Jeffrey S. Warren

Hypoxic-ischemic injury induces monocyte chemoattractant protein-1 expression in neonatal rat brain.

Monocyte chemoattractant protein-1 (MCP-1) regulates monocyte accumulation in several macrophage-dependent experimental disease models. In the neonatal brain, activated microglia accumulate rapidly after hypoxic-ischemic injury. These cells produce potentially neurotoxic factors that may contribute to the progression of injury. To determine whether MCP-1 could be one of the molecular signals that influences the microglial response to hypoxic-ischemic injury in the neonatal brain, we examined the impact of acute hypoxic-ischemic injury on MCP-1 mRNA and protein expression. Seven-day-old rats underwent right carotid artery ligation, followed by 3 hours of 8% oxygen exposure, to elicit ipsilateral forebrain hypoxic-ischemic injury. To detect MCP-1 mRNA in situ hybridization assays were performed using 35S-labeled antisense riboprobes generated from rat MCP-1 cDNA. Animals were evaluated 0, 1, 2, 4, 8, 16, 24, 48, and 120 hours after hypoxic exposure (N ≥ 3/group). Immunocytochemistry (with a polyclonal rabbit antirat MCP-1 antibody) was used to determine the anatomic and temporal distribution of MCP-1, in samples obtained 10 minutes to 5 days after hypoxic exposure (N ≥ 3/group). Monocyte chemoattractant protein-1 mRNA was first detected in periventricular regions of the lesioned hemisphere 1 hour after hypoxia-ischemia; periependymal and intraparenchymal MCP-1 mRNA expression were detected at 4 hours; hybridization signal peaked at 8 to 24 hours; and no MCP-1 mRNA was detected at 48 and 120 hours. In lesioned forebrain, MCP-1 protein expression were consistently detected at 2.5 to 48 hours after hypoxia-ischemia. Many immunoreactive cells appeared to be neurons. These results suggest that in the developing brain, MCP-1 could represent a functionally important molecular signal for the microglial response to hypoxic-ischemic injury.

Monocyte chemoattractant protein-1 is a mediator of acute excitotoxic injury in neonatal rat brain

Monocyte chemoattractant protein-1 is a chemokine with potent monocyte activating and chemotactic effects. Monocyte chemoattractant protein-1 gene and protein expression is rapidly up-regulated in response to a variety of acute and chronic central nervous system disorders. The activation and recruitment of microglia and monocytes into areas of inflammation may play a critical role in the pathogenesis of acute brain injury. Monocyte chemoattractant protein-1 could be a pathophysiologically important mediator of the microglial and monocyte responses in the brain. Using a well-characterized model of acute excitotoxic brain injury in neonatal rats, experiments were designed to evaluate whether monocyte chemoattractant protein-1 plays a role in the progression of tissue damage. Direct co-administration of recombinant monocyte chemoattractant protein-1 with the excitotoxin N-methyl-D-aspartate exacerbated injury, both in the striatum and in the hippocampus, by 55% and 167%, respectively. Complementary experiments to determine the effect of functional inhibition of monocyte chemoattractant protein-1, using an anti-monocyte chemoattractant protein-1-neutralizing antibody, revealed that co-administration of the antibody with N-methyl-D-aspartate attenuated tissue injury in the striatum and hippocampus by 57% and 39%, respectively.Together, these data suggest that monocyte chemoattractant protein-1 is a mediator of acute excitotoxic brain injury in neonatal rats and that inflammatory mechanisms contribute significantly to the pathogenesis of acute neonatal brain injury. Whether chemokines are pathophysiologically relevant mediators of neuronal injury in human neonates remains to be determined.

Review: Oxidative Injury to the Vascular Endothelium

In recent years, increasing clinical and experimental data have provided compelling evidence that neutrophil-derived oxygen radicals and their metabolites are important mediators of vascular endothelial injury. In this review, attention is directed toward in vitro, ex vivo, and in vivo experimental studies contributing to current understanding of mechanisms of oxyradical mediated endothelial damage. Although these studies may have broad significance, they appear to have particular relevance to the pathogenesis of the adult respiratory distress syndrome and other types of acute pulmonary dysfunction.

Role of O2− in neutrophil recruitment into sites of dermal and pulmonary vasculitis

Using analogous models of acute dermal vasculitis and alveolitis in rats, we have examined the role of oxygen-derived metabolities in the tissue damage associated with neutrophil influx into sites of immune complex deposition. In the lung, as previously reported, catalase and deferoxamine are highly protective, while superoxide dismutase (SOD) has a transient protective effect. The xanthine oxidase inhibitors, allopurinol, and lodoxamide, are also protective. In the skin, neither catalase (which has been covalently linked to the antibody) nor deferoxamine is protective, suggesting that H2O2 and iron are not absolutely required for the development of dermal vasculitis. In the skin, SOD, as well as the inhibitors of xanthine oxidase, have protective effects. These data suggest that the neutrophil-mediated pathways of immune complex injury in the dermal and pulmonary microvascular compartments are fundamentally different. As a measurement of neutrophil accumulation, measurements of myeloperoxidase in tissue extracts have been employed. In both the lung and skin, the protective effects of SOD and the xanthine oxidase inhibitors are paralleled by reductions in neutrophil influx into sites of injury. In contrast, catalase and deferoxamine have no effect on neutrophil accumulation. These data suggest that vascular beds in rat skin and lung are fundamentally different with respect to mechanisms of acute immune complex mediated injury. The data also provide evidence that O2- contributes significantly to the accumulation of neutrophils.

Intrapulmonary interleukin 1 mediates acute immune complex alveolitis in the rat

Interleukin 1 alpha and beta are polypeptide cytokines that possesses a wide variety of immunologic and inflammatory activities. We have examined the role of intrapulmonary interleukin 1 in the pathogenesis of acute IgG immune complex alveolitis in the rat. Intratracheal instillation of IgG anti-bovine serum albumin accompanied by intravenous infusion of bovine albumin results in acute neutrophil and complement-dependent alveolitis. Over the course of evolving lung injury there was a 12-fold increase in bronchoalveolar lavage fluid interleukin 1 activity. Intratracheal instillation of neutralizing anti-interleukin 1 beta antibodies upon induction of lung injury resulted in a dose-dependent reduction in lung injury as assessed by measurements of pulmonary hemorrhage and vascular permeability. Morphometric analysis and measurements of myeloperoxidase activities in whole lung homogenates from rats that received anti-interleukin 1 beta revealed a pronounced reduction in neutrophil recruitment compared to positive controls. Incubation of isolated alveolar macrophages with preformed IgG immune complexes resulted in dose-dependent interleukin 1 secretion. These data suggest that intrapulmonary IL-1 activity plays a role in neutrophil recruitment and is necessary for the full development of acute IgG immune complex induced lung injury in the rat.

Comparison of imputation methods for missing laboratory data in medicine

Objectives Missing laboratory data is a common issue, but the optimal method of imputation of missing values has not been determined. The aims of our study were to compare the accuracy of four imputation methods for missing completely at random laboratory data and to compare the effect of the imputed values on the accuracy of two clinical predictive models. Design Retrospective cohort analysis of two large data sets. Setting A tertiary level care institution in Ann Arbor, Michigan. Participants The Cirrhosis cohort had 446 patients and the Inflammatory Bowel Disease cohort had 395 patients. Methods Non-missing laboratory data were randomly removed with varying frequencies from two large data sets, and we then compared the ability of four methods—missForest, mean imputation, nearest neighbour imputation and multivariate imputation by chained equations (MICE)—to impute the simulated missing data. We characterised the accuracy of the imputation and the effect of the imputation on predictive ability in two large data sets. Results MissForest had the least imputation error for both continuous and categorical variables at each frequency of missingness, and it had the smallest prediction difference when models used imputed laboratory values. In both data sets, MICE had the second least imputation error and prediction difference, followed by the nearest neighbour and mean imputation. Conclusions MissForest is a highly accurate method of imputation for missing laboratory data and outperforms other common imputation techniques in terms of imputation error and maintenance of predictive ability with imputed values in two clinical predicative models.

Neutrophil adhesion to human endothelial cells is induced by the membrane attack complex: the roles of P-selectin and platelet activating factor.

A variety of inflammatory diseases are accompanied by activation of the complement system. We examined the role of the membrane attack complex (MAC) in mediating neutrophil adhesion to endothelial cells. To assemble the MAC in endothelial cell monolayers, a C5b-like molecule was created through the treatment of purified C5 with the oxidizing agent chloramine-T, followed by addition of the remaining components (C6-C9) that constitute the MAC. Use of this method abrogated potentially confounding effects mediated by other complement components (e.g., C5a). MAC assembly resulted in a rapid (30 min), concentration-dependent increase in neutrophil adherence. A monoclonal antibody directed against P-selectin inhibited MAC-mediated neutrophil adhesion. A whole cell EIA confirmed P-selectin expression after formation of the MAC. Incubation of neutrophils with the platelet-activating factor receptor antagonist, CF 3988, also significantly decreased adhesion, indicating that PAF plays a role in MAC-mediated adhesion. These results suggest that the MAC can promote neutrophil adhesion through P-selectin and PAF-mediated mechanisms.

Homocysteine Augments Cytokine-Induced Chemokine Expression in Human Vascular Smooth Muscle Cells: Implications for Atherogenesis

Hyperhomocysteinemia is an independent risk factor for atherosclerosis and atherothrombosis. While in vitro studies have revealed a number of homocysteine-mediated alterations in the thromboregulatory properties of endothelial cells, comparatively little is known about homocysteine-modulated smooth muscle cell function. We observed that exposure of human aortic smooth muscle cells to pathophysiologically relevant concentrations of homocysteine results in concentration-dependent increases in cytokine-induced MCP-1 and IL-8 secretion. RNase protection assays revealed that both MCP-1 and IL-8 mRNA concentrations are increased in homocysteine-treated smooth muscle cells when compared to cells activated with cytokines alone. Homocysteine treatment also increased cytosolic-to-nuclear translocation of the p65 and p50 subunits of the Rel/NF-κB family of transcription factors but had no effect on AP-1 activation. Cumulatively, these data suggest that homocysteine may increase monocyte recruitment into developing atherosclerotic lesions by upregulating MCP-1 and IL-8 expression in vascular smooth muscle cells.

Laboratory Test Utilization Program Structure and Impact in a Large Academic Medical Center

In 2008, the University of Michigan Health System (UMHS) created a Laboratory Test Utilization Program that included the establishment of a Laboratory Formulary Committee under the imprimatur of the Faculty Group Practice, the Office of Clinical Affairs, the Department of Pathology, and UMHS hospital administration. A critical component of the program is UM-CareLink, an order entry system for inpatients and inpatient-like venues. UM-CareLink allows very basic decision support comment prompts. Through the application of peer-reviewed medical evidence, input by medical content experts, excellent cooperation by medical staff, and close oversight by Pathology of the Sendout Laboratory, this program has led to a robust process of test utilization oversight, excellent communication with clinical services, and significant UMHS activity-adjusted reductions in laboratory expense.

Comparison of the ultrasonic scalpel to CO2 laser and electrosurgery in terms of tissue injury and adhesion formation in a rabbit model

OBJECTIVE To determine the relative effect of an ultrasonic scalpel on reproductive tissue compared with CO2 laser and electrosurgery. DESIGN Prospective, randomized animal study. SETTING University laboratory setting. ANIMALS Sixteen New Zealand White rabbits. INTERVENTION(S) A steel scalpel, an ultrasonic scalpel, a CO2 laser, or electrosurgery were used to perform an ovarian wedge resection and to remove the distal uterine horn. A 3-cm longitudinal incision also was made in the uterine horn. MAIN OUTCOME MEASURE(S) The number of 1-second bursts of needle-tip electrosurgery required for hemostasis, the depth and degree of coagulation necrosis, degree of fibrin deposition, and postoperative adhesion formation. RESULT(S) The amount of electrosurgery needed to achieve hemostasis was less for any of the four power techniques than for the steel scalpel, with the exception of the ultrasonic scalpel at level 5 when used on the ovary. The depth (range: 0.30 to 0.38 mm) and the degree of coagulation necrosis was not different for any of the power techniques. The fibrin score was greatest for the ultrasonic scalpel at level 5 in both the ovarian tissue and the uterine tissue. There was no difference in adhesion scores for the power techniques and the steel scalpel. CONCLUSION(S) The ultrasonic scalpel at level 3 is not different from either CO2 laser or electrosurgery in terms of hemostatic properties, coagulation necrosis, or adhesion formation in the rabbit model.