Biomedical subjects
Publications and source records attributed to Ronald V Maier.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The application of genome-wide expression analysis to a large-scale, multicentered program in critically ill patients poses a number of theoretical and technical challenges. We describe here an analytical and organizational approach to a systematic evaluation of the variance associated with genome-wide expression analysis specifically tailored to study human disease. We analyzed sources of variance in genome-wide expression analyses performed with commercial oligonucleotide arrays. In addition, variance in gene expression in human blood leukocytes caused by repeated sampling in the same subject, among different healthy subjects, among different leukocyte subpopulations, and the effect of traumatic injury, were also explored. We report that analytical variance caused by sample processing was acceptably small. Blood leukocyte gene expression in the same individual over a 24-h period was remarkably constant. In contrast, genome-wide expression varied significantly among different subjects and leukocyte subpopulations. Expectedly, traumatic injury induced dramatic changes in apparent gene expression that were greater in magnitude than the analytical noise and interindividual variance. We demonstrate that the development of a nation-wide program for gene expression analysis with careful attention to analytical details can reduce the variance in the clinical setting to a level where patterns of gene expression are informative among different healthy human subjects, and can be studied with confidence in human disease.
BACKGROUND: Oxidative stress results in macrophage reprogramming through the formation of focal adhesion-like complexes on lipid rafts. Although the cellular mechanisms responsible for this reprogramming remain unknown, oxidative stress is known to result in a transient increase in intracellular calcium. This transient flux is thought to occur through the membrane dissociation of the calcium-bound protein annexin VI. The purpose of this study is to clarify the source of the calcium, and determine if it is responsible for the formation of focal adhesion-like complexes during oxidative stress through the activation of calcium/calmodulin dependent protein kinase II (CaMK II). METHODS: THP-1 cells were stimulated with hydrogen peroxide. Selected cells were pretreated with methyl beta-cyclodextrin (MbetaCD), a cholesterol-depleting agent; 1,2-bis aminophenoxy ethane-N,N,N',N'-tetraacetic acid, an intracellular calcium chelator; or autocamtide 2-related inhibitory peptide, a CaMK II inhibitor. Intracellular calcium flux was determined by a Fluo-3 technique. Lipid raft and cellular protein were extracted and analyzed for active CaMK II, annexin VI, and components of focal adhesion-like complexes. RESULTS: Hydrogen peroxide exposure led to mobilization of annexin VI from lipid rafts to the cytosol, which was followed by an increase in cytosolic calcium, phosphorylation of CaMK II, and formation of focal adhesion-like complexes. Cholesterol depletion from lipid rafts attenuated all of these effects. 1,2-bis Aminophenoxy ethane-N,N,N',N'-tetraacetic acid and autocamtide 2-related inhibitory peptide pretreatment attenuated CaMK II phopshorylation and formation of focal ahdesionlike complexes. CONCLUSIONS: Macrophage reprogramming during oxidative stress occurs through the cytosolic mobilization of annexin VI from lipid rafts. As a result, bound calcium dissociates, resulting in the activation of CaMK II and the formation of focal adhesion-like complexes.
Explore the source record for details and available documents.
Platelet-activating factor (PAF) primes the macrophage proinflammatory response to inflammatory stimuli, such as lipopolysaccharide (LPS). The cellular events responsible for this priming or reprogramming remain unresolved, but may occur through an increase in cytosolic calcium, inducing calcium/calmodulin-dependent kinase (CaMK) activation. To study this, differentiated THP-1 cells were used to study the effect of CaMK II and IV inhibition on PAF-induced reprogramming of TLR4-mediated events. LPS induced p38, ERK 1/2, and JNK/SAPK phosphorylation, NF-kappaB and AP-1 activation, and TNF-alpha and IL-10 production. PAF pretreatment selectively increased LPS-induced ERK 1/2, JNK/SAPK, NF-kappaB and AP-1 activation, and TNF-alpha production. Inhibition of CaMK II prevented PAF-induced priming of these events. Inhibition of CaMK IV prevented LPS-induced ERK 1/2, JNK/SAPK, NF-kappaB and AP-1 activation, and TNF-alpha production, but increased IL-10 production with or without PAF pretreatment. Neither CaMK II nor IV inhibition had any affect on p38 activity. These data suggest that the function of CaMK II is essential for PAF-induced macrophage priming. This priming event is mediated in part by modulation of ERK 1/2, JNK/SAPK, NF-kappaB, and AP-1 activation. CaMK IV, on the other hand, is not specific for priming by PAF and appears to have a direct link in TLR4-mediated events.
We sought to determine if hypertonic saline (HTS) impacted alveolar macrophage (AM) activation and intracellular inflammatory gene signaling in a model of systemic inflammation. Rats received an intravenous administration of 4 mL/kg of 7.5% HTS or L-lactate lactated Ringer's (L-LR). They were simultaneously treated with an intraperitoneal injection of zymosan, which induces noninfectious systemic inflammation. AM were harvested by bronchoalveolar lavage 24 h after treatment. AM activation was analyzed by measurement of baseline and lipopolysaccharide (LPS)-induced TNF-alpha production. Intracellular signaling was analyzed for activation of the mitogen-activated protein kinases (MAPKs): ERK1/2, JNK, and p38. AM from HTS-treated rats produced less TNF-alpha than from L-LR-treated rats (927 +/- 335 pg/mL [SEM] vs. 3628 +/- 783 pg/mL [SEM], P = 0.001) and were also less responsive to LPS (4444 +/- 86 pg/mL [SEM] vs. 6666 +/- 91 pg/mL [SEM], P = 0.058). However, there was no difference in MAPK activation. In vivo HTS prevents excessive AM activation during systemic inflammation. This suppression is mediated through alternate pathways and does not induce the classic MAPK signaling cascade.
BACKGROUND: Several studies have demonstrated that the success rate of prehospital intubation is improved by the use of neuromuscular blocking agents (NMBAs). However, a recent study has reported that prehospital intubation with NMBAs worsens outcome after traumatic brain injury (TBI). We sought to determine the effect of the use of NMBAs to facilitate prehospital intubation on outcome after TBI. METHODS: All patients admitted to our Level I trauma center with a head Abbreviated Injury Scale score >/= 3 were identified by the trauma registry (January 1998-June 2003). Patient records were matched with prehospital databases. Patients were further stratified on the basis of prehospital Glasgow Coma Scale (GCS) score into mild (GCS score of 14/15), moderate (GCS score of 9-13), and severe (GCS score < 9) TBI. Outcome included mortality and good outcome (survival to discharge with a GCS score of 14/15). RESULTS: There were 3,052 patients who were identified as having been transported directly from the field. Complete prehospital data were available for 2,012 patients (66%). Of these, 920 were mild TBI (intubation rate, 17.4%), 293 moderate TBI (intubation rate, 57.7%), and 799 severe TBI (intubation rate, 95%). Overall, 72% of intubated patients received NMBAs. There were no significant differences in demographics or injury severity between the groups. Patients not receiving NMBAs were more likely to be hypotensive and have prehospital cardiopulmonary resuscitation (p = 0.001). The unadjusted mortality for the patients intubated with NMBAs was 25% versus 37% for those not receiving NMBAs (p < 0.001). When adjusted for confounding variables, patients intubated with NMBAs were more likely to survive (odds ratio, 0.63; 95% confidence interval, 0.41-0.97; p = 0.04) and have a good outcome (odds ratio, 1.7; 95% confidence interval, 1.2-2.6; p = 0.006) than those in the no-NMBA group. CONCLUSION: The use of NMBAs to facilitate prehospital intubation improves outcome for patients with TBI. The value of prehospital intubation for TBI remains to be determined; however, any trial evaluating nonintubation for TBI must be compared with NMBA-facilitated intubation to be valid.
Explore the source record for details and available documents.
OBJECTIVE: Increased medical staff workload has been associated with worse outcomes in several studies. Inappropriate staffing has also been implicated in the increased risk of mortality for medical patients admitted on weekends. A theoretical threshold patient load may exist, beyond which the resources are strained and patient outcomes suffer. The goal of the study was to see whether trauma patients admitted during 'high' patient-load periods, at night, or on weekends had worse outcomes. METHODS: Trauma patients admitted to a high-volume Level I trauma center from 1994 to 2002 were analyzed. Patient load was defined as a combination of the number of patients admitted and the severity of their illness. On the basis of a multivariate regression model, a probability of fatal outcome was calculated for each patient as a marker for the severity of illness. For each patient, two new variables were calculated, the number of admissions (#ad) and the average probability of fatal outcome (PFO) for the 24-hour period in which the patient was admitted (excluding the patient him- or herself). The above variables, night/d, and weekend/d were placed in a multivariate regression model. RESULTS: There were 30,686 patients. Age, mechanism of injury, Injury Severity Score, maximum head Abbreviated Injury Scale score, admission Glasgow Coma Scale score, systolic blood pressure, and intubation status were the independent predictors of mortality. This model had an outstanding predictive power, with an area under the receiver operating characteristic curve of 0.96. The mean #ad was 11 +/- 4 and PFO was 0.08 +/- 0.07. Values above the 90th percentile were considered 'high' for #ad > 17 or PFO > 0.18. There was no difference in mortality for patients admitted during high #ad (odds ratio [OR], 0.95; p = 0.7) or high PFO (OR, 0.99; p = 0.9) versus low. There was no difference in mortality if a patient was admitted on weekends versus weekdays (OR, 0.9; p = 0.2) or at night versus day (OR, 0.9; p = 0.2). There was no difference in hospital length of stay for high #ad, high PFO, nights, or weekends. CONCLUSION: At this Level I trauma center that is part of an established statewide trauma system, patient outcomes were not compromised during high-patient-load periods, at night, or on weekends.
The adherence and activation of primary human monocytes was investigated on a polyelectrolyte multilayer film containing hyaluronic acid (HA) and poly-L-lysine (PLL). The sequential layer-by-layer deposition of the multilayer film was characterized by surface plasmon resonance. Eight alternating bilayers displayed an effective thickness of 16.15 nm with a total polymer coverage of 2.10 microg/cm2. For cell studies, HA-PLL multilayers were constructed on tissue culture polystyrene (TCPS) substrates and characterized by time of flight second ion mass spectrometry (ToF-SIMS) analysis. Principal component analysis of the ToF-SIMS spectra resolved no significant difference in surface chemistry between PLL-terminated and HA-terminated multilayer surfaces. Monocyte adhesion on PLL- and HA-terminated surfaces was measured by the lactate dehydrogenase assay and showed a significant decrease in cell adhesion after 24 h incubation. Cell viability measured by Live/Dead fluorescent staining showed significant cell death in the adherent cell population over these 24 h. Tumor necrosis factor-alpha (TNF-alpha) production, a measure of monocyte activation, was quantified by ELISA and normalized to the number of adherent monocytes. The activation of monocytes on PLL-terminated and HA-terminated surfaces was nearly identical, and both surfaces had TNF-alpha levels that were 8-fold higher than TCPS. These results demonstrate that sufficient PLL had diffused into the surface layer to direct monocyte adherence and to induce cytokine activation and cell death on the HA-terminated multilayer films. The diffusion of the second multilayer component to the coating surface should, thus, be taken into account in the design of polyelectrolyte-based biomaterial coating strategies.
Explore the source record for details and available documents.
The analysis of gene expression data in clinical medicine has been plagued by the lack of a critical evaluation of accepted methodologies for the collection, processing, and labeling of RNA. In the present report, the reliability of two commonly used techniques to isolate RNA from whole blood or its leukocyte compartment was compared by examining their reproducibility, variance, and signal-to-noise ratios. Whole blood was obtained from healthy subjects and was either untreated or stimulated ex vivo with Staphylococcus enterotoxin B (SEB). Blood samples were also obtained from trauma patients but were not stimulated with SEB ex vivo. Total RNA was isolated from whole blood with the PAXgene proprietary blood collection system or from isolated leukocytes. Biotin-labeled cRNA was hybridized to Affymetrix GeneChips. The Pearson correlation coefficient for gene expression measurements in replicates from healthy subjects with both techniques was excellent, exceeding 0.985. Unsupervised analyses, including hierarchical cluster analysis, however, revealed that the RNA isolation method resulted in greater differences in gene expression than stimulation with SEB or among different trauma patients. The intraclass correlation, a measure of signal-to-noise ratio, of the difference between SEB-stimulated and unstimulated blood from healthy subjects was significantly higher in leukocyte-derived samples than in whole blood: 0.75 vs. 0.46 (P = 0.002). At the P < 0.001 level of significance, twice as many probe sets discriminated between SEB-stimulated and unstimulated blood with leukocyte isolation than with PAXgene. The findings suggest that the method of RNA isolation from whole blood is a critical variable in the design of clinical studies using microarray analyses.
The adhesion and activation of monocytes and macrophages are thought to affect the foreign body response to implanted medical devices. However, these cells interact with devices indirectly, because of the prior adsorption of proteins. Therefore, we preadsorbed several "model" biomaterial surfaces with proteins and then measured foreign body giant cell (FBGC) formation, tumor necrosis factor alpha (TNFalpha) release, and procoagulant activity. The model surfaces were tissue culture polystyrene (TCPS), untreated polystyrene (PS), and Primaria, whereas the proteins used were albumin, fibronectin, fibrinogen, and immunoglobulin. FBGC formation, TNFalpha release, and procoagulant activity of monocytes were the highest for surfaces preadsorbed with IgG. FBGC formation was lower on surfaces with adsorbed fibrinogen and fibronectin than on uncoated surfaces. TNFalpha release and procoagulant activity of monocytes were similar on surface adsorbed with fibrinogen, fibronectin, or albumin. Monocyte activation was also affected by the surface chemistry of the substrates, because FBGC formation was the highest on PS and the lowest on TCPS. Monocyte procoagulant activity was the highest on Primaria. Adsorbed proteins and surface chemistry were found to have strong effects on FBGC formation, monocyte TNFalpha release, and procoagulant activity in vitro, providing support for the idea that these same variables could affect macrophage-mediated foreign body response to biomaterials in vivo.
The ideal system for management of trauma remains controversial, especially in respect of prehospital care and regionalisation of trauma-care delivery. To explore these issues, we compare two differing trauma systems--in the USA the focus is on the trauma centre, with a lesser emphasis on prehospital care, whereas in France there is more emphasis on prehospital care coordinated by the Service d'Aide Médicale Urgente. We describe the historical developments, current structure, and major controversies with regard to trauma-care delivery in the two countries. Comparative evidence on the effectiveness of the two systems was obtained through a structured review of databases, but very little evidence permits direct comparison of outcomes across the two systems. Crude injury mortality rates and fatality rates from motor-vehicle accidents (crashes in US usage) are higher in France than in the USA, although adjustment for potential confounders is difficult. Adjusted mortality rates suggest equivalent outcomes among patients who survive to hospital, although these data are confounded by the lack of a contemporaneous comparator population. There are differences in the American and French trauma systems that might translate into measurable differences in trauma-related mortality. However, the lack of data to allow comparison of outcomes between countries significantly impedes the identification and implementation of components of a trauma system that are effective and the discarding of those that offer little benefit.
Explore the source record for details and available documents.
The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.