Derek Barclay

Multiplex Analysis of Urinary Cytokine Levels in Rat Model of Cyclophosphamide-induced Cystitis

OBJECTIVES The urinary proteome is a potential easily accessible source of biomarkers for inflammatory bladder diseases, including interstitial cystitis. In the present study, we subjected rat urine to multiplex cytokine analysis in an attempt to identify an inflammatory signature of the temporal course of cyclophosphamide (CYP)-induced cystitis. METHODS Rat urine was collected for 12 hours after CYP injection (150 mg/kg) for multiplex analysis of 14 cytokines by a multiple antigen bead assay (Luminex 100 IS). Urine from each void was collected, and the voiding frequency was determined. The bladder tissue was analyzed for cytokines levels and histologic evidence of inflammation. RESULTS Significant changes were noted in the urine levels of all cytokines with respect to baseline at 2, 4, 6, and 10 hours after CYP injection. Elevation was noted at all times for most cytokines, except for monocyte chemotactic protein-1, which had a 5-fold decrease at 2 hours. The urine and tissue levels of interleukin (IL)-1beta, IL-4, and growth-related oncogene/keratinocyte-derived chemokine correlated significantly, with a positive Spearman correlation also noted for granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1-1, IL-18, and interferon-gamma. The tissue levels for most cytokines, except for IL-2, and urinary frequency were significantly elevated in the CYP-treated rats compared with the control vehicle-treated rats. The hints of severe inflammation in the bladder indicated by the urinary cytokines were confirmed by bladder histologic examination and the tissue cytokine levels at necropsy. CONCLUSIONS The progression of CYP-induced cystitis was clearly reflected in the urine matrix by the temporal and quantitative changes in the cytokine levels. Additional delineation of urine and bladder tissue cytokine expression might yield biomarkers for cystitis.

A Two-Compartment Mathematical Model of Endotoxin-induced Inflammatory and Physiologic Alterations in Swine

Objective:To gain insights into individual variations in acute inflammation and physiology. Design:Large-animal study combined with mathematical modeling. Setting:Academic large-animal and computational laboratories. Subjects:Outbred juvenile swine. Interventions:Four swine were instrumented and subjected to endotoxemia (100 µg/kg), followed by serial plasma sampling. Measurements and Main Results:Swine exhibited various degrees of inflammation and acute lung injury, including one death with severe acute lung injury (PaO2/FIO2 ratio &mgr;200 and static compliance &mgr;10 L/cm H2O). Plasma interleukin-1&bgr;, interleukin-4, interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor-&agr;, high mobility group box-1, and NO2-/NO3- were significantly (p &mgr; .05) elevated over the course of the experiment. Principal component analysis was used to suggest principal drivers of inflammation. Based in part on principal component analysis, an ordinary differential equation model was constructed, consisting of the lung and the blood (as a surrogate for the rest of the body), in which endotoxin induces tumor necrosis factor-&agr; in monocytes in the blood, followed by the trafficking of these cells into the lung leading to the release of high mobility group box-1, which in turn stimulates the release of interleukin-1&bgr; from resident macrophages. The ordinary differential equation model also included blood pressure, PaO2, and FIO2, and a damage variable that summarizes the health of the animal. This ordinary differential equation model could be fit to both inflammatory and physiologic data in the individual swine. The predicted time course of damage could be matched to the oxygen index in three of the four swine. Conclusions:The approach described herein may aid in predicting inflammation and physiologic dysfunction in small cohorts of subjects with diverse phenotypes and outcomes.

Analysis of Serum Inflammatory Mediators Identifies Unique Dynamic Networks Associated with Death and Spontaneous Survival in Pediatric Acute Liver Failure

Background Tools to predict death or spontaneous survival are necessary to inform liver transplantation (LTx) decisions in pediatric acute liver failure (PALF), but such tools are not available. Recent data suggest that immune/inflammatory dysregulation occurs in the setting of acute liver failure. We hypothesized that specific, dynamic, and measurable patterns of immune/inflammatory dysregulation will correlate with outcomes in PALF. Methods We assayed 26 inflammatory mediators on stored serum samples obtained from a convenience sample of 49 children in the PALF study group (PALFSG) collected within 7 days after enrollment. Outcomes were assessed within 21 days of enrollment consisting of spontaneous survivors, non-survivors, and LTx recipients. Data were subjected to statistical analysis, patient-specific Principal Component Analysis (PCA), and Dynamic Bayesian Network (DBN) inference. Findings Raw inflammatory mediator levels assessed over time did not distinguish among PALF outcomes. However, DBN analysis did reveal distinct interferon-gamma-related networks that distinguished spontaneous survivors from those who died. The network identified in LTx patients pre-transplant was more like that seen in spontaneous survivors than in those who died, a finding supported by PCA. Interpretation The application of DBN analysis of inflammatory mediators in this small patient sample appears to differentiate survivors from non-survivors in PALF. Patterns associated with LTx pre-transplant were more like those seen in spontaneous survivors than in those who died. DBN-based analyses might lead to a better prediction of outcome in PALF, and could also have more general utility in other complex diseases with an inflammatory etiology.

An adequately robust early TNF-α response is a hallmark of survival following trauma/hemorrhage

Background Trauma/hemorrhagic shock (T/HS) results in cytokine-mediated acute inflammation that is generally considered detrimental. Methodology/Principal Findings Paradoxically, plasma levels of the early inflammatory cytokine TNF-α (but not IL-6, IL-10, or NO2 -/NO3 -) were significantly elevated within 6 h post-admission in 19 human trauma survivors vs. 4 non-survivors. Moreover, plasma TNF-α was inversely correlated with Marshall Score, an index of organ dysfunction, both in the 23 patients taken together and in the survivor cohort. Accordingly, we hypothesized that if an early, robust pro-inflammatory response were to be a marker of an appropriate response to injury, then individuals exhibiting such a response would be predisposed to survive. We tested this hypothesis in swine subjected to various experimental paradigms of T/HS. Twenty-three anesthetized pigs were subjected to T/HS (12 HS-only and 11 HS + Thoracotomy; mean arterial pressure of 30 mmHg for 45–90 min) along with surgery-only controls. Plasma obtained at pre-surgery, baseline post-surgery, beginning of HS, and every 15 min thereafter until 75 min (in the HS only group) or 90 min (in the HS + Thoracotomy group) was assayed for TNF-α, IL-6, IL-10, and NO2 -/NO3 -. Mean post-surgery±HS TNF-α levels were significantly higher in the survivors vs. non-survivors, while non-survivors exhibited no measurable change in TNF-α levels over the same interval. Conclusions/Significance Contrary to the current dogma, survival in the setting of severe, acute T/HS appears to be associated with an immediate increase in serum TNF-α. It is currently unclear if this response was the cause of this protection, a marker of survival, or both. This abstract won a Young Investigator Travel Award at the SHOCK 2008 meeting in Cologne, Germany.

Temporal Patterns of Circulating Inflammation Biomarker Networks Differentiate Susceptibility to Nosocomial Infection Following Blunt Trauma in Humans.

BACKGROUND Severe traumatic injury can lead to immune dysfunction that renders trauma patients susceptible to nosocomial infections (NI) and prolonged intensive care unit (ICU) stays. We hypothesized that early circulating biomarker patterns following trauma would correlate with sustained immune dysregulation associated with NI and remote organ failure. METHODS In a cohort of 472 blunt trauma survivors studied over an 8-year period, 127 patients (27%) were diagnosed with NI versus 345 trauma patients without NI. To perform a pairwise, case-control study with 1:1 matching, 44 of the NI patients were compared with 44 no-NI trauma patients selected by matching patient demographics and injury characteristics. Plasma obtained upon admission and over time were assayed for 26 inflammatory mediators and analyzed for the presence of dynamic networks. RESULTS Significant differences in ICU length of stay (LOS), hospital LOS, and days on mechanical ventilation were observed in the NI patients versus no-NI patients. Although NI was not detected until day 7, multiple mediators were significantly elevated within the first 24 hours in patients who developed NI. Circulating inflammation biomarkers exhibited 4 distinct dynamic patterns, of which 2 clearly distinguish patients destined to develop NI from those who did not. Mediator network connectivity analysis revealed a higher, coordinated degree of activation of both innate and lymphoid pathways in the NI patients over the initial 24 hours. CONCLUSIONS These studies implicate unique dynamic immune responses, reflected in circulating biomarkers that differentiate patients prone to persistent critical illness and infections following injury, independent of mechanism of injury, injury severity, age, or sex.

Central Role for MCP-1/CCL2 in Injury-Induced Inflammation Revealed by In Vitro , In Silico , and Clinical Studies

The translation of in vitro findings to clinical outcomes is often elusive. Trauma/hemorrhagic shock (T/HS) results in hepatic hypoxia that drives inflammation. We hypothesize that in silico methods would help bridge in vitro hepatocyte data and clinical T/HS, in which the liver is a primary site of inflammation. Primary mouse hepatocytes were cultured under hypoxia (1% O2) or normoxia (21% O2) for 1–72 h, and both the cell supernatants and protein lysates were assayed for 18 inflammatory mediators by Luminex™ technology. Statistical analysis and data-driven modeling were employed to characterize the main components of the cellular response. Statistical analyses, hierarchical and k-means clustering, Principal Component Analysis, and Dynamic Network Analysis suggested MCP-1/CCL2 and IL-1α as central coordinators of hepatocyte-mediated inflammation in C57BL/6 mouse hepatocytes. Hepatocytes from MCP-1-null mice had altered dynamic inflammatory networks. Circulating MCP-1 levels segregated human T/HS survivors from non-survivors. Furthermore, T/HS survivors with elevated early levels of plasma MCP-1 post-injury had longer total lengths of stay, longer intensive care unit lengths of stay, and prolonged requirement for mechanical ventilation vs. those with low plasma MCP-1. This study identifies MCP-1 as a main driver of the response of hepatocytes in vitro and as a biomarker for clinical outcomes in T/HS, and suggests an experimental and computational framework for discovery of novel clinical biomarkers in inflammatory diseases.

Mathematical modeling of posthemorrhage inflammation in mice: studies using a novel, computer-controlled, closed-loop hemorrhage apparatus.

Hemorrhagic shock (HS) elicits a global acute inflammatory response, organ dysfunction, and death. We have used mathematical modeling of inflammation and tissue damage/dysfunction to gain insight into this complex response in mice. We sought to increase the fidelity of our mathematical model and to establish a platform for testing predictions of this model. Accordingly, we constructed a computerized, closed-loop system for mouse HS. The intensity, duration, and time to achieve target MAP could all be controlled using a software. Fifty-four male C57/black mice either were untreated or underwent surgical cannulation. The cannulated mice were divided into 8 groups: (a) 1, 2, 3, or 4 h of surgical cannulation alone and b) 1, 2, 3, or 4 h of cannulation + HS (25 mmHg). MAP was sustained by the computer-controlled reinfusion and withdrawal of shed blood within ±2 mmHg. Plasma was assayed for the cytokines TNF, IL-6, and IL-10 as well as the NO reaction products NO2−/NO3−. The cytokine and NO2−/NO3− data were compared with predictions from a mathematical model of post-hemorrhage inflammation, which was calibrated on different data. To varying degrees, the levels of TNF, IL-6, IL-10, and NO2−/NO3− predicted by the mathematical model matched these data closely. In conclusion, we have established a hardware/software platform that allows for highly accurate, reproducible, and mathematically predictable HS in mice.ABBREVIATIONS-HS-hemorrhagic shock; HR-heart rate

Hemoadsorption reprograms inflammation in experimental gram-negative septic peritonitis: insights from in vivo and in silico studies.

Improper compartmentalization of the inflammatory response leads to systemic inflammation in sepsis. Hemoadsorption (HA) is an emerging approach to modulate sepsis-induced inflammation. We sought to define the effects of HA on inflammatory compartmentalization in Escherichia coli-induced fibrin peritonitis in rats. Hypothesis: HA both reprograms and recompartmentalizes inflammation in sepsis. Sprague Dawley male rats were subjected to E. coli peritonitis and, after 24 h, were randomized to HA or sham treatment (sepsis alone). Venous blood samples collected at 0, 1, 3 and 6 h (that is, 24–30 h of total experimental sepsis), and peritoneal samples collected at 0 and 6 h, were assayed for 14 cytokines along with NO2−/NO3−. Bacterial counts were assessed in the peritoneal fluid at 0 and 6 h. Plasma tumor necrosis factor (TNF)-α, interleukin (IL)-6, CXCL-1, and CCL2 were significantly reduced in HA versus sham. Principal component analysis (PCA) suggested that inflammation in sham was driven by IL-6 and TNF-α, whereas HA-associated inflammation was driven primarily by TNF-α, CXCL-1, IL-10 and CCL2. Whereas peritoneal bacterial counts, plasma aspartate transaminase levels and peritoneal IL-5, IL-6, IL-18, interferon (IFN)-γ and NO2−/NO3− were significantly lower, both CXCL-1 and CCL2 as well as the peritoneal-to-plasma ratios of TNF-α, CXCL-1 and CCL2 were significantly higher in HA versus sham, suggesting that HA-induced inflammatory recompartmentalization leads to the different inflammatory drivers discerned in part by PCA. In conclusion, this study demonstrates the utility of combined in vivo/in silico methods and suggests that HA exerts differential effects on mediator gradients between local and systemic compartments that ultimately benefit the host.

Trauma in silico: Individual-specific mathematical models and virtual clinical populations

A mathematical model of the human response to trauma replicates individual patient outcomes but predicts unexpected results in populations. A virtual large sample size Severe trauma or bleeding evokes an all-hands-on-deck immune response. When properly orchestrated, the myriad cytokines and peptides help to heal the patient. However, this process can easily go awry, and administering the right drug to compensate is a challenge. Brown and colleagues mathematically modeled the complicated immune responses from the data of 33 blunt trauma patients and then generated a larger cohort of 10,000 virtual trauma patients. This large virtual cohort predicted the reactions of smaller validation cohorts, but the surprise was that understanding the response details in a single patient did not predict how the population would act. The author’s virtual clinical trial indicated that inhibition of interleukin-6 (IL-6) produced a small survival benefit, whereas IL-1β inhibition did not help much and tumor necrosis factor–α made things worse. Trauma-induced critical illness is driven by acute inflammation, and elevated systemic interleukin-6 (IL-6) after trauma is a biomarker of adverse outcomes. We constructed a multicompartment, ordinary differential equation model that represents a virtual trauma patient. Individual-specific variants of this model reproduced both systemic inflammation and outcomes of 33 blunt trauma survivors, from which a cohort of 10,000 virtual trauma patients was generated. Model-predicted length of stay in the intensive care unit, degree of multiple organ dysfunction, and IL-6 area under the curve as a function of injury severity were in concordance with the results from a validation cohort of 147 blunt trauma patients. In a subcohort of 98 trauma patients, those with high–IL-6 single-nucleotide polymorphisms (SNPs) exhibited higher plasma IL-6 levels than those with low IL-6 SNPs, matching model predictions. Although IL-6 could drive mortality in individual virtual patients, simulated outcomes in the overall cohort were independent of the propensity to produce IL-6, a prediction verified in the 98-patient subcohort. In silico randomized clinical trials suggested a small survival benefit of IL-6 inhibition, little benefit of IL-1β inhibition, and worse survival after tumor necrosis factor–α inhibition. This study demonstrates the limitations of extrapolating from reductionist mechanisms to outcomes in individuals and populations and demonstrates the use of mechanistic simulation in complex diseases.

Gender-based reciprocal expression of transforming growth factor-β1 and the inducible nitric oxide synthase in a rat model of cyclophosphamide-induced cystitis

BackgroundThe pluripotent cytokine transforming growth factor-β1 (TGF-β1) is the central regulator of inducible Nitric Oxide Synthase (iNOS) that is responsible for nitric oxide (NO) production in inflammatory settings. Previous studies have implicated a role for NO, presumably derived from iNOS, in cyclophosphamide (CYP)-induced cystitis in the bladder. TGF-β1 is produced in latent form and requires dissociation from the latency-associated peptide (LAP) to act as primary anti-inflammatory and pro-healing modulator following tissue injury in the upper urinary tract. Since the role of TGF-β1 in lower urinary tract inflammation is currently unknown, and since gender-based differences exist in the setting of interstitial cystitis (IC), the present study examined the relationship between TGF-β1 and iNOS/NO in the pathogenesis of CYP-induced cystitis in both male and female rats.MethodsSprague-Dawley rats, 4 months of age, of either gender were given 150 mg/kg CYP intraperitoneally. Urinary and bladder tissue TGF-β1 and NO reaction products (NO2-/NO3-) were quantified as a function of time following CYP. Expression of active and latent TGF-β1 as well as iNOS in harvested bladder tissue was assessed by immunohistochemistry.ResultsFemale rats had significantly higher levels of NO2-/NO3- in urine even at baseline as compared to male rats (p < 0.001), whereas there was no gender based significant difference in urine levels of active or latent TGF-β1 prior to CYP injection. Inflammatory and cytotoxic changes were induced by CYP in the bladder of both sexes that were accompanied by differences in the urine levels of NO2-/NO3- and TGF-β1. Male rats responded to CYP with significantly lower levels of NO2-/NO3- and significantly higher levels of TGF-β1 in urine (p < 0.05) as compared to females at all time points after CYP. The urine levels of NO2-/NO3- after CYP were inversely correlated to latent and active TGF-β1 (Pearson coefficient of -0.72 and -0.69 in females and -0.89 and -0.76 in males, respectively; p < 0.01). Bladder tissue of male rats exhibited significantly higher levels of both latent and active TGF-β1 (p < 0.01) compared to female rats after CYP. TGF-β1 and iNOS protein was mostly localized in the urothelium.ConclusionThe results of this study suggest that there exists an inverse relationship between the expression of TGF-β1 and iNOS/NO2-/NO3- in CYP-inflamed bladder. The gender of the animal appears to magnify the differences in urine levels of TGF-β1 and NO2-/NO3- in this inflammatory setting. These results support the hypothesis that TGF-β1 can suppress iNOS expression associated with bladder inflammation and reduce systemic levels of NO2-/NO3-, and further suggest that this feature of TGF-β1 can be harnessed for therapy and diagnosis of interstitial cystitis.