU patients in a timely manner.

P3
High levels of phenylcarboxylic acids reflect the severity in ICU patients and affect phagocytic activity of neutrophils Background: Previously it has been shown that phenylcarboxylic acids (PhCAs) could have microbial origin in human blood [1], associated with mortality [2] and demonstrated biological activity [3]. It is known that neutrophil dysfunction is one of the key mechanisms of severe infection and sepsis development. Objectives: to determine PhCAs levels typical for different severity of bacterial infection and the ability of these clinically relevant concentrations of PhCAs to affect the neutrophil phagocytosis in-vitro. Materials and methods: Blood samples were collected from outpatients (n = 17) with local bacterial infection of skin and soft tissue (ISST) and from critically ill patients with documented infection (n = 60): 46 patients on the day of admission to ICU and 14 -in terminal state one day before death. Clinical and laboratory data, SOFA Scores in patients were matched. Serum levels of PhCAs were measured using gas chromatography with flame ionization detector. Data from the previous study in healthy adult donors (n = 72) were used as a control [4]. The effect of PhCAs on the function of peripheral blood neutrophils was assessed by measuring the number of latex beads (d = 1.5 μm) phagocytized in-vitro, blood samples from healthy volunteers (n = 30). Data were compared by Mann-Whitney U-test and Sign test, Spearman's rank correlation coefficient was defined, p < 0.05 was considered significant (STATISTICA 10). Results: Levels of PhLA, p-HPhLA and p-HPhAA in patients were significantly higher than in healthy donors, as in severe infections and local bacterial inflammatory processes (Fig. 10, Table 1). A positive correlation with serum levels of the PhCAs and SOFA were found (r = 0.645-0.740, p < 0.001). In vitro we observed a significant decrease in the number of phagocytic neutrophils by 15 % under the influence of 6.0 μM p-HPhAA and 6.0 μM PhLA. Intensity of the absorption capacity of neutrophils significantly decreased by: 14 %, 31 % under the influence of 0.6, 6.0 μM p-HPhLA, respectively; 23 % -0.6 μM PhLA; 31 %, 30 % -0.6, 6.0 μM p-HPhAA, respectively. Perhaps, the ability of bacteria to produce PhCAs is one of adaptive mechanisms to protect from effector cells of immune system. Conclusions: Serum levels of PhCAs reflect the severity in ICU's patients and reach maximum values in terminally ill patients with infection. In-vitro PhCAs are able to inhibit phagocytic activity of neutrophils in clinically significant concentrations.   Background: Mitochondrial dysfunction is inherent in many systemic pathologies, including the inflammatory syndrome and sepsis. It is assumed that mitochondrial dysfunction in sepsis is associated with the overproduction of cytokines, reactive oxygen species (ROS), and NO, which affects several enzymes and complexes of the respiratory chain. However, the role of bacterial metabolites, which can accumulate in the blood of patients with infection, is not considered. Differences in the serum level of phenolic metabolites, in septic patients, compared with healthy donors was shown earlier [1]. It was found that these compounds affect the mitochondrial respiration and ROS production, showing either the prooxidant or antioxidant effects depending on their chemical structure [2,3]. In this work we studied the effect of bacterial phenolic acids on the activity of mitochondrial oxidative enzymes in the liver and kidney.

Materials and methods:
The experiments were performed on male Wistar rats. Liver mitochondria were isolated by the standard method. Concentrated homogenates (1 g tissue/ml medium) were prepared from the liver and kidney by a rapid procedure involving cooling, punching through press, and homogenization in a medium (125 mM KCl, 30 mM HEPES, pH 7.4). The effect of phenolic acids (all from Sigma) on the oxidation activity of mitochondria (0.5 mg protein/ml) and homogenate (1 mg protein/ml) was assessed by the reduction of nitroblue tetrazolium (NBT) at a wavelength of 560 nm after a 10-min incubation in the presence of an oxidation substrate followed by the addition of 20 μl of 10 % Triton X-100. Results: Phenolic acids influenced mitochondrial oxidative activity differently depending on the chemical structure of phenolic compounds and the oxidation substrate. Benzoic, phenylacetic, and phenylpropionic acids inhibited NAD-dependent oxidation by 40 % and weakly decreased succinate oxidation, while p-hydroxyphenyllactate and p-hydroxyphenylacetate activated NBT reduction supported by succinate and glutamate oxidation (Fig. 11). Similar effects were detected on tissue homogenates and were more pronounced in the liver than in the kidney. The antioxidant N-acetylcysteine prevented inhibition, indicating the contribution of thiol groups and ROS production to the decrease in oxidative activity induced by phenolic acids.
Conclusions: Aromatic bacterial metabolites can be involved in the development of the mitochondrial failure. Some phenolic acids inhibit NAD-dependent oxidation. Their action is tissuespecific. The influence of these phenolic acids is similar in some respect to the action of proinflammatory cytokines (Fig. 12). Thus, phenolic acids may regulate the signaling action of interleukins by affecting the respiration and ROS production in mitochondria and neutrophils [3].  Background: The prognosis of patients with severe sepsis or septic shock is improving. In our center, although promoting the standardization and individualization of sepsis treatment, some patients die. We examined the better factor of the prognosis of septic patients. Materials and methods: We included the patients with severe sepsis and septic shock who admitted from the emergency department to the ICU from January 2014 to June 2015. We defined severe sepsis as patients with SOFA score 2 points worse than sepsis onset before. Gender, age, APACHE2, SOFA, time from sepsis onset to our center visits, and time from sepsis onset to the first antibiotic administration, were examined. Results: The number of patients was 60: non-survivor 18(30 %), and survivor 42(70 %). Table 2 showed characteristics of each group. As APACHE2 or SOFA was bad, life prognosis was bad. As the definitive treatment or the first antibiotic administration was earlier, life prognosis was better. Table 3 showed the way patients' visited the center. Conclusions: The number of patients was 60: non-survivor 18(30 %), and survivor 42(70 %). Table 2 showed characteristics of each group. As APACHE2 or SOFA was bad, life prognosis was bad. As the definitive treatment or the first antibiotic administration was earlier, life prognosis was better. Table 3 showed the way patients' visited the center. Background: Sepsis, a leading cause of death in the ICU, is characterized by detrimental inflammation and multiple organ failure. We reported that electrical vagal stimulation controls inflammation and improves organ function in sepsis [1][2][3]. Even sepsis accounts for only 2 % of the hospitalizations; it makes up 17 % of in-hospital deaths in the US. We reasoned that nerve stimulation may control immunological stress during surgery, a major cause contributing sepsis during hospitalization. Materials and methods: Here, we report a prospective doubleblinded pilot trial to analyze whether intraoperative transdermal nerve stimulation prevents trauma, physiological and immunological stress during surgery. Results: Transdermal nerve stimulation was performed with electroacupuncture on anesthetized patients, and blood samples were collected under anesthesia to avoid any placebo interference. Subjects with electroacupuncture required 60 % less postoperative analgesic, but they had pain scores similar to that in the control patients. Electroacupuncture prevented postoperative hyperglycemia and attenuated serum ACTH in the older and heavier group of patients. From an immunological perspective, electroacupuncture did not affect the protective immune responses to surgical trauma including the induction of IL6 and IL10. The most significant immunological effect of electroacupuncture was enhancing TGFβ1 production during surgery in the older and lighter group of patients.
Conclusions: These results suggest that intraoperative electroacupuncture can reduce postoperative use of analgesic and improve immune and stress responses to surgery in anesthetized patients. Background: Septic shock is a major cause of death among the critically ill. Incompletely understood biology has lent itself to be explored at the genome level. Availability of genome-wide expression data from different studies on septic shock empowers the quest for pathways of interest by integration and meta-analysis of multiple data sets. Materials and methods: Electronic search was performed on medical literature (PubMed) and genome-wide gene expression databases (National Centre for Biotechnology Information Gene Expression Omnibus). Human studies of transcriptomic analysis of circulating leukocytes were selected. Systematic analysis was conducted to detect pathways differentially regulated in septic shock. Additionally coexpression network analysis was conducted.
Results: Osteoclast differentiation was consistently up-regulated in septic shock. As shown in Fig. 13, principal components analysis clearly distinguishes pathway expression in septic shock from sepsis.
Conclusions: A multi-gene expression signature differentiated septic shock from the milder form of sepsis. This deepens our understanding of disease biology of septic shock and is of potential value for clinical management.  Background: The mortality rate for critically ill septic patients remains unacceptably high. Timely administration of appropriate antimicrobials is the cornerstone of management, and achieving therapeutic levels is essential for ensuring treatment efficacy and preventing the development of drug-resistant pathogens [1]. The purpose of this article is to review the literature pertaining to antibiotic dosing in septic patients on the intensive care unit, and to explore whether therapeutic drug monitoring (TDM) may help to optimise management of this important patient group. Materials and methods: A literature search was performed using Web of Science and PubMed databases. Search terms included 'antibiotic level*' , 'antibiotic dos*' , 'therapeutic drug monitoring' and 'sepsis'. The reference lists of selected papers were also reviewed for relevant articles. Results: Critically ill septic patients exhibit a multitude of pathophysiological changes which affect drug handling, making achievement of optimum antibiotic dosing difficult [2]. Ample data is available to suggest that a worrying number of these patients do not receive therapeutic antibiotic doses [3][4][5]. Notably, the 'Defining Antibiotic Levels in Intensive Care Unit Patients' study measured serum concentrations of β-lactams in 384 patients across 68 hospitals in 10 countries. In 16 % of patients concentrations did not exceed the minimum inhibitory concentration, and this correlated with a poorer clinical outcome. [6] Similarly, a study measuring serum β-lactam concentrations in 80 patients found sub-therapeutic levels in the majority [7], and a 2014 study of 30 septic patients found linezolid levels to be sub-therapeutic in 63 % and toxic in 7 % [8]. Several studies suggest that TDM may lead to significant improvements in drug concentrations [9][10], although relatively few randomised controlled trials (RCT) have been conducted. One RCT compared TDM with standard dosing in 41 patients treated with piperacillin/tazobactam. Adequate drug concentrations were found in a significantly higher proportion of patients in the intervention than the control group. Mortality, however, did not differ significantly (although this was not a primary endpoint, and the study was not powered to detect this) [11]. Similar findings were reported from an RCT comparing TDM with standard dosing in 32 patients with neutropenic sepsis treated with piperacillin/tazobactam [12].
Conclusions: Based on the available evidence, it would seem that a worryingly high proportion of patients are receiving inadequate antibiotic doses, increasing the likelihood of treatment failure and the development of drug-resistance. Therapeutic drug monitoring offers a potential solution. A number of studies have demonstrated a benefit in pharmacokinetic/pharmacodynamic parameters. It remains unclear whether this translates to an improvement in clinical outcomes, and further research is required. Background: Antimicrobial resistance has emerged as an important determinant of outcome for patients in the intensive care unit (ICU). In recent years infections due to extraintestinal pathogenic Escherichia coli (ExPEC) predominantly E.coli sequence type 131 (ST131) is of great concern due to significant morbidity and mortality [2] a major clone linked to the spread of the CTX-M-15 extended spectrum-βlactamase (ESBL) resistance is growing concern in the ICU patient's population as it has been directly related with fluoroquinolone resistance and coresistance to aminoglycosides and trimethoprime-sulfamthoxazole, consequently delays in appropriate therapy, higher costs, and increased use of "last resort" antimicrobials e.g. carbapenemase to treat life threatening infections [3,4]. E. coli ST131 lineage among ExPEC is leading cause of community as well as hospital acquired urinary tract infections (UTIs) and also frequently encountered in soft tissue infections, blood stream infections (BSIs) and neonatal meningitis [1][2][3][4]. It is of major awareness to investigate its prevalence in countries such as India and to determine antibiotic resistance, virulence factors, associated clinical risk factors and potential genetic architecture of E. coli ST131.  Background: Despite advances in medicine, identification of sepsis from non-infectious systemic inflammatory conditions such as trauma, pancreatitis remains a challenging task for clinicians. Sepsis and trauma are both found to be associated with hypocholestrolemia and inflammation. Various studies have assessed and documented the prognostic role of hypolipidemia and procalcitonin in diagnosis and prognosis of sepsis; but in the rural setting, estimation of PCT is not easily available. Hence, we elucidated the usefulness of serum lipid profile as compared to procalcitonin in diagnosis of sepsis and trauma in the ICU. Materials and methods: Serum total cholesterol (TC), high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein cholesterol(LDL-C) and PCT were measured in 121 community acquired sepsis patients and 31 trauma patients at admission to ICU. SPSS version 17 was used for statistical analysis. Mann Whitney U test was applied to determine the significance between two groups and receiver operating characteristic (ROC) curves were plotted; best cut off points were derived. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated. Results: Median levels of TC, HDL-C, LDL-C were significantly lower whereas PCT levels were significantly higher in sepsis group as compared to trauma group (Table 4).  Background: Heparin binding protein (HBP) is released from neutrophils early in the immune response, which leads to sepsis. It is a very promising predictive biomarker for the development of sepsis and plasma levels are associated with development of organ failure in patients. Heparin is a drug typically used as an anticoagulant however it has recently been shown to affect many different systems, including inflammation. HBP binds very strongly to heparin and some studies have suggested that the use of heparin can have beneficial effects in sepsis. Therefore we hypothesized that HBP plays a causative role in the development of vascular leakage and AKI in sepsis and that heparin derivatives can be used to block these effects. Materials and methods: In-vitro, cultured endothelial and renal epithelial cells were treated with recombinant HBP. Trans-endothelial electrical resistance (TEER) was used to evaluate permeability of endothelial cell monolayers. IL-6 ELISA was used to evaluate HBPinduced inflammation in renal cells. Recombinant HBP was also injected into mice and scanning electron microscopy used to evaluate plasma leakage and inflammation. Administration of different heparin derivatives was used to block HBP-induced effects on permeability and IL-6 release in vitro and plasma leakage in the lungs and kidneys in-vivo.
Results: TEER showed that HBP significantly (P < 0.001) increased the permeability of endothelial cell monolayers. HBP also induced the release of IL-6 from renal cells (P < 0.01). This suggests that HBP plays causative roles in the development of vascular leakage and renal inflammation. HBP was also injected into mice and scanning electron microscopy revealed that it induced plasma leakage in the lungs and the kidneys. Administration of different heparin derivatives blocked the ability of HBP to induce permeability (p < 0.001) and IL-6 release in-vitro (P < 0.001) and plasma leakage in the lungs and kidneys in-vivo. Conclusions: Heparin binding protein (HBP) appears to play a causative role in sepsis and heparin compounds may be potential new therapeutics in its treatment.

P13
Hypothalamic impairment is associated with vasopressin deficiency during sepsis Luis Henrique Angenendt da Costa 1 , Nilton Nascimentos dos Santos Júnior 1 , Carlos Henrique Rocha Catalão 1 , Maria José Alves da Rocha 2 Background: Clinical and experimental studies have shown many hormonal alterations during sepsis. Considering vasopressin (AVP) secretion, in the early phase of sepsis its plasma concentration is increased. However, during the pathophysiological process the plasma levels remain inadequately low, despite of persistent hypotension. One of the hypotheses suggested for this relative deficiency is the apoptosis of vasopressinergic neurons in hypothalamus. Our objective was to identify elements involved in the hypothalamic cellular death and evaluate the modifications of glial cells and blood-brainbarrier (BBB) during sepsis.  Materials and methods: Male Wistar rats (250-300 g) were submitted to sepsis by cecal ligation and puncture (CLP) or non-manipulated (naïve), as control and then divided in two groups. In the first one, they were perfused and brains were collected for immunohistochemistry. In another one they were decapitated for blood collection and further plasma interferon-gama (IFN-γ) analysis by ELISA. Brain was also removed for apoptosis-related proteins expression analysis in the hypothalamus or in the supraoptic (SON) and paraventricular (PVN) nuclei by western blot. A third one was separated for the investigation of BBB permeability. Results: Despite of increased immunostaining for CD8 and MHC-I in the SON of septic animals, we did not find evidence of cell death mediated by immune cells. In the SON and PVN of septic animals, the expression of proteins involved in the activation of the extrinsic apoptosis pathway (tBID, cleaved caspase-8) was not altered, whereas anti-apoptotic factors related to the intrinsic pathway (BCL-2, BCL-xL) were decreased. In the SON of these animals, microglia assumed a morphology related to its activation, associated with the increase of plasma IFN-γ. There was a transitory breakdown of BBB in hypothalamus after 6 hours following CLP.

Conclusions:
The results indicate that the intrinsic apoptosis pathway seems to be responsible for the cell death observed in vasopressinergic nuclei and this condition is associated with microglial activation and BBB leaking. Background: Since decades worldwide investigators are searching reliable sepsis markers for prognostic and diagnostic evaluation of critically ill patients. Our group has already assessed sCD25 and IL-10 as dependable sepsis markers. Recently presepsin (soluble CD14 subtype, sCD14-ST) has been shown to increase in plasma of patients with sepsis. Here we evaluated the role of presepsin (PRES) in predicting outcome of critical septic patients; SOFA, procalcitonin (PCT) and C-reactive protein (CRP) were also assessed in parallel. Materials and methods: Critical patients admitted to the Unit of Intensive Care (ICU) of the University Hospital of Catanzaro (Italy) were sequentially enrolled into this observational prospective study, if a sepsis was clinically suspected; healthy volunteers were also included as controls. The SOFA score was assessed at the time of ICU admission. Clinical and microbiological data including blood culture were collected periodically. Based on 28 days survival, subjects were stratified in survivors and non-survivors. Plasma and serum samples were collected at multiple time points; samples were tested for presepsin (PATHFAST Presepsin assay), procalcitonin (VIDAS BRAHMS PCT), Creactive protein and IgG4 (BNTM II System immunonephelometry). A statistical analysis was carried out by ANOVA plus PLSD Fisher's test.
Results: At the admission SOFA scores were found significantly higher (p < 0.001) in nonsurvivor patients vs. survivor subjects (Fig. 15). Levels of presepsin were significantly more elevated at T-0 (p = 0.0007) (Fig. 16), at T-1 (p < 0.0001) and at T-2 (p < 0.0001) in non-survivors vs. survivors at the same time. Presepsin concentrations were found significantly increased at T-0 (p = 0.0073), T1 (p = 0.0111) and T2 (p = 0.0167) in blood culture-positive in comparison to culture-negative patients at the same time.
In culture-positive patients at T-0 both PCT and CRP levels, were found significantly enhanced vs. Background: Bacterial sepsis in humans is a common cause of illness and death worldwide, accounting for 60-80 % of deaths in developing countries and 20-30 % of deaths in the United States [1][2][3].
Overwhelming inflammation associated with Gram-negative sepsis (endotoxemia; lipopolysaccharide [LPS]) can lead to organ failure and death, and there is a critical need to identify therapy that will target this inflammation. Recently, horses have been identified as an important, emerging model for human sepsis, given that sepsis occurs naturally and is a leading cause of illness and death in both neonatal and adult horses [4]. Similar to sepsis in humans, horses develop sepsis-associated acute lung injury, septic peritonitis from bowel disruption, and bacteremia [4][5][6][7][8]. Studies have shown that mesenchymal stem cells (MSC) can decrease lymphocyte proliferation and downregulate pro-inflammatory cascades [9,10]. Specifically, MSC have been shown to secrete IL-6 and PGE2 in response to TNF-α, which results in decreased lymphocyte proliferation and thus, downregulation of pro-inflammatory cascades [11,12]. Importantly, equine MSC are easily harvested from peripheral blood, which allows for simple isolation and minimal donor morbidity [13]. We hypothesized that addition of equine peripheral blood MSC (PB-MSC) to cultures of equine mononuclear cells would result in increased production of IL-6, PGE2 and TXA2, and inhibition of TNF-α and IL-8 production following LPS stimulation. Materials and methods: We investigated the immunomodulatory properties of PB-MSC following stimulation with 100 pg/mL of lipopolysaccharide (LPS). Equine PB-MSC were added to equine monocyte cultures at ratios of 1:1, 1:2, 1:5, 1:10, and 1:100 (monocytes:PB-MSC), and supernatants were collected 12 hours after the onset of LPS treatment. Culture supernatant concentrations of the proinflammatory cytokines TNF-α, IL-6 and IL-8 and the eicosanoids PGE2 and TXA2 were evaluated by enzyme-linked immunosorbent assays (ELISA).
Results: As expected, the addition of equine PB-MSC to equine monocyte cultures resulted in increased concentrations of IL-6, PGE2 and TXA2, and decreased concentrations of TNF-α at 12 hours post-LPS stimulation (Fig. 17). There was no effect of equine PB-MSCs on IL-8 concentrations 12 hours post-LPS stimulation (data not shown).
Conclusions: Equine PB-MSC appear to respond in vitro to LPSstimulated equine monocytes by facilitating increases in cytokines that suppress lymphocyte proliferation and inhibit selective proinflammatory cytokines. Subsequent studies will evaluate lymphocyte responses in-vitro, as well as the safety and efficacy of equine PB-MSC in an in-vivo equine model of endotoxemia. These results have important implications for treatment of sepsis in horses and other species.