Regulation of mitochondrial function by hypoxia and inflammation in sepsis: A putative role for hypoxia inducible factor

Sepsis-related organ failure is the leading cause of mortality in European intensive care units (ICU). Although the inflammatory cascade of mediators in response to infection is well known, the relationships between regional inflammation, microvascular heterogeneity, hypoxia and hypoxia-inducible gene expression, and finally, organ dysfunction, are unknown. Growing evidence suggests that not only low oxygen supply to the tissues secondary to macrovascular and microvascular alterations, but also altered cellular oxygen utilization is involved in the development of multiorgan dysfunction [1]–[3]. Microbial products and innate and adaptive dysregulated immune response to infection directly affect parenchymal cells of organs and may contribute to multiorgan dysfunction.

Postoperative sepsis in infants below 6 months of age

BACKGROUND: Sepsis is a threatening postoperative complication especially in small infants. Regarding the advances in perinatal medicine, its incidence is unknown to date. We aimed to investigate the incidence, risk factors, laboratory findings and outcome of postoperative sepsis in infants younger than 6 months old. METHODS: We examined postoperative sepsis in babies below 6 months of age during a 4-year period at a tertiary pediatric institution. RESULTS: The rate of postoperative sepsis was 6.9%. Laparotomy with enterotomy, thoracotomy and diaphragmatic hernia repair (P<0.05, respectively) as well as low postnatal age and long operation time (P<0.001, respectively) were correlated with the incidence of sepsis. Significant independent predictors for the development of sepsis were the presence of a central venous catheter and perioperative antibiotic treatment (P<0.001, respectively). Coagulase negative Staphylococci were the major infecting organism associated with postoperative sepsis, accounting for 53% of monomicrobial infections. Complete blood counts with differential were not different between infants with sepsis and controls, who had undergone the same surgical procedures. Outcome was favorable in all cases; however, the length of hospital stay was significantly longer in sepsis patients (P<0.05). CONCLUSIONS: Postoperative sepsis syndrome is a frequent complication in infants below 6 months of age and causes significant prolongation of hospital stay. Adequate prevention and therapeutic strategies warrant further prospective investigations.

Effect of fluid resuscitation on mortality and organ function in experimental sepsis models

Introduction Several recent studies have shown that a positive fluid balance in critical illness is associated with worse outcome. We tested the effects of moderate vs. high-volume resuscitation strategies on mortality, systemic and regional blood flows, mitochondrial respiration, and organ function in two experimental sepsis models. Methods 48 pigs were randomized to continuous endotoxin infusion, fecal peritonitis, and a control group (n = 16 each), and each group further to two different basal rates of volume supply for 24 hours [moderate-volume (10 ml/kg/h, Ringer's lactate, n = 8); high-volume (15 + 5 ml/kg/h, Ringer's lactate and hydroxyethyl starch (HES), n = 8)], both supplemented by additional volume boli, as guided by urinary output, filling pressures, and responses in stroke volume. Systemic and regional hemodynamics were measured and tissue specimens taken for mitochondrial function assessment and histological analysis. Results Mortality in high-volume groups was 87% (peritonitis), 75% (endotoxemia), and 13% (controls). In moderate-volume groups mortality was 50% (peritonitis), 13% (endotoxemia) and 0% (controls). Both septic groups became hyperdynamic. While neither sepsis nor volume resuscitation strategy was associated with altered hepatic or muscle mitochondrial complex I- and II-dependent respiration, non-survivors had lower hepatic complex II-dependent respiratory control ratios (2.6 +/- 0.7, vs. 3.3 +/- 0.9 in survivors; P = 0.01). Histology revealed moderate damage in all organs, colloid plaques in lung tissue of high-volume groups, and severe kidney damage in endotoxin high-volume animals. Conclusions High-volume resuscitation including HES in experimental peritonitis and endotoxemia increased mortality despite better initial hemodynamic stability. This suggests that the strategy of early fluid management influences outcome in sepsis. The high mortality was not associated with reduced mitochondrial complex I- or II-dependent muscle and hepatic respiration.

Pancreatic stone protein is highly increased during posttraumatic sepsis and activates neutrophil granulocytes

OBJECTIVES: The level of pancreatic stone protein/regenerating protein (PSP/reg), a secretory protein produced in the pancreas, increases dramatically during pancreatic disease. However, after stress (e.g., anesthesia), PSP/reg levels are increased transiently in animals without pancreatic injury. Therefore, we aimed to determine whether PSP/reg is an acute-phase protein after nonpancreatic trauma. PATIENTS: Eighty-three polytraumatic patients without pancreatic damage. MEASUREMENTS AND MAIN RESULTS: We compared serum PSP/reg levels from polytraumatic patients without pancreatic damage with those in healthy controls (n = 38). C-reactive protein, interleukin-6, procalcitonin, and leukocyte numbers were also compared. The expression of CD62L and CD11b on neutrophils after exposure to PSP/reg was analyzed by flow cytometry. Thirty-three patients (39%) developed sepsis, 32 (38%) had local infections, and 18 (21%) had no infections. At admission, PSP/reg serum levels (10.2 [6.2-14.5] ng/mL; median [interquartile range]) were comparable with those in healthy controls (10.4 [7.5-12.3] ng/mL). During hospital stay, PSP/reg levels were elevated significantly in patients with sepsis (146.4 ng/mL) and in patients with infections (111.4 ng/mL) compared with patients without infections (22.8 ng/mL). Furthermore, binding of fluorescein isothiocyanate-labeled recombinant PSP/reg to human neutrophils was demonstrated. Recombinant PSP/reg elicited a dose-dependent shedding of L-selectin (CD62L) and upregulation of beta2-integrin (CD11b) in neutrophils, which indicates that PSP/reg activates neutrophils. CONCLUSIONS: We conclude that PSP/reg is up-regulated in blood after trauma, and the PSP/reg level is related to the severity of inflammation. Furthermore, PSP/reg binds to and activates neutrophils. Therefore, PSP/reg might be an acute-phase protein that could serve as a marker for posttraumatic complications.

A MIF haplotype is associated with the outcome of patients with severe sepsis: a case control study

BACKGROUND: Macrophage migration inhibitory factor (MIF) plays an important regulatory role in sepsis. In the promoter region a C/G single nucleotide polymorphism (SNP) at position -173 (rs755622) and a CATT5-8 microsatellite at position -794 are related to modified promoter activity. The purpose of the study was to analyze their association with the incidence and outcome of severe sepsis. METHODS: Genotype distributions and allele frequencies in 169 patients with severe sepsis, 94 healthy blood donors and 183 postoperative patients without signs of infection or inflammation were analyzed by real time PCR and Sequence analysis. All included individuals were Caucasians. RESULTS: Genotype distribution and allele frequencies of severe sepsis patients were comparable to both control groups. However, the genotype and allele frequencies of both polymorphisms were associated significantly with the outcome of severe sepsis. The highest risk of dying from severe sepsis was detectable in patients carrying a haplotype with the alleles -173 C and CATT7 (p = 0.0005, fisher exact test, RR = 1,806, CI: 1.337 to 2.439). CONCLUSION: The haplotype with the combination of the -173 C allele and the -794 CATT7 allele may not serve as a marker for susceptibility to sepsis, but may help identify septic patients at risk of dying.

Potential clinical utility of polymerase chain reaction in microbiological testing for sepsis

OBJECTIVES: To evaluate the potential improvement of antimicrobial treatment by utilizing a new multiplex polymerase chain reaction (PCR) assay that identifies sepsis-relevant microorganisms in blood. DESIGN: Prospective, observational international multicentered trial. SETTING: University hospitals in Germany (n = 2), Spain (n = 1), and the United States (n = 1), and one Italian tertiary general hospital. PATIENTS: 436 sepsis patients with 467 episodes of antimicrobial treatment. METHODS: Whole blood for PCR and blood culture (BC) analysis was sampled independently for each episode. The potential impact of reporting microorganisms by PCR on adequacy and timeliness of antimicrobial therapy was analyzed. The number of gainable days on early adequate antimicrobial treatment attributable to PCR findings was assessed. MEASUREMENTS AND MAIN RESULTS: Sepsis criteria, days on antimicrobial therapy, antimicrobial substances administered, and microorganisms identified by PCR and BC susceptibility tests. RESULTS: BC diagnosed 117 clinically relevant microorganisms; PCR identified 154. Ninety-nine episodes were BC positive (BC+); 131 episodes were PCR positive (PCR+). Overall, 127.8 days of clinically inadequate empirical antibiotic treatment in the 99 BC+ episodes were observed. Utilization of PCR-aided diagnostics calculates to a potential reduction of 106.5 clinically inadequate treatment days. The ratio of gainable early adequate treatment days to number of PCR tests done is 22.8 days/100 tests overall (confidence interval 15-31) and 36.4 days/100 tests in the intensive care and surgical ward populations (confidence interval 22-51). CONCLUSIONS: Rapid PCR identification of microorganisms may contribute to a reduction of early inadequate antibiotic treatment in sepsis.

Group B Streptococcus colonization in pregnancy: prevalence and prevention strategies of neonatal sepsis

Early onset neonatal sepsis due to Group B streptococci (GBS) is responsible for severe morbidity and mortality of newborns. While different preventive strategies to identify women at risk are being recommended, the optimal strategy depends on the incidence of GBS-sepsis and on the prevalence of anogenital GBS colonization. We therefore aimed to assess the Group B streptococci prevalence and its consequences on different prevention strategies. We analyzed 1316 pregnant women between March 2005 and September 2006 at our institution. The prevalence of GBS colonization was determined by selective cultures of anogenital smears. The presence of risk factors was analyzed. In addition, the direct costs of screening and intrapartum antibiotic prophylaxis were estimated for different preventive strategies. The prevalence of GBS colonization was 21%. Any maternal intrapartum risk factor was present in 37%. The direct costs of different prevention strategies have been estimated as follows: risk-based: 18,500 CHF/1000 live births, screening-based: 50,110 CHF/1000 live births, combined screening- and risk-based: 43,495/1000 live births. Strategies to prevent GBS-sepsis in newborn are necessary. With our colonization prevalence of 21%, and the intrapartum risk profile of women, the screening-based approach seems to be superior as compared to a risk-based approach.

[Mitochondrial dysfunction during sepsis, impact and possible regulating role of hypoxia-inducible factor-1alpha]

There is a direct correlation between the development of the multiple organ dysfunction syndrome (MODS) and the elevated mortality associated with sepsis. The mechanisms responsible for MODS development are being studied, however, the main efforts regarding MODS evaluation have focused on oxygen delivery optimization and on the modulation of the characteristic inflammatory cascade of sepsis, all with negative results. Recent studies have shown that there is development of tissue acidosis, even when there are normal oxygen conditions and limited presence of tissue cellular necrosis or apoptosis, which would indicate that cellular energetic dysfunction may be a central element in MODS pathogenesis. Mitochondrias are the main source of cellular energy, central regulators of cell death and the main source for reactive oxygen species. Several mechanisms contribute to mitochondrial dysfunction during sepsis, that is blockage of pyruvate entry into the Krebs cycle, oxidative phosphorylation substrate use in other enzymatic complexes, enzymatic complex inhibition and membrane damage mediated by oxidative stress, and reduction in mitochondrial content. Hypoxia-inducible factor-1alpha (HIF-1alpha) is a nuclear transcription factor with a central role in the regulation of cellular oxygen homeostasis. Its induction under hypoxic conditions is associated to the expression of hundreds of genes that coordinate the optimization of cellular oxygen delivery and the cellular energy metabolism. HIF-1alpha can also be stabilized under normoxic condition during inflammation and this activation seems to be associated with a prominent pro-inflammatory profile, with lymphocytes dysfunction, and to a reduction in cellular oxygen consumption. Further studies should establish a role for HIF-1alpha as a therapeutic target.

Thromboelastometry for the assessment of coagulation abnormalities in early and established adult sepsis: a prospective cohort study

INTRODUCTION: The inflammatory response to an invading pathogen in sepsis leads to complex alterations in hemostasis by dysregulation of procoagulant and anticoagulant factors. Recent treatment options to correct these abnormalities in patients with sepsis and organ dysfunction have yielded conflicting results. Using thromboelastometry (ROTEM(R)), we assessed the course of hemostatic alterations in patients with sepsis and related these alterations to the severity of organ dysfunction. METHODS: This prospective cohort study included 30 consecutive critically ill patients with sepsis admitted to a 30-bed multidisciplinary intensive care unit (ICU). Hemostasis was analyzed with routine clotting tests as well as thromboelastometry every 12 hours for the first 48 hours, and at discharge from the ICU. Organ dysfunction was quantified using the Sequential Organ Failure Assessment (SOFA) score. RESULTS: Simplified Acute Physiology Score II and SOFA scores at ICU admission were 52 +/- 15 and 9 +/- 4, respectively. During the ICU stay the clotting time decreased from 65 +/- 8 seconds to 57 +/- 5 seconds (P = 0.021) and clot formation time (CFT) from 97 +/- 63 seconds to 63 +/- 31 seconds (P = 0.017), whereas maximal clot firmness (MCF) increased from 62 +/- 11 mm to 67 +/- 9 mm (P = 0.035). Classification by SOFA score revealed that CFT was slower (P = 0.017) and MCF weaker (P = 0.005) in patients with more severe organ failure (SOFA >or= 10, CFT 125 +/- 76 seconds, and MCF 57 +/- 11 mm) as compared with patients who had lower SOFA scores (SOFA <10, CFT 69 +/- 27, and MCF 68 +/- 8). Along with increasing coagulation factor activity, the initially increased International Normalized Ratio (INR) and prolonged activated partial thromboplastin time (aPTT) corrected over time. CONCLUSIONS: Key variables of ROTEM(R) remained within the reference ranges during the phase of critical illness in this cohort of patients with severe sepsis and septic shock without bleeding complications. Improved organ dysfunction upon discharge from the ICU was associated with shortened coagulation time, accelerated clot formation, and increased firmness of the formed blood clot when compared with values on admission. With increased severity of illness, changes of ROTEM(R) variables were more pronounced.

Arterial blood pressure during early sepsis and outcome

OBJECTIVE: To evaluate the association between arterial blood pressure (ABP) during the first 24 h and mortality in sepsis. DESIGN: Retrospective cohort study. SETTING: Multidisciplinary intensive care unit (ICU). PATIENTS AND PARTICIPANTS: A total of 274 septic patients. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Hemodynamic, and laboratory parameters were extracted from a PDMS database. The hourly time integral of ABP drops below clinically relevant systolic arterial pressure (SAP), mean arterial pressure (MAP), and mean perfusion pressure (MPP = MAP - central venous pressure) levels was calculated for the first 24 h after ICU admission and compared with 28-day-mortality. Binary and linear regression models (adjusted for SAPS II as a measure of disease severity), and a receiver operating characteristic (ROC) analysis were applied. The areas under the ROC curve were largest for the hourly time integrals of ABP drops below MAP 60 mmHg (0.779 vs. 0.764 for ABP drops below MAP 55 mmHg; P < or = 0.01) and MPP 45 mmHg. No association between the hourly time integrals of ABP drops below certain SAP levels and mortality was detected. One or more episodes of MAP < 60 mmHg increased the risk of death by 2.96 (CI 95%, 1.06-10.36, P = 0.04). The area under the ROC curve to predict the need for renal replacement therapy was highest for the hourly time integral of ABP drops below MAP 75 mmHg. CONCLUSIONS: A MAP level > or = 60 mmHg may be as safe as higher MAP levels during the first 24 h of ICU therapy in septic patients. A higher MAP may be required to maintain kidney function.

Mesenteric lymphangitis and sepsis due to RTX toxin-producing Actinobacillus spp in 2 foals with hypothyroidism-dysmaturity syndrome

Actinobacillus suis-like organisms (ASLOs) have been isolated from the genital, respiratory, and digestive tracts of healthy adult horses, horses with respiratory disease, and septic foals. Two foals with congenital hypothyroidism-dysmaturity syndrome from separate farms developed ASLO infection. At necropsy, both had contracted carpal flexor tendons, thyroid hyperplasia, and thrombotic and necrotizing mesenteric lymphangitis and lymphadenitis; one foal also had mandibular prognathism. Numerous ASLOs were isolated from tissues from both foals, including intestine. Biochemical testing and mass spectrometric analysis of the two Actinobacillus isolates did not allow unequivocal identification. Comparative genetic analysis was done on these and similar isolates, including phylogeny based on 16S rRNA, rpoB and recN genes, as well as RTX (repeat in toxin) toxin typing of apxIA-apxIVA and aqxA genes. One isolate was identified as Actinobacillus suis sensu stricto, based on the presence of apxIA and apxIIA but not aqxA, whereas the other isolate had aqxA but neither apxIA nor apxIIA, consistent with A equuli ssp haemolyticus. Based on genotypic analysis of the isolates included for comparison, 3 of 3 equine ASLOs and 2 of 5 A equuli isolates were reclassified as A equuli subsp haemolyticus, emphasizing the importance of toxin genotyping in accurate classification of actinobacilli.

Mesenchymal stromal cells improve survival during sepsis in the absence of heme oxygenase-1: the importance of neutrophils

The use of mesenchymal stromal cells (MSCs) for treatment of bacterial infections, including systemic processes like sepsis, is an evolving field of investigation. This study was designed to investigate the potential use of MSCs, harvested from compact bone, and their interactions with the innate immune system, during polymicrobial sepsis induced by cecal ligation and puncture (CLP). We also wanted to elucidate the role of endogenous heme oxygenase (HO)-1 in MSCs during a systemic bacterial infection. MSCs harvested from the bones of HO-1 deficient (-/-) and wild-type (+/+) mice improved the survival of HO-1(-/-) and HO-1(+/+) recipient mice when administered after the onset of polymicrobial sepsis induced by CLP, compared with the administration of fibroblast control cells. The MSCs, originating from compact bone in mice, enhanced the ability of neutrophils to phagocytize bacteria in vitro and in vivo and to promote bacterial clearance in the peritoneum and blood after CLP. Moreover, after depleting neutrophils in recipient mice, the beneficial effects of MSCs were entirely lost, demonstrating the importance of neutrophils for this MSC response. MSCs also decreased multiple organ injury in susceptible HO-1(-/-) mice, when administered after the onset of sepsis. Taken together, these data demonstrate that the beneficial effects of treatment with MSCs after the onset of polymicrobial sepsis is not dependent on endogenous HO-1 expression, and that neutrophils are crucial for this therapeutic response.