Steve E. Calvano

Genomic responses in mouse models poorly mimic human inflammatory diseases

A cornerstone of modern biomedical research is the use of mouse models to explore basic pathophysiological mechanisms, evaluate new therapeutic approaches, and make go or no-go decisions to carry new drug candidates forward into clinical trials. Systematic studies evaluating how well murine models mimic human inflammatory diseases are nonexistent. Here, we show that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another. Among genes changed significantly in humans, the murine orthologs are close to random in matching their human counterparts (e.g., R2 between 0.0 and 0.1). In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases.

A network-based analysis of systemic inflammation in humans

Oligonucleotide and complementary DNA microarrays are being used to subclassify histologically similar tumours, monitor disease progress, and individualize treatment regimens. However, extracting new biological insight from high-throughput genomic studies of human diseases is a challenge, limited by difficulties in recognizing and evaluating relevant biological processes from huge quantities of experimental data. Here we present a structured network knowledge-base approach to analyse genome-wide transcriptional responses in the context of known functional interrelationships among proteins, small molecules and phenotypes. This approach was used to analyse changes in blood leukocyte gene expression patterns in human subjects receiving an inflammatory stimulus (bacterial endotoxin). We explore the known genome-wide interaction network to identify significant functional modules perturbed in response to this stimulus. Our analysis reveals that the human blood leukocyte response to acute systemic inflammation includes the transient dysregulation of leukocyte bioenergetics and modulation of translational machinery. These findings provide insight into the regulation of global leukocyte activities as they relate to innate immune system tolerance and increased susceptibility to infection in humans.

Human Toll-Like Receptor 4 Mutations but Not CD14 Polymorphisms Are Associated with an Increased Risk of Gram-Negative Infections

Human toll-like receptor 4 (hTLR4) and CD14 are known to be components of the lipopolysaccharide receptor complex. Our study investigated the association between TLR4 mutations (Asp299Gly and Thr399Ile) and CD14 polymorphism(s) with outcome in an intensive care unit (ICU) population at risk for sepsis. By use of a polymerase chain reaction-based restriction fragment-length polymorphism analysis technique, the hTLR4 gene was altered in 14 (18%) of 77 ICU patients (all positive for systemic inflammatory response syndrome) and in 5 (13%) of 39 volunteers. There was a significantly higher incidence of gram-negative infection among patients with the mutations (11 [79%] of 14), compared with that in the wild-type population (11 [17%] of 63; P=.004). No association between CD14 polymorphism(s) and the incidence of infection or outcome was observed. These findings indicate that hTLR4 mutations are associated with an increased incidence of gram-negative infections in critically ill patients in a surgical setting.

Acute Inflammatory Response to Endotoxin in Mice and Humans

ABSTRACT Endotoxin injection has been widely used to study the acute inflammatory response. In this study, we directly compared the inflammatory responses to endotoxin in mice and humans. Escherichia coli type O113 endotoxin was prepared under identical conditions, verified to be of equal biological potency, and used for both mice and humans. The dose of endotoxin needed to induce an interleukin-6 (IL-6) concentration in plasma of ∼1,000 pg/ml 2 h after injection was 2 ng/kg of body weight in humans and 500 ng/kg in mice. Healthy adult volunteers were injected intravenously with endotoxin, and male C57BL/6 mice (n = 4 to 12) were injected intraperitoneally with endotoxin. Physiological, hematological, and cytokine responses were determined. Endotoxin induced a rapid physiological response in humans (fever, tachycardia, and slight hypotension) but not in mice. Both mice and humans exhibited lymphopenia with a nadir at 4 h and recovery by 24 h. The levels of tumor necrosis factor (TNF) and IL-6 in plasma peaked at 2 h and returned to baseline levels by 4 to 6 h. IL-1 receptor antagonist RA and TNF soluble receptor I were upregulated in both mice and humans but were upregulated more strongly in humans. Mice produced greater levels of CXC chemokines, and both mice and humans exhibited peak production at 2 h. These studies demonstrate that although differences exist and a higher endotoxin challenge is necessary in mice, there are several similarities in the inflammatory response to endotoxin in mice and humans.

Quantitative proteome analysis of human plasma following in vivo lypopolysaccharide administration using 16O/18O labeling and the accurate mass and time tag approach

Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes postdigestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional LC-FTICR mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC elution time AMT tag data base was initially generated using MS/MS following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag data base contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion of high abundance proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses, and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration.

High Dynamic Range Characterization of the Trauma Patient Plasma Proteome

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this “divide-and-conquer” strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3654 different proteins with 1494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 “classic” cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.

Peripheral blood leukocyte kinetics following in vivo lipopolysaccharide (LPS) administration to normal human subjects. Influence of elicited hormones and cytokines.

Lipopolysaccharide (LPS, endotoxin) administration to human subjects elicits significant elevations in plasma cachectin/TNF, epinephrine, and cortisol. This study examined the temporal relationship between changes in blood leukocyte subsets and plasma mediators following endotoxin administration to normal human subjects. A five-minute intravenous infusion of purified LPS (20 units/kg Escherichia coli) was administered to 12 healthy volunteers. Blood samples were obtained at varying intervals after infusion and analyzed for differential cell counts and lymphocyte subsets (CD2, CD3, CD4, CD8, CD20, and HLA-DR) by flow microfluorimetry, and also assayed for plasma cachectin/TNF, epinephrine, and cortisol. Plasma cachectin/TNF was significantly elevated at 75 and 90 minutes after infusion with a peak concentration of 261 +/- 115 pg/ml noted 75 minutes after infusion. A significant plasma epinephrine elevation of 181 +/- 75 pg/ml was demonstrated one hour after infusion, while significant elevations in plasma cortisol were noted from one to five hours after infusion with a peak level of 34 +/- 3 micrograms/dl three hours after infusion. A profound monocytopenia (p less than 0.01) was noted one hour after infusion. Temporally associated with the rise in plasma cortisol was a reversal of the early granulocytopenia to a significant granulocytosis (p less than 0.01 versus preinfusion mean), whereas a marked lymphocytopenia (p less than 0.01) was observed from one to six hours after infusion. During the period of hypercortisolemia, CD2, CD3, and CD4 lymphocyte percentages were decreased (p less than 0.01) while CD20 and HLA-DR lymphocyte percentages were increased (p less than 0.01). There was a small percentage decrease in CD8 lymphocytes from one to 24 hours after infusion (p less than 0.01), although relative to the one-hour nadir, there was a significant rise in the percentage during the time of elevated plasma cortisol concentrations. A six-hour infusion of epinephrine (30 ng/kg/min) administered to six healthy volunteers resulted in a monocytosis (p less than 0.05) and granulocytosis (p less than 0.01) without a change in lymphocyte number or lymphocyte subset percentage. Previous reports have shown that in vivo corticosteroid infusion causes a prominent granulocytosis, monocytopenia, and lymphocytopenia with a decrease in the percentages of CD3 and CD4 lymphocytes. The peripheral blood leukocyte dynamics documented in the current study are similar to patterns observed following in vivo corticosteroid administration. This study suggests that the acute adrenocortical response to endotoxemia primarily mediates the subsequent changes in leukocyte subsets.

Increased neutrophil mobilization and decreased chemotaxis during cortisol and epinephrine infusions.

Although hormones are putative mediators of neutrophil changes after injury, the effects of trauma-induced levels of plasma cortisol and epinephrine on circulating neutrophils have not been reported in humans. The dynamics of PMN mobilization and chemotaxis were evaluated during 19 infusions of epinephrine or cortisol or a combined infusion of both hormones in ten normal volunteers. Basal levels of epinephrine and cortisol increased during infusions to levels consistent with those reported following severe injury. Circulating neutrophil counts increased in parallel with plasma cortisol levels. Epinephrine mobilized the entire marginated pool of neutrophils, and the neutrophil half-life was extended from a normal of 6.6 hours to 10.4 hours by cortisol. Chemotaxis after six hours of epinephrine infusion was reduced compared with baseline chemotaxis. In four volunteers who had a second infusion of cortisol, chemotaxis was significantly depressed ten days after the infusion compared with baseline. From these data we conclude that stress levels of epinephrine mobilize the marginated pool of granulocytes into the circulating pool in a linear fashion, and cortisol raises the half-life of circulating neutrophils. Reduced neutrophil chemotaxis seen as a consequence of these infusions could account for some of the increased susceptibility to infection that occurs after major trauma.

Modeling the influence of circadian rhythms on the acute inflammatory response.

A wide variety of modeling techniques have been applied towards understanding inflammation. These models have broad potential applications, from optimizing clinical trials to improving clinical care. Models have been developed to study specific systems and diseases, but the effect of circadian rhythms on the inflammatory response has not been modeled. Circadian rhythms are normal biological variations obeying the 24-h light/dark cycle and have been shown to play a critical role in the treatment and progression of many diseases. Several of the key components of the inflammatory response, including cytokines and hormones, have been observed to undergo significant diurnal variations in plasma concentration. It is hypothesized that these diurnal rhythms are entrained by the cyclic production of the hormones cortisol and melatonin, as stimulated by the central clock in the suprachiasmatic nucleus. Based on this hypothesis, a mathematical model of the interplay between inflammation and circadian rhythms is developed. The model is validated by its ability to reproduce diverse sets of experimental data and clinical observations concerning the temporal sensitivity of the inflammatory response.

In vivo endotoxin synchronizes and suppresses clock gene expression in human peripheral blood leukocytes

Objectives:The intravenous administration of a bolus dose of endotoxin to healthy human subjects triggers acute systemic inflammatory responses that include cytokine production and dynamic changes in gene expression in peripheral blood leukocytes. This study sought to determine the state of clock gene expression in human peripheral blood leukocytes, and leukocyte subpopulations, challenged with in vivo endotoxin at two circadian/diurnal phases of the clock. Design:Clinical and laboratory investigation. Setting:University-based research laboratory and clinical research center. Subjects:Human volunteers. Interventions:Human subjects were administered a standard dose of endotoxin (2 ng/kg) or saline at either 0900 or 2100 hrs. Blood samples were collected at selected time points pre- and postinfusion. Measurements and Main Results:Clock gene expression was determined in human peripheral blood leukocytes, neutrophils, and monocytes by quantitative real-time polymerase chain reaction. The fold change for each gene was determined by use of the 2−&Dgr;&Dgr;CT method. We show that endotoxin causes profound suppression of circadian clock gene expression, clearly manifested in human peripheral blood leukocytes, neutrophils, and monocytes. Clock, Cry1-2, Per3, CSNK1 ϵ, Rora, and Rev-erb gene expression were all reduced by 80% to 90% with the nadir between 3 and 6 hrs postinfusion. Per1 and Per2 reached an expression nadir between 13 and 17 hrs postinfusion. The levels of plasma interleukin-6 and tumor necrosis factor peaked and then returned to baseline within 6 hrs. In contrast, clock gene expression remained suppressed for up to 17 hrs irrespective of the phase of the clock at the time of the endotoxin challenge. Endotoxin did not perturb the melatonin secretory rhythm. Conclusions:Circadian clock gene expression in peripheral blood leukocytes is dramatically altered and possibly uncoupled from the activity of the central clock during periods of acute systemic inflammation. The realignment of the central and peripheral clocks may constitute a previously unappreciated key factor affecting recovery from disease in humans.