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OBJECTIVES To train a model to predict vasopressor use in ICU patients with sepsis and optimize external performance across hospital systems using domain adaptation, a transfer learning approach. DESIGN Observational cohort study. SETTING Two academic medical centers from January 2014 to June 2017. PATIENTS Data were analyzed from 14,512 patients (9,423 at the development site and 5,089 at the validation site) who were admitted to an ICU and met Center for Medicare and Medicaid Services definition of severe sepsis either before or during the ICU stay. Patients were excluded if they never developed sepsis, if the ICU length of stay was less than 8 hours or more than 20 days or if they developed shock up to the first 4 hours of ICU admission. MEASUREMENTS AND MAIN RESULTS Forty retrospectively collected features from the electronic medical records of adult ICU patients at the development site (four hospitals) were used as inputs for a neural network Weibull-Cox survival model to derive a prediction tool for future need of vasopressors. Domain adaptation updated parameters to optimize model performance in the validation site (two hospitals), a different healthcare system over 2,000 miles away. The cohorts at both sites were randomly split into training and testing sets (80% and 20%, respectively). When applied to the test set in the development site, the model predicted vasopressor use 4-24 hours in advance with an area under the receiver operator characteristic curve, specificity, and positive predictive value ranging from 0.80 to 0.81, 56.2% to 61.8%, and 5.6% to 12.1%, respectively. Domain adaptation improved performance of the model to predict vasopressor use within 4 hours at the validation site (area under the receiver operator characteristic curve 0.81 [CI, 0.80-0.81] from 0.77 [CI, 0.76-0.77], p < 0.01; specificity 59.7% [CI, 58.9-62.5%] from 49.9% [CI, 49.5-50.7%], p < 0.01; positive predictive value 8.9% [CI, 8.5-9.4%] from 7.3 [7.1-7.4%], p < 0.01). CONCLUSIONS Domain adaptation improved performance of a model predicting sepsis-associated vasopressor use during external validation.

Supplemental Digital Content is available in the text., OBJECTIVES: To provide updated information on the burdens of sepsis during acute inpatient admissions for Medicare beneficiaries. DESIGN: Analysis of paid Medicare claims via the Centers for Medicare and Medicaid Services DataLink Project. SETTING: All U.S. acute-care hospitals, excluding federally operated hospitals (Veterans Administration and Defense Health Agency). Patients: All Medicare beneficiaries, January 2012—February 2020, with an explicit sepsis diagnostic code assigned during an inpatient admission. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The count of Medicare Part A/B (fee-for-service) plus Medicare Advantage inpatient sepsis admissions rose from 981,027 (CY2012) to 1,700,433 (CY 2019). The proportion of total admissions with sepsis in the Medicare Advantage population rose from 21.43% to 35.39%, reflecting the increasing beneficiary proportion enrolled in Medicare Advantage. In CY2019, 6-month mortality rates in Medicare fee-for-service beneficiaries for sepsis continued to decline, but remained high: 59.9% for septic shock, 35.5% for severe sepsis, 30.8% for sepsis attributed to a specific organism, and 26.5% for unspecified sepsis. Total fee-for-service-only inpatient hospital costs rose from $17.79B (CY2012) to $22.98B (CY2019). We estimated that the aggregate cost of sepsis hospital care for the entire U.S. population was at least $57.47B in 2019. Inclusion of 14 months’ (January 2019—February 2020) newer data exposed new trends: the cost per patient, number of admissions, and fraction of patients with sepsis labeled as present on admission inflected around November 2015, coincident with the change to International Classification of Diseases, 10th Edition, and introduction of the Severe Sepsis and Septic Shock Management Bundle (SEP-1) metric. CONCLUSIONS: Sepsis among Medicare beneficiaries precoronavirus disease 2019 imposed immense burdens upon patients, their families, and the taxpayers.

BACKGROUND Flow cytometry (FCM) is a rapid diagnostic tool for monitoring immune cell function. We sought to determine if assessment of cell phenotypes using standardized FCM could be used to identify nosocomial infection after trauma. METHODS Prospective study of trauma patients at a Level I center from 2014 to 2018. Clinical and FCM data were collected within 24 hours of admission. Random forest (RF) models were developed to estimate the risk of severe sepsis (SS), organ space infection (OSI), and ventilator-associated pneumonia (VAP). Variables were selected using backward elimination and models were validated with leave-one-out. RESULTS One hundred and thirty-eight patients were included (median age, 30 years [23-44 years]; median Injury Severity Score, 20 (14-29); 76% (105/138) Black; 60% (83/138) gunshots). The incidence of SS was 8.7% (12/138), OSI 16.7% (23/138), and VAP 18% (25/138). The final RF SS model resulted in five variables (RBCs transfused in first 24 hours; absolute counts of CD56- CD16+ lymphocytes, CD4+ T cells, and CD56 bright natural killer [NK] cells; percentage of CD16+ CD56+ NK cells) that identified SS with an AUC of 0.89, sensitivity of 0.98, and specificity of 0.78. The final RF OSI model resulted in four variables (RBC in first 24 hours, shock index, absolute CD16+ CD56+ NK cell counts, percentage of CD56 bright NK cells) that identified OSI with an AUC of 0.76, sensitivity of 0.68, and specificity of 0.82. The RF VAP model resulted in six variables (Sequential [Sepsis-related] Organ Failure Assessment score: Injury Severity Score; CD4- CD8- T cell counts; percentages of CD16- CD56- NK cells, CD16- CD56+ NK cells, and CD19+ B lymphocytes) that identified VAP with AUC of 0.86, sensitivity of 0.86, and specificity of 0.83. CONCLUSIONS Combined clinical and FCM data can assist with early identification of posttraumatic infections. The presence of NK cells supports the innate immune response that occurs during acute inflammation. Further research is needed to determine the functional role of these innate cell phenotypes and their value in predictive models immediately after injury. LEVEL OF EVIDENCE Prognostic, level III.

Importance Sepsis is a common syndrome with substantial morbidity and mortality. A combination of vitamin C, thiamine, and corticosteroids has been proposed as a potential treatment for patients with sepsis. Objective To determine whether a combination of vitamin C, thiamine, and hydrocortisone every 6 hours increases ventilator- and vasopressor-free days compared with placebo in patients with sepsis. Design, setting, and participants Multicenter, randomized, double-blind, adaptive-sample-size, placebo-controlled trial conducted in adult patients with sepsis-induced respiratory and/or cardiovascular dysfunction. Participants were enrolled in the emergency departments or intensive care units at 43 hospitals in the United States between August 2018 and July 2019. After enrollment of 501 participants, funding was withheld, leading to an administrative termination of the trial. All study-related follow-up was completed by January 2020. Interventions Participants were randomized to receive intravenous vitamin C (1.5 g), thiamine (100 mg), and hydrocortisone (50 mg) every 6 hours (n = 252) or matching placebo (n = 249) for 96 hours or until discharge from the intensive care unit or death. Participants could be treated with open-label corticosteroids by the clinical team, with study hydrocortisone or matching placebo withheld if the total daily dose was greater or equal to the equivalent of 200 mg of hydrocortisone. Main outcomes and measures The primary outcome was the number of consecutive ventilator- and vasopressor-free days in the first 30 days following the day of randomization. The key secondary outcome was 30-day mortality. Results Among 501 participants randomized (median age, 62 [interquartile range {IQR}, 50-70] years; 46% female; 30% Black; median Acute Physiology and Chronic Health Evaluation II score, 27 [IQR, 20.8-33.0]; median Sequential Organ Failure Assessment score, 9 [IQR, 7-12]), all completed the trial. Open-label corticosteroids were prescribed to 33% and 32% of the intervention and control groups, respectively. Ventilator- and vasopressor-free days were a median of 25 days (IQR, 0-29 days) in the intervention group and 26 days (IQR, 0-28 days) in the placebo group, with a median difference of -1 day (95% CI, -4 to 2 days; P = .85). Thirty-day mortality was 22% in the intervention group and 24% in the placebo group. Conclusions and relevance Among critically ill patients with sepsis, treatment with vitamin C, thiamine, and hydrocortisone, compared with placebo, did not significantly increase ventilator- and vasopressor-free days within 30 days. However, the trial was terminated early for administrative reasons and may have been underpowered to detect a clinically important difference. Trial registration ClinicalTrials.gov Identifier: NCT03509350.

ObjectiveTo measure heart rate variability metrics in critically ill COVID-19 patients with comparison to all-cause critically ill sepsis patients.Design and patientsRetrospective analysis of COVID-19 patients admitted to an ICU for at least 24h at any of Emory Healthcare ICUs between March and April 2020. The comparison group was a cohort of all-cause sepsis patients prior to COVID-19 pandemic.Interventionsnone.MeasurementsContinuous waveforms were captured from the patient monitor. The EKG was then analyzed for each patient over a 300 second (s) observational window, that was shifted by 30s in each iteration from admission till discharge. A total of 23 HRV metrics were extracted in each iteration. We use the Kruskal–Wallis and Steel–Dwass tests (p < 0.05) for statistical analysis and interpretations of HRV multiple measures.ResultsA total of 141 critically-ill COVID-19 patients met inclusion criteria, who were compared to 208 patients with all-cause sepsis. Demographic parameters were similar apart from a high proportion of African-Americans in the COVID-19 cohort. Three non-linear markers, including SD1:SD2, sample entropy, approximate entropy and four linear features mode of Beat-to-Beat interval (NN), Acceleration Capacity (AC), Deceleration Capacity (DC), and pNN50, were statistical significance between more than one binary combinations of the sub-groups (comparing survivors and non-survivors in both the COVID-19 and sepsis cohorts). The three nonlinear features and AC, DC, and NN (mode) were statistically significant across all four combinations. Temporal analysis of the main markers showed low variability across the 5 days of analysis, compared with sepsis patients.ConclusionsHeart rate variability is broadly implicated across patients infected with SARSCoV-2, and admitted to the ICU for critical illness. Comparing these metrics to patients with all-cause sepsis suggests a unique set of expressions that differentiate this viral phenotype. This finding could be investigated further as a potential biomarker to predict poor outcome in this patient population, and could also be a starting point to measure potential autonomic dysfunction in COVID-19.

Supplemental Digital Content is available in the text., Objectives: To provide contemporary estimates of the burdens (costs and mortality) associated with acute inpatient Medicare beneficiary admissions for sepsis. Design: Analysis of paid Medicare claims via the Centers for Medicare & Medicaid Services DataLink Project. Setting: All U.S. acute care hospitals, excluding federally operated hospitals (Veterans Administration and Defense Health Agency). Patients: All Medicare beneficiaries, 2012–2018, with an inpatient admission including one or more explicit sepsis codes. Interventions: None. Measurements and Main Results: Total inpatient hospital and skilled nursing facility admission counts, costs, and mortality over time. From calendar year (CY)2012–CY2018, the total number of Medicare Part A/B (fee-for-service) beneficiaries with an inpatient hospital admission associated with an explicit sepsis code rose from 811,644 to 1,136,889. The total cost of inpatient hospital admission including an explicit sepsis code for those beneficiaries in those calendar years rose from $17,792,657,303 to $22,439,794,212. The total cost of skilled nursing facility care in the 90 days subsequent to an inpatient hospital discharge that included an explicit sepsis code for Medicare Part A/B rose from $3,931,616,160 to $5,623,862,486 over that same interval. Precise costs are not available for Medicare Part C (Medicare Advantage) patients. Using available federal data sources, we estimated the aggregate cost of inpatient admissions and skilled nursing facility admissions for Medicare Advantage patients to have risen from $6.0 to $13.4 billion over the CY2012–CY2018 interval. Combining data for fee-for-service beneficiaries and estimates for Medicare Advantage beneficiaries, we estimate the total inpatient admission sepsis cost and any subsequent skilled nursing facility admission for all (fee-for-service and Medicare Advantage) Medicare patients to have risen from $27.7 to $41.5 billion. Contemporary 6-month mortality rates for Medicare fee-for-service beneficiaries with a sepsis inpatient admission remain high: for septic shock, approximately 60%; for severe sepsis, approximately 36%; for sepsis attributed to a specific organism, approximately 31%; and for unspecified sepsis, approximately 27%. Conclusion: Sepsis remains common, costly to treat, and presages significant mortality for Medicare beneficiaries.

Objectives To distinguish characteristics of Medicare beneficiaries who will have an acute inpatient admission for sepsis from those who have an inpatient admission without sepsis, and to describe their further trajectories during and subsequent to those inpatient admissions. Design Analysis of paid Medicare claims via the Centers for Medicare and Medicaid Services DataLink Project. Setting All U.S. acute care hospitals, excepting federal hospitals (Veterans Administration and Defense Health Agency). Patients Medicare beneficiaries, 2012-2018, with an inpatient hospital admission including one or more explicit sepsis codes. Interventions None. Measurements and main results Prevalent diagnoses in the year prior to the inpatient admission; healthcare contacts in the week prior to the inpatient admission; discharges, transfers, readmissions, and deaths (trajectories) for 6 months following discharge from the inpatient admission. Beneficiaries with no sepsis inpatient hospital admission for a year prior to an index hospital admission for sepsis were nearly indistinguishable by accumulated diagnostic codes from beneficiaries who had an index hospital admission without sepsis. Although the timing of healthcare services in the week prior to inpatient hospital admission was similar among beneficiaries who would be admitted for sepsis versus those whose inpatient admission did not include a sepsis code, the setting differed: beneficiaries destined for a sepsis admission were more likely to have received skilled nursing or unskilled nursing (e.g., nursing aide for activities of daily living) care. In contrast, comparing beneficiaries who had been free of any inpatient admission for an entire year and then required an inpatient admission, acute inpatient stays that included a sepsis code led to more than three times as many deaths within 1 week of discharge, with more admissions to skilled nursing facilities and fewer discharges to home. Comparing all beneficiaries who were admitted to a skilled nursing facility after an inpatient hospital admission, those who had sepsis coded during the index admission were more likely to die in the skilled nursing facility; more likely to be readmitted to an acute inpatient hospital and subsequently die in that setting; or if they survive to discharge from the skilled nursing facility, they are more likely to go next to a custodial nursing home. Conclusions Although Medicare beneficiaries destined for an inpatient hospital admission with a sepsis code are nearly indistinguishable by other diagnostic codes from those whose admissions will not have a sepsis code, their healthcare trajectories following the admission are worse. This suggests that an inpatient stay that included a sepsis code not only identifies beneficiaries who were less resilient to infection but also signals increased risk for worsening health, for mortality, and for increased use of advanced healthcare services during and postdischarge along with an increased likelihood of an inpatient hospital readmission.

Supplemental Digital Content is available in the text., Objective: To evaluate the impact of sepsis, age, and comorbidities on death following an acute inpatient admission and to model and forecast inpatient and skilled nursing facility costs for Medicare beneficiaries during and subsequent to an acute inpatient sepsis admission. Design: Analysis of paid Medicare claims via the Centers for Medicare & Medicaid Services DataLink Project (CMS) and leveraging the CMS-Hierarchical Condition Category risk adjustment model. Setting: All U.S. acute care hospitals, excepting federal hospitals (Veterans Administration and Defense Health Agency). Patients: All Part A/B (fee-for-service) Medicare beneficiaries with an acute inpatient admission in 2017 and who had no inpatient sepsis admission in the prior year. Interventions: None. Measurements and Main Results: Logistic regression models to determine covariate risk contribution to death following an acute inpatient admission; conventional regression to predict Medicare beneficiary sepsis costs. Using the Hierarchical Condition Category risk adjustment model to illuminate influence of illness on outcome of inpatient admissions, representative odds ratios (with 95% CIs) for death within 6 months of an admission (referenced to beneficiaries admitted but without the characteristic) are as follows: septic shock, 7.27 (7.19–7.35); metastatic cancer and acute leukemia (Hierarchical Condition Category 8), 6.76 (6.71–6.82); all sepsis, 2.63 (2.62–2.65); respiratory arrest (Hierarchical Condition Category 83), 2.55 (2.35–2.77); end-stage liver disease (Hierarchical Condition Category 27), 2.53 (2.49–2.56); and severe sepsis without shock, 2.48 (2.45–2.51). Models of the cost of sepsis care for Medicare beneficiaries forecast arise approximately 13% over 2 years owing the rising enrollments in Medicare offset by the cost of care per admission. Conclusions: A sepsis inpatient admission is associated with marked increase in risk of death that is comparable to the risks associated with inpatient admissions for other common and serious chronic illnesses. The aggregate costs of sepsis care for Medicare beneficiaries will continue to increase.

Sepsis is among the leading causes of morbidity, mortality, and cost overruns in critically ill patients. Early intervention with antibiotics improves survival in septic patients. However, no clinically validated system exists for real-time prediction of sepsis onset. We aimed to develop and validate an Artificial Intelligence Sepsis Expert algorithm for early prediction of sepsis.Observational cohort study.Academic medical center from January 2013 to December 2015.Over 31,000 admissions to the ICUs at two Emory University hospitals (development cohort), in addition to over 52,000 ICU patients from the publicly available Medical Information Mart for Intensive Care-III ICU database (validation cohort). Patients who met the Third International Consensus Definitions for Sepsis (Sepsis-3) prior to or within 4 hours of their ICU admission were excluded, resulting in roughly 27,000 and 42,000 patients within our development and validation cohorts, respectively.None.High-resolution vital signs time series and electronic medical record data were extracted. A set of 65 features (variables) were calculated on hourly basis and passed to the Artificial Intelligence Sepsis Expert algorithm to predict onset of sepsis in the proceeding T hours (where T = 12, 8, 6, or 4). Artificial Intelligence Sepsis Expert was used to predict onset of sepsis in the proceeding T hours and to produce a list of the most significant contributing factors. For the 12-, 8-, 6-, and 4-hour ahead prediction of sepsis, Artificial Intelligence Sepsis Expert achieved area under the receiver operating characteristic in the range of 0.83-0.85. Performance of the Artificial Intelligence Sepsis Expert on the development and validation cohorts was indistinguishable.Using data available in the ICU in real-time, Artificial Intelligence Sepsis Expert can accurately predict the onset of sepsis in an ICU patient 4-12 hours prior to clinical recognition. A prospective study is necessary to determine the clinical utility of the proposed sepsis prediction model.

Purpose: To explore potential design for pharmacogenomics trials in sepsis, we investigate the interaction between pharmacogenomic biomarkers and response to drotrecogin alfa (activated) (DrotAA). This trial was designed to validate whether previously identified improved response polymorphisms (IRPs A and B) were associated with an improved response to DrotAA in severe sepsis. Methods: Patients with severe sepsis at high risk of death, who received DrotAA or not, with DNA available were included and matched to controls adjusting for age, APACHE II or SAPS II, organ dysfunction, ventilation, medical/surgical status, infection site, and propensity score (probability that a patient would have received DrotAA given their baseline characteristics). Independent genotyping and two-phase data transfer mitigated bias. The primary analysis compared the effect of DrotAA in IRP+ and IRP− groups on in-hospital 28-day mortality. Secondary endpoints included time to death in hospital; intensive care unit (ICU)-, hospital-, and ventilator-free days; and overall DrotAA treatment effect on mortality. Results: Six hundred and ninety-two patients treated with DrotAA were successfully matched to 1935 patients not treated with DrotAA. Genotyping was successful for 639 (DrotAA) and 1684 (nonDrotAA) matched patients. The primary hypothesis of a genotype-by-treatment interaction (assessed by conditional logistic regression analysis) was not significant (P = 0.30 IRP A; P = 0.78 IRP B), and there was no significant genotype by treatment interaction for any secondary endpoint. Conclusions: Neither IRP A nor IRP B predicted differential response to DrotAA on in-hospital 28-day mortality. ClinicalTrials.gov registration NCT01486524

Purpose Using administrative codes and minimal physiologic and laboratory data, we sought a high-specificity identification strategy for patients whose sepsis initially appeared during their ICU stay. Materials and methods We studied all patients discharged from an academic hospital between September 1, 2013 and October 31, 2014. Administrative codes and minimal physiologic and laboratory criteria were used to identify patients at high risk of developing the onset of sepsis in the ICU. Two clinicians then independently reviewed the patient record to verify that the screened-in patients appeared to become septic during their ICU admission. Results Clinical chart review verified sepsis in 437/466 ICU stays (93.8%). Of these 437 encounters, only 151 (34.6%) were admitted to the ICU with neither SIRS nor evidence of infection and therefore appeared to become septic during their ICU stay. Conclusions Selected administrative codes coupled to SIRS criteria and applied to patients admitted to ICU can yield up to 94% authentic sepsis patients. However, only 1/3 of patients thus identified appeared to become septic during their ICU stay. Studies that depend on high-intensity monitoring for description of the time course of sepsis require clinician review and verification that sepsis initially appeared during the monitoring period.

Purpose Management of sepsis in critically ill patients remains difficult and requires prolonged intensive care. Genetic testing has been proposed as a strategy to identify patients at risk for adverse outcome of critical illnesses. Therefore, we wished to determine the influence of heredity on predisposition to poor outcome and on duration of ventilator support of intensive care unit (ICU) patients. Methods A study was conducted from July 2001 to December 2005 in heterogeneous population of patients from 12 US ICUs represented by the Genetic Predisposition to Severe Sepsis (GenPSS) archive. In 1057 Caucasian critically ill patients with SAPS II probability of survival of >0.2 in the US, six functional single nucleotide polymorphisms in relation to inflammatory cytokines and innate immunity (rs1800629, rs16944, rs1800795, rs1800871, rs2569190, and rs909253) were evaluated in terms of mortality and ventilator free days. Results The AA homozygote of TNF (−308) (rs1800629) was most over-represented in the deceased patient group ( P = 0.015 with recessive model). The carriage of the TNF (−308)*AA genotype showed significantly higher odds ratio of 2.67(1.29–5.55) ( P = 0.008) after adjustment with the covariates. However, the presence of 1, 2, or 3 acute organ dysfunctions was larger prognostic factors for the adverse outcome (OR(95%CI) = 2.98(2.00–4.45), 4.01(2.07–7.77), or 19.95(4.99–79.72), P TNF (−308)*AA patient significantly diverged from that of TNF (−308)*(GG + GA) ((AA v GG + GA), Adjusted HR(95%CI) = 2.53(1.11–5.79) with Cox regression, P = 0.028). Conclusions TNF (−308)*AA is significantly associated with susceptibility to adverse outcome and to longer ventilator duration. Therefore, heredity likely affects both predisposition to ICU prognosis as well as the resource utilization.

The sepsis syndrome is an old phenomenon. A destructive response to a system disturbance, it manifests as widespread inflammation. Over the past two centuries, biomedical research has identified triggers and described components of the pathways that underlie the sepsis syndrome. Attempts at translating these findings into preventive and therapeutic interventions have met with varying levels of success. In this chapter, we examine the history of sepsis science through an engineering lens. Patterned attempts to intervene in the natural history of the sepsis syndrome will be discussed in parallel with similar, hypothetical adjustments made to a model system from the engineering canon. This juxtaposition will facilitate our review of the history of sepsis science. Using the logic of systems engineering and network science, we propose a way forward.

Sepsis is primarily a disease of the aged, with increased incidence and mortality occurring in aged hosts. Heat shock protein (HSP) 70 plays an important role in both healthy aging and the stress response to injury. The purpose of this study was to determine the role of HSP70 in mediating mortality and the host inflammatory response in aged septic hosts. Sepsis was induced in both young (6- to 12-wk-old) and aged (16- to 17-mo-old) HSP70−/− and wild-type (WT) mice to determine whether HSP70 modulated outcome in an age-dependent fashion. Young HSP70−/− and WT mice subjected to cecal ligation and puncture, Pseudomonas aeruginosa pneumonia, or Streptococcus pneumoniae pneumonia had no differences in mortality, suggesting HSP70 does not mediate survival in young septic hosts. In contrast, mortality was higher in aged HSP70−/− mice than aged WT mice subjected to cecal ligation and puncture (p = 0.01), suggesting HSP70 mediates mortality in sepsis in an age-dependent fashion. Compared with WT mice, aged septic HSP70−/− mice had increased gut epithelial apoptosis and pulmonary inflammation. In addition, HSP70−/− mice had increased systemic levels of TNF-α, IL-6, IL-10, and IL-1β compared with WT mice. These data demonstrate that HSP70 is a key determinant of mortality in aged, but not young hosts in sepsis. HSP70 may play a protective role in an age-dependent response to sepsis by preventing excessive gut apoptosis and both pulmonary and systemic inflammation.

Objective Pathogens that cause pneumonia may be treated in a targeted fashion by antibiotics, but if this therapy fails, then treatment involves only nonspecific supportive measures, independent of the inciting infection. The purpose of this study was to determine whether host response is similar after disparate infections with similar mortalities. Design Prospective, randomized controlled study. Setting Animal laboratory in a university medical center. Interventions Pneumonia was induced in FVB/N mice by either Streptococcus pneumoniae or two different concentrations of Pseudomonas aeruginosa. Plasma and bronchoalveolar lavage fluid from septic animals was assayed by a microarray immunoassay measuring 18 inflammatory mediators at multiple time points. Measurements and main results The host response was dependent on the causative organism as well as kinetics of mortality, but the pro-inflammatory and anti-inflammatory responses were independent of inoculum concentration or degree of bacteremia. Pneumonia caused by different concentrations of the same bacteria, Pseudomonas aeruginosa, also yielded distinct inflammatory responses; however, inflammatory mediator expression did not directly track the severity of infection. For all infections, the host response was compartmentalized, with markedly different concentrations of inflammatory mediators in the systemic circulation and the lungs. Hierarchical clustering analysis resulted in the identification of five distinct clusters of the host response to bacterial infection. Principal components analysis correlated pulmonary macrophage inflammatory peptide-2 and interleukin-10 with progression of infection, whereas elevated plasma tumor necrosis factor sr2 and macrophage chemotactic peptide-1 were indicative of fulminant disease with >90% mortality within 48 hrs. Conclusions Septic mice have distinct local and systemic responses to Streptococcus pneumoniae and Pseudomonas aeruginosa pneumonia. Targeting specific host inflammatory responses induced by distinct bacterial infections could represent a potential therapeutic approach in the treatment of sepsis.

To investigate the significance of functional polymorphisms of inflammatory response genes by analysis of a large population of patients, both with and without severe sepsis, and representative of the diverse populations (geographic diversity, physician diversity, clinical treatment diversity) that would be encountered in critical care clinical practice.: Collaborative case-control study conducted from July 2001 to December 2005.A heterogeneous population of patients from 12 U.S. intensive care units represented by the Genetic Predisposition to Severe Sepsis archive.A total of 854 patients with severe sepsis and an equal number of mortality, age, gender, and race-matched patients also admitted to the intensive care unit without evidence of any infection (matched nonseptic controls).We developed assays for six functional single nucleotide polymorphisms present before the first codon of tumor necrosis factor at -308, IL1B at -511, IL6 at -174, IL10 at -819, and CD14 at -159, and in the first intron of LTA (also known as tumor necrosis factor-B) at +252 (LTA[+252]). The Project IMPACT critical care clinical database information management system developed by the Society of Critical Care Medicine and managed by Tri-Analytics and Cerner Corporation was utilized. Template-directed dye-terminator incorporation assay with fluorescence polarization detection was used as a high-throughput genotyping strategy. Fifty-three percent of the patients were male with 87.3% and 6.4% of Caucasian and African American racial types, respectively. Overall mortality was 35.1% in both severe sepsis and matched nonseptic control patients group. Average ages (standard deviation) of the severe sepsis and matched nonseptic control patients were 63.0 (16.05) and 65.0 (15.58) yrs old, respectively. Among the six single nucleotide polymorphisms, LTA (+252) was most overrepresented in the septic patient group (% severe sepsis; AA 45.6: AG 51.1: GG 56.7, p = .005). Furthermore, the genetic risk effect was most pronounced in males, age60 yrs (p = .005).LTA(+252) may influence predisposition to severe sepsis, a predisposition that is modulated by gender and age. Although the genetic influences can be overwhelmed by both comorbid factors and acute illness in individual cases, population studies suggest that this is an influential biological pathway modulating risk of critical illnesses.

Systemic administration of epidermal growth factor (EGF) decreases mortality in a murine model of septic peritonitis. Although EGF can have direct healing effects on the intestinal mucosa, it is unknown whether the benefits of systemic EGF in peritonitis are mediated through the intestine. Here, we demonstrate that enterocyte-specific overexpression of EGF is sufficient to prevent intestinal barrier dysfunction and improve survival in peritonitis. Transgenic FVB/N mice that overexpress EGF exclusively in enterocytes ( IFABP-EGF) and wild-type (WT) mice were subjected to either sham laparotomy or cecal ligation and puncture (CLP). Intestinal permeability, expression of the tight junction proteins claudins-1, -2, -3, -4, -5, -7, and -8, occludin, and zonula occludens-1; villus length; intestinal epithelial proliferation; and epithelial apoptosis were evaluated. A separate cohort of mice was followed for survival. Peritonitis induced a threefold increase in intestinal permeability in WT mice. This was associated with increased claudin-2 expression and a change in subcellular localization. Permeability decreased to basal levels in IFABP-EGF septic mice, and claudin-2 expression and localization were similar to those of sham animals. Claudin-4 expression was decreased following CLP but was not different between WT septic mice and IFABP-EGF septic mice. Peritonitis-induced decreases in villus length and proliferation and increases in apoptosis seen in WT septic mice did not occur in IFABP-EGF septic mice. IFABP-EGF mice had improved 7-day mortality compared with WT septic mice (6% vs. 64%). Since enterocyte-specific overexpression of EGF is sufficient to prevent peritonitis-induced intestinal barrier dysfunction and confers a survival advantage, the protective effects of systemic EGF in septic peritonitis appear to be mediated in an intestine-specific fashion.

Lymphocytes help determine whether gut epithelial cells proliferate or differentiate but are not known to affect whether they live or die. Here, we report that lymphocytes play a controlling role in mediating gut epithelial apoptosis in sepsis but not under basal conditions. Gut epithelial apoptosis is similar in unmanipulated Rag-1−/− and wild-type (WT) mice. However, Rag-1−/− animals have a 5-fold augmentation in gut epithelial apoptosis following cecal ligation and puncture (CLP) compared to septic WT mice. Reconstitution of lymphocytes in Rag-1−/− mice via adoptive transfer decreases intestinal apoptosis to levels seen in WT animals. Subset analysis indicates that CD4+ but not CD8+, γδ, or B cells are responsible for the antiapoptotic effect of lymphocytes on the gut epithelium. Gut-specific overexpression of Bcl-2 in transgenic mice decreases mortality following CLP. This survival benefit is lymphocyte dependent since gut-specific overexpression of Bcl-2 fails to alter survival when the transgene is overexpressed in Rag-1−/− mice. Further, adoptively transferring lymphocytes to Rag-1−/− mice that simultaneously overexpress gut-specific Bcl-2 results in improved mortality following sepsis. Thus, sepsis unmasks CD4+ lymphocyte control of gut apoptosis that is not present under homeostatic conditions, which acts as a key determinant of both cellular survival and host mortality.—Stromberg, P. E., Woolsey, C. A., Clark, A. T., Clark, J. A., Turnbull, I. R., McConnell, K. W., Chang, K. C., Chung, C.-S., Ayala, A., Buchman, T. G., Hotchkiss, R. S., Coopersmith, C. M. CD4+ lymphocytes control gut epithelial apoptosis and mediate survival in sepsis.

BACKGROUND Age influences outcome of sepsis and septic shock. The mechanism of this age-dependent vulnerability to sepsis remains largely unknown. Because much of the mortality and morbidity associated with sepsis and septic shock is the result of severe derangements in the cardiovascular system, it is possible that the myocardium responds to injury in a developmentally influenced manner. We hypothesized that analysis of cardiac RNA expression profiles may differentiate between the myocardial response to sepsis in young and old mice. METHODS AND RESULTS Sixteen FVB/N male mice were stratified based on age. Young animals were 6 wks old, correlating to 4 to 6 human years, and aged animals were 20 months old correlating to 70 to 80 human years. Animals underwent either cecal ligation and puncture to produce polymicrobial sepsis or a sham operation. Both ventricles were excised after kill at 24 hrs. There were 53 genes that differed in RNA abundance between the four groups (false discovery rate of 0.005, p < 0.00001). Additionally, four genes were associated with an age-dependent response to sepsis: CYP2B2 (cytochrome P450, family 2, subfamily B, polypeptide 6), VGLL2 (vestigial like 2), and PAH (phenylalanine hydroxylase). The fourth gene is an expressed sequence tag, the function of which is related to the cytochrome P450 family. These genes play roles in phenylalanine, tyrosine, tryptophan, and fatty acid metabolism. CONCLUSIONS This report describes the transcriptional response of the heart to sepsis. In addition, our findings suggest that these differences are in part age-dependent and serve as hypothesis generation.

Staphylococcus aureus is the most common cause of healthcare-associated pneumonia. Despite the significant morbidity and mortality associated with the disease, animal models of S. aureus pneumonia are rare.We examined the pathogenicity of four different strains of S. aureus (both methicillin-sensitive and -resistant as well as Panton-Valentine leukocidin-positive and -negative) in four strains of immunocompetent inbred and outbred mice (FVB/N, C57Bl/6, BALB/c, ND4; n = 148). The immunological basis for the development of murine S. aureus pneumonia was then determined by selectively depleting neutrophils, lymphocytes, or pulmonary macrophages prior to the onset of infection. An additional cohort of animals was rendered immunosuppressed by induction of abdominal sepsis via cecal ligation and puncture 2, 4, or 7 d prior to the onset of pneumonia.Nearly all immunocompetent mice survived, regardless of which strain of S. aureus was used or which strain of mouse was infected. Among animals with immune depletion or prior immunosuppression, survival was decreased only following neutrophil depletion (26% versus 90% alive at 7 d, P0.0001). Compared to immunocompetent animals, neutrophil-depleted mice with S. aureus pneumonia had delayed pulmonary bacterial clearance at 16 and 40 h but had no difference in levels of bacteremia. Neutrophil-depleted mice also had elevated levels of pulmonary monocyte chemotactic protein-1 (822 pg/mL versus 150 pg/mL, P0.05). In contrast, pulmonary histological appearance was similar in both groups as was dry/wet lung weight.These results suggest that neutrophils play a critical role in the host response to S. aureus pneumonia, and the survival differences observed in neutrophil-depleted mice are associated with alterations in bacterial clearance and pulmonary cytokine response.

Elevated interleukin (IL)-6 levels correlate with increased mortality following sepsis. IL-6 levels >14,000 pg/ml drawn 6 hours following cecal ligation and puncture (CLP) are associated with 100% mortality in ND4 mice, even if antibiotic therapy is initiated 12 hours after the septic insult. The first aim of this study was to see if earlier institution of antibiotic therapy could improve overall survival in septic mice and rescue the subset of animals predicted to die based upon high IL-6 levels. Mice (n=184) were subjected to CLP, had IL-6 levels drawn six hours later and then were randomized to receive imipenem, a broad spectrum antimicrobial agent, beginning six or twelve hours post-operatively. Overall one-week survival improved from 25.5% to 35.9% with earlier administration of antibiotics (p14,000 pg/ml, 25% survived if imipenem was started at 6 hours, while none survived if antibiotics were started later (p14,000 pg/ml. These results demonstrate that earlier systemic therapy can improve outcome in a subset of mice predicted to die in sepsis, but we are unable to demonstrate any benefit in similar animals using targeted therapy directed at IL-6.

Background Sepsis, iron loading, and aging cause independent increases in gut epithelial and splenic apoptosis. It is unknown how their combination will affect apoptosis and systemic cytokine levels. Materials and Methods Hfe−/− mice (a murine homologue of hemochromatosis) abnormally accumulate iron in their tissues. Aged (24–26 months) or mature (16–18 months) Hfe−/− mice and wild type (WT) littermates were subjected to cecal ligation and puncture (CLP) or sham laparotomy. Intestine, spleen, and blood were harvested 24 h later and assessed for apoptosis and cytokine levels. Results Gut epithelial and splenic apoptosis were low in both aged septic and sham Hfe−/− mice, regardless of the amount of iron in their diet. Mature septic WT mice had increased apoptosis compared to age-matched sham WT mice. Mature septic Hfe−/− mice had similar levels of intestinal cell death to age-matched septic WT mice but higher levels of splenic apoptosis. Apoptosis was significantly lower in septic aged Hfe−/− mice than septic mature Hfe−/− animals. Interleukin-6 was elevated in septic aged Hfe−/− mice compared to sham mice. Conclusions Although sepsis, chronic iron dysregulation, and aging each increase gut and splenic apoptosis, their combination yields cell death levels similar to sham animals despite the fact that aged Hfe−/− mice are able to mount an inflammatory response following CLP and mature Hfe−/− mice have elevated sepsis-induced apoptosis. Combining sepsis with two risk factors that ordinarily increase cell death and increase mortality in CLP yields an apoptotic response that could not have been predicted based upon each element in isolation.

Both aging and sepsis independently increase splenic and gut epithelial apoptosis. Sepsis-induced apoptosis in either cell type is also associated with increased mortality in young mice. We sought to determine whether age alters sepsis-induced splenic and gut epithelial cell death. Young (2 months) and aged (22 months) male ND4 mice were subjected to either single-puncture cecal ligation and puncture (CLP) with a 23-gauge needle or sham laparotomy. Apoptosis was assessed 24 hours later in the spleen and gut epithelium by active caspase 3 and hematoxylin and eosin staining. Aged septic mice had increased splenic apoptosis compared with either young septic animals or aged sham animals (15 vs. 7 vs. 5 apoptotic cells/high-powered field, P < 0.05). Similarly, aged septic animals had an elevation in gut epithelial cell death compared with either young septic or aged sham mice (33 vs. 16 vs. 6 apoptotic cells/100 contiguous crypts, P < 0.05). Elevated intestinal cell death was not associated with changes in either gut proliferation or cell division. To verify that the increase in splenic apoptosis seen in septic aged animals was not strain specific, double-puncture CLP with a 25-gauge needle or sham laparotomy was performed on young (4 months) or aged (24 months) C57BL/6 male mice. Similar to results seen in outbred animals, aged septic animals in this inbred strain had increased splenic apoptosis compared with either young septic animals or aged sham animals (23 vs. 7 vs. 4 apoptotic cells/ high powered field, P < 0.05). These results indicate that although infection and aging each independently cause an increase in splenic and gut epithelial apoptosis, their combination leads to a disproportionate increase in cell death in these rapidly dividing cell populations,and potentially plays a role in the marked increase in mortality seen with aging in sepsis.

Despite having dysregulated iron metabolism, critically ill patients may receive exogenous iron for the treatment of anemia. Iron is associated with increased tissue apoptosis and may facilitate bacterial growth. We hypothesized that exogenous iron administration given after the onset of sepsis would lead to increased mortality rate. To discriminate between elevated cell death and bacterial overgrowth as potential mediators of mortality, we examined gut epithelial and lymphocyte apoptosis and systemic bacterial counts in animals given iron supplementation after the onset of sepsis.Prospective, randomized, controlled study.Animal laboratory in a university medical center.Male C57BL/6 mice, 6-10 wks old.C57BL/6 mice were subjected to cecal ligation and puncture (CLP), a well-accepted model of intra-abdominal sepsis, followed by daily subcutaneous injections of either 1 mL of iron dextran (5 mg/mL) or 0.9% NaCl for a total of five doses. Animals (n = 78) were followed for survival for 8 days. Separate cohorts (n = 76) were killed 24 or 48 hrs after cecal ligation and puncture or sham laparotomy and were assayed for gut epithelial and splenic apoptosis as well as for quantitative blood cultures.Eight-day survival was 7% in animals that received iron and 26% in mice that received 0.9% NaCl (p.005). Iron supplementation after cecal ligation and puncture increased apoptosis by both active caspase 3 and hematoxylin and eosin staining in both the intestinal epithelium and spleen at 24 hrs (p.05). Iron supplementation after sham laparotomy did not cause mortality or elevated apoptosis. Quantitative blood cultures revealed no detectable differences between septic animals that received iron and those that received 0.9% NaCl.High-dose iron supplementation with iron dextran after the onset of sepsis significantly increases mortality rate in this animal model. Iron-induced mortality may be mediated by an increase in gut epithelial and splenic apoptosis, whereas severity of bacteremia does not appear to play a causative role.

Genetically identical mice have a heterogeneous response to antibiotic therapy in sepsis, with only a subset deriving therapeutic benefit. We sought to determine whether the severity of a septic insult correlates with the survival benefit conferred by antibiotics. We also sought to determine whether antibiotics given 12 h after injury alter survival in animals predicted to die based upon high interleukin (IL)-6 levels drawn 6 h earlier. Adult male ND4 mice (n = 363) were subjected to double-puncture cecal ligation and puncture (CLP) with a 19-, 21-, or 23-gauge needle. Animals were randomized to receive imipenem or 0.9% NaCl every 12 h after CLP for 5 days. Ten-day survival was 16%, 26%, and 52%, respectively, for untreated animals. Antibiotics decreased the absolute risk of death 17% to 23% regardless of injury severity. In a separate cohort, mice (n = 37) were subjected to single or double-puncture CLP with a 21-gauge needle. IL-6 levels were assayed 6 h postoperatively and mice were followed for survival. Levels greater than 14,000 pg/mL were identified as predicting a 100% mortality (7/7 animals dead). A third set of mice (n = 94) then underwent double-puncture CLP with either 21-, 23-, or 25-gauge needle and had IL-6 levels measured in a similar fashion. Animals were randomized to receive imipenem or 0.9% NaCl beginning 12 h postoperatively (6 h after IL-6 levels were drawn) and continued for 5 days or until death. Although antibiotics decreased mortality overall, all animals with IL-6 levels greater than 14,000 pg/mL (n = 13) died, regardless of whether they received antibiotics or the gauge of needle used. These results indicate that antibiotics improve outcome in murine sepsis, regardless of injury severity. Furthermore, there is a threshold IL-6 level that can be identified 6 h after sepsis above which animals are destined to die, and antibiotic treatment does not alter their outcome.

Gut epithelial apoptosis is increased in human studies and animal models of noninfectious inflammation and sepsis. Elevated intestinal cell death appears to be physiologically significant in sepsis. Previous studies demonstrate that overexpression of the antiapoptotic protein Bcl-2 in the gut epithelium of transgenic mice is associated with improved survival from Pseudomonas aeruginosa pneumonia and cecal ligation and puncture. The functional significance of elevated gut apoptosis in noninfectious inflammation has not been examined. We hypothesized that intestinal apoptosis would be detrimental to survival in noninfectious critical illness. To address this issue, acute lung injury (ALI) was induced with intratracheal injection of lipopolysaccharide (LPS, 800 microg) in wild-type (WT) FVB/N mice and transgenic mice that overexpress Bcl-2 in their intestinal epithelium. Guts were harvested at 12, 24, 48, and 72 h and assessed for apoptosis by both hematoxylin and eosin and active caspase-3 staining in 100 contiguous crypts. ALI increased gut epithelial apoptosis 12 h after LPS instillation compared with shams (P < 0.01), whereas overexpression of Bcl-2 decreased intestinal apoptosis compared with WT animals with ALI when assayed by active caspase-3 (P < 0.05). Plasma levels of tumor necrosis factor alpha, interleukin (IL)-6, and IL-10 were similar between WT and transgenic animals with ALI, both of which had elevated IL-10 levels at 12 h and elevated IL-6 levels at 24 h compared with sham animals. In a separate experiment, transgenic and WT animals with ALI were followed for mortality to determine whether gut overexpression of Bcl-2 conferred a survival advantage. Survival at 10 days was 73% in WT animals (n = 33) and 65% in Bcl-2 animals (n = 23, P = ns). These results indicate that while gut epithelial apoptosis is elevated in multiple models of critical illness, prevention of intestinal cell death by overexpression of Bcl-2 is associated with a disparate survival effect between sepsis and noninfectious inflammation.

Iron metabolism is dysregulated in critically ill patients. A mouse model of dysregulated iron metabolism was used to examine the consequence of iron loading upon sepsis. Mice deleted in the hfe gene (hfe-/-) abnormally accumulate iron in tissue; defects in the human hfe gene are clinically expressed as hemochromatosis. Hfe-/- mice and wild-type counterparts were randomized to receive either high- or low-iron diets for 2 weeks. After iron loading, mice were subjected to cecal ligation and puncture (CLP), a clinically relevant animal model of intra-abdominal sepsis. A preliminary (but underpowered) study suggested that iron-loaded hfe-/- mice had increased mortality as compared with hfe-/- mice fed a low-iron diet. There was no difference between wild-type and hfe-/- mice fed a low-iron diet or between wild-type mice fed hihg- and low-iron diets. A subsequent, appropriately powered study showed that iron-loaded hfe-/- mice had significantly higher mortality from intra-abdominal sepsis than hfe-/- mice fed a low-iron diet. Iron loading was confirmed through chemical assay of iron concentration in hepatic tissue. Animals with dysregulated iron handling, loaded with iron and subjected to CLP, had double the mortality of animals with normal iron levels. Critical care patients often have altered iron metabolism. In clinical practice, critically ill patients may receive iron through direct administration and the transfusion of blood products. Iron therapy may adversely affect the clinical outcome from sepsis.

Although coagulation abnormalities may partly underlie the physiologic derangements of the sepsis syndrome, anticoagulant therapies have produced mixed results on survival in clinical studies. We hypothesized that a meta-analysis of clinical trials of anticoagulants in sepsis may provide insight as to the therapeutic utility of targeting the clotting cascade in this syndrome. We searched electronic databases and reviewed bibliographies of pertinent articles to identify controlled clinical studies in which anticoagulants had been administered as adjunctive therapy to patients with sepsis. After establishing statistical homogeneity, odds ratios (OR; with 95% confidence intervals [CI]) for effect of these agents on mortality and bleeding complications were determined using Mantel-Haenszel methodology. Potential for publication bias was assessed by the use of a statistical test of funnel plot asymmetry. Weighted linear regression was performed to examine the effect of control group mortality rate on treatment efficacy. We identified 11 studies that satisfied our inclusion criteria. Collectively, these studies enrolled 4690 patients (range of 29-2314) and examined three agents: antithrombin III (2659 patients), tissue factor pathway inhibitor (210 patients), and activated protein C (1821 patients). After establishing statistical homogeneity (P > 0.10, chi-square), we found that the OR (with 95% CI) for effect on mortality for these agents, relative to control treatment, was 0.8692 (0.7519-1.006). Weighted linear regression analysis was consistent with a control group mortality dependent effect for these agents (P = 0.02). Only five of the studies reported bleeding complications. Pooling the results of these five studies (4376 patients) resulted in an OR (with 95% CI) of 1.70 (1.40-2.07) relative to control treatment for bleeding risk. Anticoagulants as adjuvant therapy do not appear to improve outcome in sepsis and are associated with a significant risk of bleeding complications. To the extent that their treatment effect is dependent upon disease severity, the safety and efficacy of these agents may be enhanced by refinement in techniques of clinical stratification.

To evaluate whether the up-regulation in sepsis-induced gut epithelial apoptosis is balanced by an increase in intestinal proliferation and to assess mechanisms affecting the gut's regenerative response to overwhelming infection.Prospective, randomized, controlled study.Animal laboratory in a university medical center.Mice were subjected to intratracheal injection of Pseudomonas aeruginosa and killed between 1.5 and 24 hrs after induction of pneumonia-induced sepsis to assess for gut epithelial proliferation and cell division and for apoptosis. Animals were compared with sham-operation controls, septic transgenic mice that overexpress Bcl-2 throughout their small intestinal epithelium, and septic p53-/- mice.Proliferation and cell division were assessed by measuring S-phase and M-phase cells in intestinal crypts. The number of S-phase cells showed a progressive decline at all time points measured, with a 5-fold decrease in proliferation between control animals and septic mice 24 hrs after intratracheal injection of pathogenic bacteria (p.0001). In contrast, cells in M-phase remained constant for the first 12 hrs after the onset of sepsis, but increased nearly 50% at 24 hrs after instillation of P. aeruginosa (p.005). Both the decrease in S-phase cells and the increase in M-phase cells were partially suppressible in Bcl-2 overexpressors, but cellular proliferation and division were similar between septic p53-/- and p53+/+ mice. Crypt apoptosis was increased at all time points, with maximal death occurring between 12 and 24 hrs.Sepsis from P. aeruginosa pneumonia induces a p53-independent decrease in gut epithelial proliferation. Despite an increase in sepsis-induced intestinal apoptosis, there is no compensatory increase in intestinal epithelial proliferation, and there is evidence of a cell cycle block with an accumulation of cells in M-phase. Decreasing gut apoptosis by overexpression of Bcl-2 is associated with a partial reversal of the effect of sepsis on the cell cycle.

The incidence and mortality of sepsis increase with age, consequently, 80% of the clinical mortality from sepsis occurs in patients over age 65. Despite this aged clinical population, most research models of sepsis use 6- to 16-week-old mice as patient surrogates. This age range of mice corresponds to human ages 10 to 17 years. To assess the influence of age on rodent CLP and on antibiotic therapy, we studied young (4 month), mature (12 month), and aged (24 month) mice. Male C57BL/6 mice (n = 27-30 in each age group) were subjected to cecal ligation and puncture (CLP), two punctures with a 25-gauge needle. Mice were observed untreated for 10 days. Young mice had 20% mortality, mature mice had 70% mortality (P = 0.0013 vs. young), and aged mice had 75% mortality (P = 0.0001 vs. young). To assess the effects of age on antibiotic therapy, mice were subjected to CLP as above (n = 38-40 in each age group). Mice were then randomized to treatment with intraperitoneal injections of ceftriaxone and metronidazole or normal saline. Therapy was initiated 12 h after CLP, and injections were repeated every 12 h for 7 days. Young mice saw a 56% decrease in mortality from CLP with antibiotic therapy (P = 0.001), and mature mice had a 30% decrease in mortality (P = 0.06). Aged mice saw no benefit from antibiotic therapy. We also compared plasma cytokine levels between young and aged mice after CLP. When compared with young mice, aged mice had higher levels of IL-6 and TNF-alpha 24 h after CLP. However, high IL-6 was predictive of mortality at any age. Mice appear to have age-dependent responses to intra-abdominal sepsis and to appropriate therapy.

Patients with sepsis have impaired host defenses that contribute to the lethality of the disorder. Recent work implicates lymphocyte apoptosis as a potential factor in the immunosuppression of sepsis. If lymphocyte apoptosis is an important mechanism, specific subsets of lymphocytes may be more vulnerable. A prospective study of lymphocyte cell typing and apoptosis was conducted in spleens from 27 patients with sepsis and 25 patients with trauma. Spleens from 16 critically ill nonseptic (3 prospective and 13 retrospective) patients were also evaluated. Immunohistochemical staining showed a caspase-9-mediated profound progressive loss of B and CD4 T helper cells in sepsis. Interestingly, sepsis did not decrease CD8 T or NK cells. Although there was no overall effect on lymphocytes from critically ill nonseptic patients (considered as a group), certain individual patients did exhibit significant loss of B and CD4 T cells. The loss of B and CD4 T cells in sepsis is especially significant because it occurs during life-threatening infection, a state in which massive lymphocyte clonal expansion should exist. Mitochondria-dependent lymphocyte apoptosis may contribute to the immunosuppression in sepsis by decreasing the number of immune effector cells. Similar loss of lymphocytes may be occurring in critically ill patients with other disorders.

Sepsis is a systemic response to severe infection. Clinical sepsis is defined as an infection-induced syndrome including at least two of the features of systemic inflammatory response syndrome: fever or hypothermia (oral temperature .38°C or ,36°C); leukocytosis (.12,000 WBC/mm) or leukopenia (,4,000 WBC/ mm); tachycardia (heart rate .90 beats/minute); and tachypnea (.24 breaths/minute). The causes of the sepsis syndrome are both complex and obscure. Despite advances in the diagnosis and treatment of infectious disease, the appearance of a systemic inflammatory response remains a common sequela of bacterial, viral, or fungal infection. The progression from sepsis to severe sepsis (sepsis with dysfunction of one organ), to multiple organ dysfunction syndrome (MODS), and then to septic death marks steps that typically require escalation of treatment. Although patients with severe sepsis have been treated experimentally with antiinflammatory and immunomodulatory agents, no single agent has reduced the overall mortality. Sepsis remains a major unsolved health problem. The incidence of sepsis has increased during the last 20 years, with more than 500,000 patients developing sepsis each year in the US. The mortality rate is between 35% and 45%. Half of these patients die from a condition that independently predisposes to sepsis (such as severe injury), but at least 100,000 deaths are caused by sepsis alone. Sepsis is the 13th leading cause of death in the US, with a cost of $5 to $10 billion a year. The morbidity and mortality of sepsis were historically ascribed to delayed diagnosis and inadequate antimicrobial therapy. Although diagnostic delays and inadequate therapy undoubtedly occur, many appropriately treated patients fail to recover. More recently, attention has turned to the host response as a determinant of outcomes. Such patient-specific responses, in turn, reflect both heritable (or genetic) characteristics and acquired illnesses. The purpose of this article is to discuss what is known concerning the heritable characteristics of the genetic predisposition to sepsis. Particular heritable characteristics may be causal or, alternatively, represent associations with human disease. The distinction is important. The surgeon occasionally confronts inborn “errors” in single genes that alter protein structure or expression in a way that profoundly affects physiology. For example, the single amino acid substitution that changes ordinary adult Hemoglobin A to Hemoglobin S accounts for the pathology of sickle cell anemia. The surgeon also encounters acquired abnormalities in specific genes and their proteins, particularly abnormalities in regulatory tumor suppressor genes and their proteins. For example, colon mucosa sustaining spontaneous mutagenesis acquires a series of variants in specific genes that collectively facilitate the transition from normal to malignant epithelium. In both of these examples, causality has been demonstrated by fulfillment of modern expressions of Koch’s postulates using genetic manipulation: correction of the molecular abnormality (genotype) corrects the physiology (phenotype). Sepsis is different. Human genetics has not, and likely will not, identify a single or even several genes whose structure or expression fully determines predisposition to or outcomes from sepsis; sepsis alters the expression of hundreds of genes in dozens of tissues. Experimental attempts to modify expression or effect of particular genes using biologic therapeutics, such as the soluble tumor necrosis factor receptor and the interleukin 1 receptor antagonist in order to modify outcomes from clinical sepsis, have failed. This article does not address causality of sepsis. Rather, it examines associations between particular genetic markers (DNA sequences) and sepsis.

Background. Nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS or NOS2) has been implicated in the hypotension, organ failure, and death that complicate sepsis. To avoid the confounding effects and limitations of iNOS inhibitors, we used iNOS gene knockout mice to examine the effect of inducible NO production in a model of polymicrobial abdominal sepsis treated with antibiotics. We hypothesized that iNOS gene deficiency would significantly alter outcome. Methods. C57BL6 wild-type (control) and congenic iNOS knockout mice were studied concurrently. Under halothane anesthesia, the ceca were ligated with 4-0 silk suture and punctured twice with a 26-gauge needle (cecal ligation and puncture, CLP). Survival was followed for 7 days, after which necropsies were performed in surviving animals. In an accompanying study examining the acute effects of sepsis, organ injury at 18 hours after CLP as determined by histology and the degree of cell death by apoptosis were examined with the use of hematoxylin and eosin (H&E) and TUNEL staining and two-channel fluorescence-activated cell sorter (FACS) analysis. Results. Sham laparotomy produced no lethality in either knockout (n = 3) or wild-type (n = 3) animals. Compared with survival in controls (n = 20), survival after CLP in iNOS knockout mice (n = 21) was significantly decreased (P

The purpose of this study was to determine whether apoptosis is a major mechanism of cell death in patients with sepsis. The activities of caspase-3 and the antiapoptotic protein, BCL-2, were investigated also.A prospective study of 20 patients who died of sepsis and multiple organ dysfunction was performed. The control group of 16 patients consisted of critically ill, nonseptic patients who were evaluated either prospectively (7) or retrospectively (9). In addition, normal colon sections from seven patients who had bowel resections were included. Apoptosis was evaluated in hematoxylin and eosin-stained specimens by deoxyuridine triphosphate nick end-labeling (TUNEL) and by DNA gel electrophoresis.Two academic medical centers.Critically ill patients.In septic patients, apoptosis was detected in diverse organs by all three methods with a predominance in lymphocytes and intestinal epithelial cells. Hematoxylin and eosin-stained specimens from septic patients demonstrated at least focal apoptosis in 56.3% of spleens, 47.1% of colons, and 27.7% of ileums. Indirect evidence of lymphocyte apoptosis in septic patients included extensive depletion of lymphocytes in white pulp and a marked lymphocytopenia in 15 of 19 patients. Hematoxylin and eosin from nonseptic patients' tissues revealed a low level of apoptosis in one patient only. The TUNEL method increased in positivity with a delay in tissue fixation and was highly positive in many tissues from both septic and nonseptic patients. Immunohistochemical staining for active caspase-3 showed a marked increase in septic vs. nonseptic patients (p.01), with25% to 50% of cells being positive focally in the splenic white pulp of six septic but in no nonseptic patients.We conclude that caspase-3-mediated apoptosis causes extensive lymphocyte apoptosis in sepsis and may contribute to the impaired immune response that characterizes the disorder.

Two challenges (intraperitoneal lipopolysaccharide (LPS) administration and cecal ligation and puncture (CLP)) and two strains of mice (LPS-normoresponder (C3H/HeN) and LPS-hyporesponder (C3H/HeJ)) were used to investigate pathways of cell injury. After intraperitoneal administration of LPS, endotoxin was absorbed into the bloodstream (HeN, 10.4 +/- 9.4 x 10(4) EU/mL; HeJ, 14.7 +/- 6.0 x 10(4) EU/mL), but as expected, only C3H/HeN mice produced serum tumor necrosis factor (TNF) (HeN, 2.5 +/- 2.0 x 10(3)pg/mL; HeJ, 87.0 +/- 38.7 pg/mL). Gel electrophoretic analysis of DNA extracted from six organs demonstrated the apoptotic "ladder" only in the thymus and only in the HeN mice. When the mice were challenged with CLP, both HeN and HeJ produced a small amount of serum TNF (HeN, 5.8 +/- 3.5 x 10(2) pg/mL; HeJ, 2.2 +/- 2.5 x 10(2) pg/mL) and both strains had very mild endotoxemia (HeN, 23.4 +/- 3.8 EU/mL; HeJ, 27.9 +/- 10.1 EU/mL). The DNA fragmentation pattern characteristic of apoptosis was observed not only in thymus but also in spleen, lung, and Peyer's patch of gut of both strains. This organ-specific pattern was more pronounced in the thymus of HeN mice; otherwise, the organ-specific patterns were similar for HeN and HeJ mice challenged by CLP but absent in those same organs when those same mice were challenged with LPS. The data suggest the existence not only of an endotoxin-driven activation for thymic apoptosis, but also of an endotoxin-independent, TNF-independent pathway activating widespread apoptosis in the murine CLP model of sepsis.

Objectives The Surviving Sepsis Campaign suggests giving hydrocortisone to septic patients only if their "blood pressure is poorly responsive to fluid resuscitation and vasopressor therapy." Because the definition of "poorly responsive" is not provided, the purpose of this study was to identify prescribing triggers for hydrocortisone in septic shock. Design Retrospective chart review of patients with septic shock over 17 months, who received hydrocortisone, followed by a survey of all intensivists who attended in the study ICUs to determine whether provider attitudes matched clinical practice. Setting Eight ICUs in an academic hospital and a hybrid academic/community hospital. Patients A total of 155 patients with septic shock in whom vasopressors were initiated and hydrocortisone was prescribed. Measurements and main results Ninety-nine patients (64%) were already receiving two vasopressors before hydrocortisone was prescribed. An additional 22 patients were on a single high-dose vasopressor prior to corticosteroid initiation. Of patients who survived to have their hydrocortisone dose changed, 57% had their corticosteroids tapered, whereas 43% were abruptly discontinued. Seventy-six percent of patients were no longer on vasopressors when the first dosing change was made. Twenty-seven out of 36 intensivists (75%) completed the survey. The majority (72%) defined "poorly responsive to vasopressors" as the presence of two vasopressors, and 70% stated that they required patients to be off vasopressors prior to altering the corticosteroid dose. Conclusions Significant variability exists when corticosteroids are prescribed for septic shock, with the most common interpretation in our institution of "poorly responsive to fluid resuscitation and vasopressor therapy" being the presence of two vasopressors. The method and timing of corticosteroid discontinuation also differed among providers. Self-described prescribing patterns from intensivists closely matched their actual behavior, suggesting variability is due to differing interpretations of the guidelines themselves, rather than a deficit in knowledge translation.

Several disease states (e.g., disseminated inflammation) demonstrate physiologic stability : they resolve only slowly and are resistant to both specific and symptomatic therapies. The existence of multiple stable physiologic states, including both health and disease states, is not anticipated by classical, linear descriptions of physiologic control mechanisms. Multiple stable states are, however, predicted by a simple nonlinear model in which the resistance to perturbation derives from interconnections among the model's elements. If the stability of selected disease states derives from nonlinear interactions among cells, tissues, and organs, then some therapies aimed at supporting or normalizing performance of specific organs may be misdirected.

Objective To determine whether human endotoxemia is associated with a loss of the physiologic beat-to-beat variability of heart rate. Design Prospective, randomized, crossover, single-blind study. Setting Clinical research center in a federal, nonuniversity hospital. Subjects Healthy volunteers. Interventions Intravenous administration of reference (Escherichia coli) endotoxin or saline placebo, with or without previous administration of oral ibuprofen. Measurements and main results Electrocardiograms were continuously recorded and digitized using series of 1000 beat epochs of R-R intervals over 8 hrs. Analyses included measures in the time domain (standard deviation), frequency domain (power spectra), and a measure of regularity (approximate entropy). Endotoxin administration was associated with loss of variability by all measures. This loss of variability remained significant even with administration of ibuprofen, which blocked the development of fever and endotoxin-related symptoms. Conclusions Infusion of endotoxin into human volunteers causes loss of heart rate variability, as measured by standard deviation and power spectra, as well as an increase in heart rate regularity, as measured by approximate entropy. Changes in approximate entropy occurred earlier than changes in other heart rate variability measures and may be a useful means of detecting early sepsis. This reduction in regularity is consistent with a model in which the pathogenesis of multiple organ system dysfunction syndrome involves the physiologic uncoupling of vital organ systems.

Introduction Advances in technology have made extensive monitoring of patient physiology the standard of care in intensive care units (ICUs). While many systems exist to compile these data, there has been no systematic multivariate analysis and categorization across patient physiological data. The sheer volume and complexity of these data make pattern recognition or identification of patient state difficult. Hierarchical cluster analysis allows visualization of high dimensional data and enables pattern recognition and identification of physiologic patient states. We hypothesized that processing of multivariate data using hierarchical clustering techniques would allow identification of otherwise hidden patient physiologic patterns that would be predictive of outcome. Methods Multivariate physiologic and ventilator data were collected continuously using a multimodal bioinformatics system in the surgical ICU at San Francisco General Hospital. These data were incorporated with non-continuous data and stored on a server in the ICU. A hierarchical clustering algorithm grouped each minute of data into 1 of 10 clusters. Clusters were correlated with outcome measures including incidence of infection, multiple organ failure (MOF), and mortality. Results We identified 10 clusters, which we defined as distinct patient states. While patients transitioned between states, they spent significant amounts of time in each. Clusters were enriched for our outcome measures: 2 of the 10 states were enriched for infection, 6 of 10 were enriched for MOF, and 3 of 10 were enriched for death. Further analysis of correlations between pairs of variables within each cluster reveals significant differences in physiology between clusters. Conclusions Here we show for the first time the feasibility of clustering physiological measurements to identify clinically relevant patient states after trauma. These results demonstrate that hierarchical clustering techniques can be useful for visualizing complex multivariate data and may provide new insights for the care of critically injured patients.

Objective: Whereas most septic patients have an underlying comorbidity, most animal models of sepsis use mice that were healthy before the onset of infection. Malignancy is the most common comorbidity associated with sepsis. The purpose of this study was to determine whether mice with cancer have a different response to sepsis than healthy animals. Design. Prospective, randomized controlled study. Setting: Animal laboratory in a university medical center. Subjects: C57BI/6 mice. Interventions: Animals received a subcutaneous injection of either 250,000 cells of the transplantable pancreatic adenocarcinoma cell line Pan02 (cancer) or phosphate-buffered saline (healthy). Three weeks later, mice given Pan02 cells had reproducible, nonmetastatic tumors. Both groups of mice then underwent intratracheal injection of either Pseudomonas aeruginosa (septic) or 0.9% NaCl (sham). Animals were killed 24 hrs postoperatively or followed-up 7 days for survival. Measurements and Main Results: Mice with cancer and healthy mice appeared similar when subjected to sham operation, although cancer animals had lower levels of T- and B-lymphocyte apoptosis. Septic mice with cancer had increased mortality compared to previously healthy septic mice subjected to the identical injury (52% vs. 28%; p = .04). This was associated with increased bacteremia but no difference in local pulmonary infection. Septic mice with cancer also had increased intestinal epithelial apoptosis. Although sepsis induced an increase in T- and B-lymphocyte apoptosis in all animals, septic mice with cancer had decreased T- and B-lymphocyte apoptosis compared to previously healthy septic mice. Serum and pulmonary cytokines, lung histology, complete blood counts, and intestinal proliferation were similar between septic mice with cancer and previously healthy septic mice. Conclusions. When subjected to the same septic insult, mice with cancer have increased mortality compared to previously healthy animals. Decreased systemic bacterial clearance and alterations in intestinal epithelial and lymphocyte apoptosis may help explain this differential response.

Critical illness trials involving genetic data collection are increasingly commonplace and pose challenges not encountered in less acute settings, related in part to the precipitous, severe and incapacitating nature of the diseases involved. We performed a systematic literature review to understand the nature of such studies conducted to date, and to consider, from an ethical perspective, potential barriers to future investigations. We identified 79 trials enrolling 24 499 subjects. Median (interquartile range) number of participants per study was 263 (116.75-430.75). Of these individuals, 16 269 (66.4%) were Caucasian, 1327 (5.4%) were African American, 1707 (7.0%) were Asian Pacific Islanders and 139 (0.6%) were Latino. For 5020 participants (20.5%), ethnicity was not reported. Forty-eight studies (60.8%) recruited subjects from single centers and all studies examined a relatively small number of genetic markers. Technological advances have rendered it feasible to conduct clinical studies using high-density genome-wide scanning. It will be necessary for future critical illness trials using these approaches to be of greater scope and complexity than those so far reported. Empirical research into issues related to greater ethnic inclusivity, accuracy of substituted judgment and specimen stewardship may be essential for enabling the conduct of such trials.

Aging is associated with increased inflammation following sepsis. The purpose of this study was to determine whether this represents a fundamental age-based difference in the host response or is secondary to the increased mortality seen in aged hosts.Prospective, randomized controlled study.Animal laboratory in a university medical center.Young (6-12 weeks) and aged (20-24 months) FVB/N mice.Mice were subjected to 2 x 25 or 1 x 30 cecal ligation and puncture (CLP).Survival was similar in young mice subjected to 2 x 25 CLP and aged mice subjected to 1 x 30 CLP (p = 0.15). Young mice subjected to 1 x 30 CLP had improved survival compared with the other groups (p0.05). When injury was held constant but mortality was greater, both systemic and peritoneal levels of tumor necrosis factor-alpha, interleukin (IL)-6, IL-10, and monocyte chemotactic protein-1 were elevated 24 hours after CLP in aged animals compared with young animals (p0.05). When mortality was similar but injury severity was different, there were no significant differences in systemic cytokines between aged mice and young mice. In contrast, peritoneal levels of tumor necrosis factor-alpha, IL-6, and IL-10 were higher in aged mice subjected to 1 x 30 CLP than young mice subjected to 2 x 25 CLP despite their similar mortalities (p0.05). There were no significant differences in either bacteremia or peritoneal cultures when animals of different ages sustained similar injuries or had different injuries with similar mortalities.Aged mice are more likely to die of sepsis than young mice when subjected to an equivalent insult, and this is associated with increases in both systemic and local inflammation. There is an exaggerated local but not systemic inflammatory response in aged mice compared with young mice when mortality is similar. This suggests that systemic processes that culminate in death may be age independent, but the local inflammatory response may be greater with aging.

Targeted IL-10 therapy improves survival in preclinical models of critical illness, and intestine-specific IL-10 decreases inflammation in models of chronic Inflammatory disease. We therefore sought to determine whether intestine-specific overexpression of IL-10 would improve survival in sepsis. Transgenic mice that overexpress IL-10 in their gut epithelium (Fabpi-IL-10 mice) and wild-type (WT) littermates (n = 127) were subjected to cecal ligation and puncture with a 27-gauge needle. The 7-day survival rate was 45% in transgenic animals and 30% in WT animals (P < or = 0.05). Systemic levels of IL-10 were undetectable in both groups of animals under basal conditions and were elevated to a similar degree in septic animals regardless of whether they expressed the transgene. Local parameter of injury, including gut epithelial apoptosis, intestinal permeability, peritoneal lavage cytokines, and stimulated cytokines from intraepithelial lymphocytes, were similar between transgenic and WT mice. However, in stimulated splenocytes, proinflammatory cytokines monocyte chemoattractant protein 1 (189 +/- 43 vs. 40 +/- 8 pg/mL) and IL-6 (116 +/- 28 vs. 34 +/- 9 pg/mL) were lower in Fabpi-IL-10 mice than WT littermates despite the intestine-specific nature of the transgene (P < 0.05). Cytokine levels were similar in blood and bronchoalveolar lavage fluid between the 2 groups, as were circulating LPS levels. Transgenic mice also had lower white blood cell counts associated with lower absolute neutrophil counts (0.5 +/- 0.1 vs. 1.0 +/- 0.2 10(3)/mm3; P < 0.05). These results indicate that gut-specific overexpression of IL-10 improves survival in a murine model of sepsis, and interactions between the intestinal epithelium and the systemic immune system may play a role in conferring this survival advantage.

Caspases mediate essential key proteolytic events in inflammatory cascades and the apoptotic cell death pathway. Human caspases functionally segregate into two distinct subfamilies: those involved in cytokine maturation (caspase-1, -4 and -5) and those involved in cellular apoptosis (caspase-2, -3, -6, -7, -8, -9 and -10)1,2. Although caspase-12 is phylogenetically related to the cytokine maturation caspases, in mice it has been proposed as a mediator of apoptosis induced by endoplasmic reticulum stress including amyloid-β cytotoxicity, suggesting that it might contribute to the pathogenesis of Alzheimer's disease3. Here we show that a single nucleotide polymorphism in caspase-12 in humans results in the synthesis of either a truncated protein (Csp12-S) or a full-length caspase proenzyme (Csp12-L). The read-through single nucleotide polymorphism encoding Csp12-L is confined to populations of African descent and confers hypo-responsiveness to lipopolysaccharide-stimulated cytokine production in ex vivo whole blood, but has no significant effect on apoptotic sensitivity. In a preliminary study, we find that the frequency of the Csp12-L allele is increased in African American individuals with severe sepsis. Thus, Csp12-L attenuates the inflammatory and innate immune response to endotoxins and in doing so may constitute a risk factor for developing sepsis.

Injury will equal or surpass communicable disease in the year 2020 as the number one cause of lost disability-adjusted life-years worldwide. The major cause of "late death" after trauma is organ dysfunction, commonly as a complication of shock or sepsis. The pathophysiology of injury-induced organ dysfunction is poorly characterized but has been linked to systemic inflammation as a result of infection (either obvious or occult) or massive tissue injury (systemic inflammatory response syndrome, SIRS). Subsequent complications of organ dysfunction, including death, may also stem from immunosuppression characteristic of what has been called the counter-regulatory anti-inflammatory response syndrome (CARS). At the cellular level, injurious stimuli trigger adaptive stress responses that include changes in gene expression. Multiple organ dysfunction syndrome (MODS) is the summation of these stress responses to severe systemic injury, integrated at the cellular, organ, and host levels. We hypothesize that a complete understanding at the molecular level of the stress responses induced by injury will aid in the development of therapeutic strategies for treating MODS in the critically ill surgical patient. This paper reviews recent data from our Cellular Injury and Adaptation Laboratory relevant to our understanding of MODS pathophysiology, particularly as it relates to stress-induced cell death by apoptosis. Our data suggest that inhibition of stress-induced apoptosis may improve survival after severe injury.

DNA microarrays allow genome-wide assessment of changes in relative messenger RNA abundance and thus can be used to monitor changes in gene expression. The aim of this series of experiments was to gain experience in sepsis gene expression profiling in a well-accepted model of murine polymicrobial abdominal sepsis and begin characterizing (in the parlance of genomics) the sepsis "transcriptome."Prospective animal study.University-based animal research facility.SUBJECTS C57BL/6 mice.After induction of general anesthesia, cecal ligation and puncture were performed to induce peritonitis and polymicrobial sepsis. The control group had sham laparotomy only. Three samples of spleen and liver were collected from septic and sham animals at 24 hrs after laparotomy. Changes in expression were measured for 588 annotated mouse genes by using a commercially available complementary DNA microarray kit.Broad-scale gene expression profiles were characterized for septic liver and spleen and compared with sham controls. The analytical tools used included commercially available software packages and a novel analysis program. Very little overlap was observed in the septic gene expression profiles of these two organs. Most of the genes identified have previously been linked to regulation of the inflammatory response; importantly, however, some have not. In addition, hierarchical cluster analysis showed that cecal ligation and puncture at 24 hrs induced coordinate expression of genes that alter cell signaling and survival pathways in spleen, consistent with previously published reports of sepsis-induced splenocyte apoptosis. The current limitations of microarray analysis as reflected in these studies are also discussed.Microarray technology provides a powerful new tool for rapidly analyzing tissue-specific changes in gene expression induced by sepsis in animal models. To our knowledge, these data constitute the first report on the use of microarrays to determine the sepsis transcriptome.

Sepsis continues to be a common source of morbidity and mortality in critically ill patients. Single nucleotide polymorphisms (SNPs) present in genes encoding inflammatory mediators have been associated with predisposition and outcome in this syndrome. The use of high throughput SNP analysis in large epidemiological studies is necessary to more fully understand the genetic underpinnings of this disease. We adapted template-directed dye-terminator incorporation with fluorescence polarization detection (TDI-FP) to the analysis of eight SNPs implicated in mediating the sepsis syndrome: TNF-alpha (-308), TNF-alpha (-238), TNF-beta (+250), IL-1beta (+3953), IL-6 (-174), IL-10 (-592), plasminogen activator inhibitor-1 (PAI-1 (-675)), and TLR4 299 (+1032). Optimization of PCR, amplicon purification, and template-directed dye-terminator incorporation reactions were necessary to achieve acceptable performance characteristics for these assays. Sequence validated samples served as controls. Using this method we were able to assign genotype in 99.3% of assays and identified 64 unique genotypes in samples obtained from 90 individuals. TDI-FP is a flexible and robust method of SNP detection that can be optimized in a systematic fashion. This method has potential advantages compared with other high throughput genotyping techniques and appears well suited to clinical situations requiring analysis of large numbers of samples.