Geoffrey M. Silver

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.

Toxic epidermal necrolysis: does immunoglobulin make a difference?

Experimental evidence implicates Fas ligand-mediated keratinocyte apoptosis as an underlying mechanism of toxic epidermal necrolysis syndrome (TEN). In vitro studies indicate a potential role for immunoglobulin (Ig) therapy in blocking Fas ligand signaling, thus reducing the severity of TEN. Anecdotal reports have described successful treatment of TEN patients with Ig; however, no study to date has analyzed outcome data in a large series of patients treated with Ig using institutional controls. The SCORTEN severity-of-illness score ranks severity and predicts prognosis in TEN patients using age, heart rate, TBSA slough, history of malignancy, and admission blood urea nitrogen, serum bicarbonate, and glucose levels. A retrospective chart review was performed that included all patients treated for TEN at our burn center since 1997. Ig therapy was instituted for all patients with biopsy-proven TEN beginning in January 2000. Twenty-one TEN patients were treated before Ig (no-Ig group), and 24 patients have been treated with Ig. SCORTEN data were collected, as well as length of stay (LOS) and status upon discharge. Each patient was given a SCORTEN of 0 to 6, with 1 point each for age greater than 40, TBSA slough greater than 10%, history of malignancy, admission BUN greater than 28 mg/dl, HCO3 less than 20 mg/dl, and glucose greater then 252 mg/dl. Outcome was compared between patients treated with Ig and without Ig. Overall mortality for patients treated before Ig was 28.6% (6/21), and with Ig, mortality was 41.7%% (10/24). There was no significant difference in age or TBSA slough. The average SCORTEN between the groups was equivalent (2.2 in no-Ig group vs 2.7 in Ig group, P = 0.3), and no group of patients with any SCORTEN score showed a significant benefit from Ig therapy. Overall LOS as well as LOS for survivors was longer in the Ig group. This series represents the largest single-institution analysis of TEN patient outcome after institution of Ig therapy. Our data do not show a significant improvement in mortality for TEN patients treated with Ig at any level of severity and may indicate a potential detriment in using Ig. Ig should not be given to TEN patients outside of a clinical trial. A multicenter, prospective, double-blinded randomized trial is necessary and urgently indicated to determine whether Ig therapy is beneficial or harmful in the care of TEN patients.

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.

Cell-specific expression and pathway analyses reveal alterations in trauma-related human T cell and monocyte pathways.

Monitoring genome-wide, cell-specific responses to human disease, although challenging, holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements, including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations, combined with cell-specific pathway analyses, offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes), monocyte (2,801 genes), and total leukocyte (3,437 genes) transcriptomes, with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1, CD152, NRP-1, and Lag3) and concomitant decreases in stimulatory receptors (CD28, CD4, and IL-2Rα). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus, genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly, application of cell separation, genome-wide expression, and cell-specific pathway analyses can be used to discover pathway alterations in human disease.

Adverse Clinical Outcomes Associated With Elevated Blood Alcohol Levels at the Time of Burn Injury

Elevated blood alcohol content (BAC) on admission is associated with poorer outcomes, larger burns and more inhalation injury. This study’s purpose was to examine the effects of alcohol through a matched case-controlled study, measuring early and extended markers of clinical outcomes. The hypothesis was that patients with an elevated admission BAC would require more resuscitation and have a longer hospital stay. Admissions 16 to 75 years of age with 15 to 75% TBSA and admission BACs were identified. Patients with BAC >30 mg/dl (BAC+, cases) were matched with patients with undetectable BAC (BAC−, controls), according to age, sex, TBSA, inhalation injury and mechanism. Screening identified 258 patients, 146 with admission BACs. Twenty-seven had a BAC ≥ 30 mg/dl. There were 24 matched pairs. At 24 hours, BAC+ group had larger acute physiology and chronic health evaluation II scores (23.33 vs 18.75, P < .05), fluid requirements (5.25 vs 3.82 L (cc/kg/TBSA), P < .05), and base deficit (11.15 vs 7.15, P < .05). The duration of mechanical ventilation (14.85 vs 4.23 days, P < .05), intensive care unit length of stay (22.85 vs 9.38, P < .05), hospital length of stay (28.95 vs 15.68, P < .05), and mean hospital charges (

The effect of granulocyte colony-stimulating factor (G-CSF) upon burn-induced defective neutrophil chemotaxis.

239,507 vs

Polymorphonuclear neutrophil chemiluminescence in whole blood from blunt trauma patients with multiple injuries.

144,598, P < .05) were increased in the BAC+ patients. Despite matched baseline injury characteristics, elevated BAC was associated with poorer short term and extended clinical outcomes, illustrating the impact of alcohol intoxication on physiologic derangement after burn injury.

Standard operating procedures for the clinical management of patients enrolled in a prospective study of inflammation and the host response to thermal injury

Severe thermal injury results in impairment of granulocyte production and function. The ability to improve the functional capacity of neutrophils could contribute to a reduced morbidity and mortality from sepsis following thermal injury. Previous studies from this laboratory have shown that rhG-CSF increases the number of femoral marrow granulocyte progenitor cells and circulating neutrophils as well as the survival rate following burn wound infection. The studies reported here examine the effect of in-vivo administration of rhG-CSF on neutrophil chemotaxis following a burn injury and also following superimposed Pseudomonas burn wound sepsis in mice. Casein-elicited peritoneal neutrophils were harvested 72 hours after burn injury and 24 hours after infection. Chemotaxis was assessed using microchemotaxis chambers and 10(-5) M fMet-Phe as a chemoattractant. The number of neutrophils that migrated into the filter was used as an index of directed chemotaxis. Burn injury resulted in depressed chemotaxis compared with sham or sham/G-CSF-treated animals (p less than 0.05). Administration of rhG-CSF to burned animals resulted in a level of neutrophil chemotaxis comparable with that in control animals. The presence of a burn wound infection caused no further impairment of chemotaxis. Administration of rhG-CSF to animals with a burn wound infection resulted in improved chemotaxis compared with sham, burned, and burned/infected animals. The beneficial effect of G-CSF following burn wound infections from this and previous studies appears to be a combination of expanded numbers of myeloid elements and preservation of their function.

A silver-coated antimicrobial barrier dressing used postoperatively on meshed autografts : A dressing comparison study

BACKGROUND Studies using isolated polymorphonuclear neutrophils (PMNs) indicate that trauma is associated with altered function of PMNs. Because isolation of PMNs can itself alter the function of these cells, we examined the relationships among measures of injury severity and several indices of PMN function using whole blood samples from trauma patients. METHODS Whole blood samples were obtained from 12 blunt trauma patients with multiple injuries in the intensive care unit of a Level I trauma center within 24 hours of admission and from 11 healthy volunteers. Samples were assayed for PMN chemiluminescence (CL) in response to a complement receptor 3 (CR3)-dependent agonist and for CD11b (CR3) expression. Common clinical parameters were correlated with CL and CR3 expression. RESULTS The CL ratio (i.e., unprimed/primed CL) was significantly correlated with initial base deficit (BD), Injury Severity Score (ISS), CR3 expression, units of packed red blood cells transfused during the interval before blood sampling, and length of intensive care unit stay (survivors only). In contrast, BD did not correlate with units of red blood cells transfused or length of stay. Similarly, ISS did not correlate with length of stay. CONCLUSION Significant correlations were observed between CL ratios and CR3 expression, ISS, initial BD, length of stay, and units of blood given. These data suggest that measuring CL produced by PMNs in whole blood is a potentially useful way to assess injury severity. Whereas the initial BD and ISS are indicators of how badly injured a patient is at the time of entry into a trauma center, the CL ratio may be a more useful indicator of both injury severity and prognosis.

Microfluidic Leukocyte Isolation for Gene Expression Analysis in Critically Ill Hospitalized Patients

As part of the National Institutes of General Medical Sciences (NIGMS)-funded Inflammation and the Host Response to Injury study, participating investigators created a database, a clinical data collection protocol, and web-based case report form. To obtain high-quality clinical endpoints for correlation with genomic data, a uniform approach to patient management between centers was required. Standard operating procedures (SOPs) were generated to minimize variability and promote a uniform standard of patient care. The SOPs are necessary to enable validation of the clinical endpoints to be used for comparison with genomic and proteomic information derived from samples of blood and tissue obtained from thermally injured patients. Participating investigators identified areas of potential practice variation and developed a set of SOPs based on available data and sound clinical principles. In the absence of sufficient clinical data to identify a single management strategy, SOPs were designed to apply the best approach to management without interfering with local standards of care. The data- collection instrument, or case report form, was constructed concurrently with the SOPs. Wherever possible, the case report form was modified to collect data that might resolve controversial management issues. Modifications in management strategies that were necessary for children are delineated as needed. Data queries and site visits were conducted to audit compliance. SOPs for 10 areas of clinical care were developed. The institution of the SOPs required minor changes in clinical practice patterns and personnel training but did not require participating centers to procure new technology or alter the utilization of clinical resources significantly. The SOPs represent current management strategies applied to the study population to reduce variation in patient management. The SOPs are easily adaptable to other burn-related clinical protocols as well as to the routine daily management burn patients.