Long wavelength light exposure reduces systemic inflammation coagulopathy, and acute organ injury following polytrauma in mice.

BACKGROUND: Evidence suggests that variation in light exposure strongly influences the dynamic of inflammation, coagulation, and the immune system. Polytrauma induces systemic inflammation that can lead to end-organ injury. Here, we hypothesize that alterations in light exposure influence post-trauma inflammation, coagulopathy, and end-organ injury. METHODS: Study Type: Original Research Article. Level of Evidence: Basic Science (Level IV).C57BL/6 mice underwent a validated polytrauma and hemorrhage model performed following 72 hours of exposure to red (617 nm, 1,700lux), blue (321 nm, 1,700lux), and fluorescent white light (300lux) (n = 6-8/group). The animals were sacrificed at 6 h post-trauma. Plasma samples were evaluated and compared for pro-inflammatory cytokine expression levels, coagulation parameters, markers of liver and renal injury, and histological changes (Carstairs staining). One-way ANOVA statistical tests were applied to compare study groups. RESULTS: Pre-exposure to long-wavelength red light significantly reduced the inflammatory response at 6 hours post-polytrauma compared to blue and ambient light, as evidenced by decreased levels of IL-6, MCP-1 (both p < 0.001), liver injury markers (ALT, p < 0.05), and kidney injury markers (cystatin C, p < 0.01). Additionally, Carstairs staining of organ tissues revealed milder histological changes in the red light-exposed group, indicating reduced end-organ damage. Furthermore, PT was significantly lower (p < 0.001) and fibrinogen levels were better maintained (p < 0.01) in the red light-exposed mice compared to those exposed to blue and ambient light. CONCLUSION: Prophylactic light exposure can be optimized to reduce systemic inflammation, coagulopathy and minimize acute organ injury following polytrauma. Understanding the mechanisms by which light exposure attenuates inflammation may provide a novel strategy to reducing trauma related morbidity.

MACROPHAGE SWITCHING: POLARIZATION AND MOBILIZATION AFTER TRAUMA.

ABSTRACT: Introduction: Trauma alters the immune response in numerous ways, affecting both the innate and adaptive responses. Macrophages play an important role in inflammation and wound healing following injury. We hypothesize that macrophages mobilize from the circulation to the site of injury and secondary sites after trauma, with a transition from proinflammatory (M1) shortly after trauma to anti-inflammatory (M2) at later time points. Methods: C57Bl6 mice (n = 6/group) underwent a polytrauma model using cardiac puncture/hemorrhage, pseudofemoral fracture, and liver crush injury. The animals were killed at several time points: uninjured, 24 h, and 7 days. Peripheral blood mononuclear cells, spleen, liver nonparenchymal cells, and lung were harvested, processed, and stained for flow cytometry. Macrophages were identified as CD68 + ; M1 macrophages were identified as iNOS + ; M2 macrophages as arginase 1 + . Results: We saw a slight presence of M1 macrophages at baseline in peripheral blood mononuclear cells (6.6%), with no significant change at 24 h and 7 days after polytrauma. In contrast, the spleen has a larger population of M1 macrophages at baseline (27.7%), with levels decreasing at 24 h and 7 days after trauma (20.6% and 12.6%, respectively). A similar trend is seen in the lung where at baseline 14.9% of CD68 + macrophages are M1, with subsequent continual decrease reaching 8.7% at 24 h and 4.4% at 7 days after polytrauma. M1 macrophages in the liver represent 14.3% of CD68 + population in the liver nonparenchymal cells at baseline. This percentage increases to 20.8% after trauma and decreases at 7 days after polytrauma (13.4%). There are few M2 macrophages in circulating peripheral blood mononuclear cells and in spleen at baseline and after trauma. The percentage of M2 macrophages in the lungs remains constant after trauma (7.2% at 24 h and 9.2% at 7 days). In contrast, a large proportion of M2 macrophages are seen in the liver at baseline (36.0%). This percentage trends upward and reaches 45.6% acutely after trauma and drops to 21.4% at 7 days. The phenotypic changes in macrophages seen in the lungs did not correlate with a functional change in the ability of the macrophages to perform oxidative burst, with an increase from 2.0% at baseline to 22.1% at 7 days after polytrauma ( P = 0.0258). Conclusion: Macrophage phenotypic changes after polytrauma are noted, especially with a decrease in the lung M1 phenotype and a short-term increase in the M2 phenotype in the liver. However, macrophage function as measured by oxidative burst increased over the time course of trauma, which may signify a change in subset polarization after injury not captured by the typical macrophage phenotypes.

Bilateral Central Retinal Vein Occlusion as a First Presentation of Multiple Myeloma: A Case Report.

INTRODUCTION: Acute presentation of multiple myeloma in the emergency department (ED) is an uncommon yet life-threatening clinical entity. CASE REPORT: A 42-year-old male presented to the ED with severe generalized fatigue and vision changes most notable in his left eye. Bilateral central retinal vein occlusion (CRVO) was diagnosed on dilated fundus exam in the ED. CONCLUSION: The most common cause of CRVO in adults over age 50 is vascular disease, but in younger adults, conditions of systemic inflammation or hyperviscosity must be considered. Diagnosis of CRVO requires emergent ophthalmology consultation and further treatment with phototherapy, steroids, and potentially anti-vascular endothelial growth factor. Ultimately, patients require hematology/oncology and ongoing management of acute hyperviscosity syndrome. We present this case to increase awareness surrounding this diagnosis among emergency physicians. Multiple myeloma should be considered in young patients who present to the ED with bilateral CRVO, acute renal failure, and symptomatic anemia.

Platelet HMGB1 in Platelet-Rich Plasma (PRP) promotes tendon wound healing.

Platelet-rich plasma (PRP) is a widely used autologous treatment for tendon injuries in clinics. Platelets (PLTs) are a major source of high mobility group box1 (HMGB1) that is gaining attention as a chemoattractant that can recruit stem cells to the wound area to enhance healing of injured tissues; however, the contribution of PLT HMGB1 in wounded tendon healing remains unexplored. This study investigated the effect of PLT HMGB1 within PRP on tendon healing using PLT HMGB1 knockout (KO) and GFP mice. A window defect was created in the patellar tendons of both groups of mice, and wounds were treated with either saline, PRP isolated from PLT HMGB1-KO mice, or PRP isolated from GFP mice. Seven days post-treatment, animals were sacrificed and analyzed by gross inspection, histology, and immunostaining for characteristic signs of tendon healing and repair. Our results showed that in comparison to mice treated with PRP from PLT HMGB1-KO mice, wounds treated with PRP from GFP mice healed faster and exhibited a better organization in tendon structure. Mice treated with PRP from PLT HMGB1-KO mice produced tendon tissue with large premature wound areas and low cell densities. However, wounds of PLT HMGB1-KO mice showed better healing with PRP from HMGB1-KO mice compared to saline treatment. Moreover, wounds treated with PRP from GFP mice had increased extracellular HMGB1, decreased CD68, increased stem cell markers CD146 and CD73, and increased collagen III protein expression levels compared to those treated with PRP from PLT HMGB1-KO mice. Thus, PLT HMGB1 within PRP plays an important role in tendon wound healing by decreasing inflammation, increasing local HMGB1 levels, and recruiting stem cells to the wound area in the tendon. Our findings also suggest that the efficacy of PRP treatment for tendon injuries in clinics may depend on PLT HMGB1 within PRP preparations.

Moderate and intensive mechanical loading differentially modulate the phenotype of tendon stem/progenitor cells in vivo.

To examine the differential mechanobiological responses of specific resident tendon cells, we developed an in vivo model of whole-body irradiation followed by injection of either tendon stem/progenitor cells (TSCs) expressing green fluorescent protein (GFP-TSCs) or mature tenocytes expressing GFP (GFP-TNCs) into the patellar tendons of wild type C57 mice. Injected mice were subjected to short term (3 weeks) treadmill running, specifically moderate treadmill running (MTR) and intensive treadmill running (ITR). In MTR mice, both GFP-TSC and GFP-TNC injected tendons maintained normal cell morphology with elevated expression of tendon related markers collagen I and tenomodulin. In ITR mice injected with GFP-TNCs, cells also maintained an elongated shape similar to the shape found in normal/untreated control mice, as well as elevated expression of tendon related markers. However, ITR mice injected with GFP-TSCs showed abnormal changes, such as cell morphology transitioning to a round shape, elevated chondrogenic differentiation, and increased gene expression of non-tenocyte related genes LPL, Runx-2, and SOX-9. Increased gene expression data was supported by immunostaining showing elevated expression of SOX-9, Runx-2, and PPARγ. This study provides evidence that while MTR maintains tendon homeostasis by promoting the differentiation of TSCs into TNCs, ITR causes the onset of tendinopathy development by inducing non-tenocyte differentiation of TSCs, which may eventually lead to the formation of non-tendinous tissues in tendon tissue after long term mechanical overloading conditions on the tendon.

Preoperative Predictors of 30-Day Mortality and Prolonged Length of Stay after Above-Knee Amputation.

BACKGROUND: The above-knee amputation (AKA) is an operation of last resort with high postoperative morbidity and mortality. This study identifies preoperative risk factors predictive of both 30-day mortality and extended length of stay (LOS) in AKA patients. METHODS: Two hundred ninety-five AKA patients from 2004 to 2013 from a single institution were retrospectively reviewed using a deidentified electronic medical record. Rationally selected factors potentially influencing 30-day mortality and LOS were chosen, including demographics, etiologies, vascular surgical history, lifestyle factors, comorbidities, and laboratory values. Variables trending with one of the end points on bivariate analysis (P ≤ 0.10) were entered into multivariate forward stepwise regression models to determine independence as a risk factor (P ≤ 0.05). Subgroup analysis of AKA patients without a traumatic, burn, or malignant etiology was similarly conducted. RESULTS: Within the 295 patient cohort, 60% of the patients were male, 18% were African American, mean age was 58 years and mean body mass index was 28 kg/m(2). The 30-day mortality rate was 9%, and mean postoperative LOS of discharged patients was 9.3 days. Upon logistic regression, thrombocytopenia (platelet count < 250 × 10(6)/mL, P < 0.001, odds ratio 6.1) and preoperative septic shock (P = 0.02, odds ratio 5.1) were identified as independent risk factors for 30-day mortality. Upon linear regression, burn etiology (P < 0.001, B = 15.8 days), leukocytosis (white blood cell count > 12 × 10(6)/mL, P < 0.001, B = 6.2 days), and guillotine amputation (P < 0.001, B = 7.6 days) were independently associated with prolonged LOS. Excluding patients with AKAs due to trauma, burn, or malignancy, only thrombocytopenia (platelet count < 250 × 10(6)/mL, P < 0.001, odds ratio 10.2) and leukocytosis (white blood cell count > 12 × 10(6)/mL, P = 0.01, B = 5.2 days) were independent risk factors for in-hospital mortality and prolonged LOS, respectively. CONCLUSIONS: Preoperative septic shock and thrombocytopenia are independent risk factors for 30-day mortality after AKA, while burn etiology, leukocytosis, and guillotine amputation contribute to prolonged LOS. Awareness of these risk factors may help enhance both preoperative decision making and expectations of the hospital admission.

Effect of injection augmentation on need for framework surgery in unilateral vocal fold paralysis.

OBJECTIVES/HYPOTHESIS: To determine whether injection augmentation reduces the likelihood of ultimately needing definitive framework surgery in unilateral vocal fold paralysis (UVFP) patients. STUDY DESIGN: Retrospective cohort study. METHODS: All patients diagnosed with UVFP (2008-2012) at the academic center were identified. The time from symptom onset to presentation to either community otolaryngologist and/or academic center, as well as any directed treatment(s), were recorded. Stepwise, multivariate logistic regression analysis was used to determine whether injection augmentation independently affected odds of needing definitive, framework surgery among patients who were seen within 9 months of symptom onset and had not undergone any prior rehabilitative procedures. RESULTS: Cohort consisted of 633 patients (55% female, 80% Caucasian, median age 60 years) with UVFP. The majority of etiologies were either surgery (48%) or idiopathic (37%). Duration to presentation at community otolaryngologist was shorter than to the academic center (median 2 vs. 6 months). Overall, less than half of UVFP patients had any operation (46%). Multivariate logistic regression found that earlier injection augmentation did not affect odds of ultimately undergoing framework surgery (odds ratio 1.13; confidence interval, 0.92-1.40; P = 0.23). CONCLUSION: Nearly half of UVFP patients do not require any rehabilitative procedure. When indicated, early injection augmentation is effective at temporarily alleviating associated symptoms but does not reduce likelihood of needing a definitive framework operation in patients with UVFP. Understanding practice patterns and fostering early detection and treatment may improve quality of life in this patient population.

Long-term mortality associated with acute kidney injury in children following congenital cardiac surgery.

OBJECTIVE: Children undergoing congenital cardiac surgery (CCS) are at increased risk for acute kidney injury (AKI) due to a number of factors. Recent evidence suggests AKI may influence mortality beyond the immediate postoperative period and hospitalization. We sought to determine the association between renal failure and longer-term mortality in children following CCS. METHODS: Our Study population included all patients that underwent cardiac surgery at our institution during a period of 3 years from 2004 through 2006. The primary definition of acute renal injury was based on pRIFLE using estimated creatinine clearance (pRIFLE eCCL). RESULTS: Predictors of mortality. Age, single ventricle status, and renal failure as defined by pRIFLE stage F were associated with mortality. The hazard ratio for a patient with renal failure as defined by pRIFLE stage F was 3.82 (CI 1.89-7.75). Predictors of AKI as defined by pRIFLE. Duration of cardiopulmonary bypass (CPB) and age were the only variables associated with pRIFLE by univariate analysis. However, in the ordinal or survival model, age was the only variable associated with renal failure as defined by pRIFLE. As patient age increases from 0.30 to 3.5 years, the risks of having renal injury (pRIFLE stage I) or failure (pRIFLE stage F) decreases (OR 0.44, CI 0.21-0.94). CONCLUSION: Mortality risk following CCS is increased in younger patients and those experiencing postoperative renal failure as defined by pRIFLE for a period of time that extends well beyond the immediate postoperative period and the time of hospitalization.

Catalytic and functional roles of conserved amino acids in the SET domain of the S. cerevisiae lysine methyltransferase Set1.

In S. cerevisiae, the lysine methyltransferase Set1 is a member of the multiprotein complex COMPASS. Set1 catalyzes mono-, di- and trimethylation of the fourth residue, lysine 4, of histone H3 using methyl groups from S-adenosylmethionine, and requires a subset of COMPASS proteins for this activity. The methylation activity of COMPASS regulates gene expression and chromosome segregation in vivo. To improve understanding of the catalytic mechanism of Set1, single amino acid substitutions were made within the SET domain. These Set1 mutants were evaluated in vivo by determining the levels of K4-methylated H3, assaying the strength of gene silencing at the rDNA and using a genetic assessment of kinetochore function as a proxy for defects in Dam1 methylation. The findings indicate that no single conserved active site base is required for H3K4 methylation by Set1. Instead, our data suggest that a number of aromatic residues in the SET domain contribute to the formation of an active site that facilitates substrate binding and dictates product specificity. Further, the results suggest that the attributes of Set1 required for trimethylation of histone H3 are those required for Pol II gene silencing at the rDNA and kinetochore function.

Direct comparison of 20-hour IV, 36-hour oral, and 72-hour oral acetylcysteine for treatment of acute acetaminophen poisoning.

There is no general consensus among clinicians on the superior route or duration of treatment with N-acetylcysteine (NAC) for acute acetaminophen (APAP) poisoning, and head-to-head studies comparing intravenous (IV) and oral NAC have not been done. Recent 20-hour IV NAC protocol failures in the United States prompted some to question its safety. Our objective was to determine if treatment with the 20-hour IV NAC protocol results in clinical outcomes different from the longer 36-hour oral or 72-hour oral NAC protocols in cases of acute APAP poisoning. We performed a retrospective analysis of all consecutive cases of acute APAP overdose where NAC treatment was initiated within 8 hours of ingestion between January 1, 2002, and December 31, 2007. Outcomes were survival, transplant, and death; secondary outcomes were based on King's College Criteria; interrater reliability was calculated with a kappa score. Out of 4642 cases of APAP overdose, 795 met study inclusion criteria: 213 were treated with 20-hour IV protocol, 213 with the 36-hour oral protocol, and 369 with the 72-hour oral protocol. The mean age in these groups was 25 years [95% confidence interval (CI): 22-26], 26 years (95%CI: 23-29), and 27 years (95%CI: 25-28), respectively. The mean 4-hour APAP concentration was 199 µg/mL (95%CI: 188-211), 174 µg/mL (95%CI: 164-184), and 205 µg/mL (95%CI: 195-216), respectively. No cases of transplant or death occurred, and secondary outcomes were rare. When administered within 8 hours of acute APAP poisoning, the 20-hour IV treatment protocol was as effective as the longer 36-hour oral and 72-hour oral treatment protocols. Further study is needed to determine outcome differences between IV and oral NAC when treatment is initiated >8 hours after overdose or in cases of coingestion with other drugs.

Bacillus anthracis peptidoglycan stimulates an inflammatory response in monocytes through the p38 mitogen-activated protein kinase pathway.

We hypothesized that the peptidoglycan component of B. anthracis may play a critical role in morbidity and mortality associated with inhalation anthrax. To explore this issue, we purified the peptidoglycan component of the bacterial cell wall and studied the response of human peripheral blood cells. The purified B. anthracis peptidoglycan was free of non-covalently bound protein but contained a complex set of amino acids probably arising from the stem peptide. The peptidoglycan contained a polysaccharide that was removed by mild acid treatment, and the biological activity remained with the peptidoglycan and not the polysaccharide. The biological activity of the peptidoglycan was sensitive to lysozyme but not other hydrolytic enzymes, showing that the activity resides in the peptidoglycan component and not bacterial DNA, RNA or protein. B. anthracis peptidoglycan stimulated monocytes to produce primarily TNFalpha; neutrophils and lymphocytes did not respond. Peptidoglycan stimulated monocyte p38 mitogen-activated protein kinase and p38 activity was required for TNFalpha production by the cells. We conclude that peptidoglycan in B. anthracis is biologically active, that it stimulates a proinflammatory response in monocytes, and uses the p38 kinase signal transduction pathway to do so. Given the high bacterial burden in pulmonary anthrax, these findings suggest that the inflammatory events associated with peptidoglycan may play an important role in anthrax pathogenesis.

Identification of lanthionine synthase C-like protein-1 as a prominent glutathione binding protein expressed in the mammalian central nervous system.

Proteomic experiments were performed to identify novel glutathione (GSH) binding proteins expressed in the mammalian central nervous system. Bovine brain lysate was affinity purified using an immobilized glutathione-Sepharose column. Proteins that bound the immobilized glutathione were eluted with free glutathione and identified by one- and two-dimensional electrophoresis coupled with mass spectrometric analysis of tryptic fragments. Major proteins purified by this technique were glutathione S-transferase-mu (GST-mu) and GST-pi and lanthionine synthase C-like protein-1 (LanCL1). LanCL1 is a mammalian homologue of a prokaryotic enzyme responsible for the synthesis of thioether (lanthionine) cross-links within nascent polypeptide chains, yielding macrocyclic proteins with potent microbicidal activity. An antibody against LanCL1 was generated and applied to immunochemical studies of spinal cord tissue from SOD1G93A transgenic mice, a model for amyotrophic lateral sclerosis (ALS), wherein LanCL1 expression was found to be increased at presymptomatic stages of the disease. These results indicate LanCL1 is a glutathione binding protein possibly significant to neurodegenerative disease.

On the relation of oxidative stress to neuroinflammation: lessons learned from the G93A-SOD1 mouse model of amyotrophic lateral sclerosis.

The central nervous system (CNS) presents both challenges and opportunities to researchers of redox biochemistry. The CNS is sensitive to oxidative damage during aging or disease; excellent transgenic models of specific neurodegenerative diseases have been created that reproduce oxidative stress components of the corresponding human disorder. Mouse models of familial amyotrophic lateral sclerosis (ALS) based on overexpressed mutant human Cu, Zn-superoxide dismutase (SOD1) are cases in point. These animals experience predictably staged, age-dependent motor neuron degeneration with profound cellular and biochemical damage to nerve fibers and spinal cord tissue. Severe protein and lipid oxidation occurs in these animals, apparently as an indirect consequence of protein aggregation or cytopathic protein-protein interactions, as opposed to aberrant redox catalysis by the mutant enzyme. Recent studies of G93A-SOD1 mice and rats suggest that oxidative damage is part of an unmitigated neuroinflammatory reaction, possibly arising in combination from mitochondrial dysfunction plus pathophysiologic activation of both astrocytes and microglia. Lesions to redox signal-transduction pathways in mutant SOD1+ glial cells may stimulate broad-spectrum upregulation of proinflammatory genes, including arachidonic acid-metabolizing enzymes [e.g., cyclooxygenase-II (COX-II) and 5-lipoxygenase (5LOX)]; nitric oxide synthase (NOS) isoforms; cytokines (particularly tumor necrosis factor alpha, TNF-alpha); chemokines; and immunoglobulin Fc receptors (FcgammaRs). The integration of these processes creates a paracrine milieu inconsistent with healthy neural function. This review summarizes what has been learned to date from studies of mutant SOD1 transgenic animals and demonstrates that the G93A-SOD1 mouse in particular is a robust laboratory for the study of neuroinflammation and redox biochemistry.

Mitochondrial dysfunction in choline deficiency-induced apoptosis in cultured rat hepatocytes.

Our recent studies have demonstrated that generation of ROS is associated with choline deficiency (CD)-induced apoptosis in CWSV-1 cells, an immortalized rat hepatocyte that becomes tumorigenic by stepwise culturing in decreasing levels of choline. In the present study, we investigated the effect of CD on loss of mitochondrial membrane potential (MMP), using the JC-1 probe by FASCAN assay. Our data demonstrate that MMP in CD-cultured cells was decreased in a time- and dose-dependent manner and that significant disruption occurred at 24 h, relative to high choline (HC, 70 microM) cultured cells. In order to investigate further the relationship among the CD-induced ROS, MMP collapse, and apoptosis, we examined the effects of different inhibitors on ROS production, MMP disruption, and apoptosis in CD or HC-cultured CWSV-1 cells. These data indicate that the disruption of MMP is an upstream event in CD-induced apoptosis, and mitochondrial dysfunction plays a key role in mediating CD-induced apoptosis in CWSV-1 cells.

The arachidonic acid 5-lipoxygenase inhibitor nordihydroguaiaretic acid inhibits tumor necrosis factor alpha activation of microglia and extends survival of G93A-SOD1 transgenic mice.

Familial forms of amyotrophic lateral sclerosis (ALS) can be caused by mutations in copper, zinc-superoxide dismutase (SOD1). Mice expressing SOD1 mutants demonstrate a robust neuroinflammatory reaction characterized, in part, by up-regulation of tumor necrosis factor alpha (TNFalpha) and its primary receptor TNF-RI. In an effort to identify small molecule inhibitors of neuroinflammation useful in treatment of ALS, a microglial culture system was established to identify TNFalpha antagonists. Walker EOC-20 microglia cells were stimulated with recombinant TNFalpha, with or without inhibitors, and the cell response was indexed by NO2- output. Three hundred and fifty-five rationally selected compounds were included in this bioassay. The arachidonic acid 5-lipoxygenase (5LOX) and tyrosine kinase inhibitor nordihydroguaiaretic acid (NDGA), a natural dicatechol, was one of the most potent non-cytotoxic antagonists tested (IC50 8 +/- 3 microm). Investigation of the G93A-SOD1 mouse model for ALS revealed increased message and protein levels of 5LOX at 120 days of age. Oral NDGA (2500 p.p.m.) significantly extended lifespan and slowed motor dysfunction in this mouse, when administration was begun relatively late in life (90 days). NDGA extended median total lifespan of G93A-SOD1 mice by 10%, and life expectancy following start of treatment was extended by 32%. Disease-associated gliosis and cleaved microtubule-associated tau protein, an indicator of axon damage, were likewise reduced by NDGA. Thus, TNFalpha antagonists and especially 5LOX inhibitors might offer new opportunities for treatment of ALS.

Reactive oxygen species in choline deficiency-induced apoptosis in rat hepatocytes.

Choline deficiency (CD) is involved in hepatocellular carcinoma and CD-induced apoptosis may be implicated in cellular malignant transformation. In this report, we studied the effects of choline deficiency on generation of reactive oxygen species (ROS) using the fluorescent probe dichlorodihydrofluorescein diacetate and the possible role of ROS on CD-induced apoptosis in cultured CWSV-1 cells, an immortalized rat hepatocyte. This cell line is reported to become tumorigenic by step-wise culturing in lower levels of choline. Our data demonstrate that CD induces a time- and dose-dependent increase in ROS in CWSV-1 cells. The increase in ROS production may be related to dysfunction of the mitochondrial respiratory chain. Our data also demonstrated that ROS generation occurred before CD-induced apoptosis, suggesting ROS may play a key role in signaling CD-induced apoptosis in CWSV-1 cells.

New perspectives on vitamin E: gamma-tocopherol and carboxyelthylhydroxychroman metabolites in biology and medicine.

Vitamin E (alpha-tocopherol or alphaT) has long been recognized as a classic free radical scavenging antioxidant whose deficiency impairs mammalian fertility. In actuality, alpha-tocopherol is one member of a class of phytochemicals that are distinguished by varying methylation of a chroman head group. Early studies conducted between 1922 and 1950 indicated that alpha-tocopherol was specific among the tocopherols in allowing fertility of laboratory animals. The unique vitamin action of alphaT, combined with its prevalence in the human body and the similar efficiency of tocopherols as chain-breaking antioxidants, led biologists to almost completely discount the "minor" tocopherols as topics for basic and clinical research. Recent discoveries have forced a serious reconsideration of this conventional wisdom. New and unexpected biological activities have been reported for the desmethyl tocopherols, such as gamma-tocopherol, and for specific tocopherol metabolites, most notably the carboxyethyl-hydroxychroman (CEHC) products. The activities of these other tocopherols do not map directly to their chemical antioxidant behavior but rather reflect anti-inflammatory, antineoplastic, and natriuretic functions possibly mediated through specific binding interactions. Moreover, a nascent body of epidemiological data suggests that gamma-tocopherol is a better negative risk factor for certain types of cancer and myocardial infarction than is a alpha-tocopherol. The potential public health implications are immense, given the extreme popularity of alphaT supplementation which can unintentionally deplete the body of gamma-tocopherol. These findings may or may not signal a major paradigm shift in free radical biology and medicine. The data argue for thorough experimental and epidemiological reappraisal of desmethyl tocopherols, especially within the contexts of cardiovascular disease and cancer biology.

Message and protein-level elevation of tumor necrosis factor alpha (TNF alpha) and TNF alpha-modulating cytokines in spinal cords of the G93A-SOD1 mouse model for amyotrophic lateral sclerosis.

Recent data indicate that certain pro-inflammatory cytokines are transcriptionally upregulated in the spinal cords of G93A-SOD1 mice, a model of amyotrophic lateral sclerosis (ALS). We previously showed that the receptor for tumor necrosis factor alpha (TNF-R1) was notably elevated at late presymptomatic as well as symptomatic phases of disease (J. Neurochem. 82 (2002) 365). We now extend these findings by showing that message for TNFalpha, as well as mRNA for interferon gamma (IFNgamma) and transforming growth factor beta1/2 (TGFbeta1, TGFbeta2), is simultaneously increased. Furthermore, TNFalpha protein is significantly increased in G93A-SOD1 mouse spinal cords, as are protein levels for interleukin-6 (IL6), IFNgamma, and the chemokines RANTES (CCL5) and KC. The interaction of TNFalpha, IL6, and IFNgamma proteins was modeled in vitro using Walker EOC-20 murine microglia with nitrite (NO(2)(-)) efflux as a quantitative index of cell response. TNFalpha alone caused robust NO(2)(-) flux, while IL6 had a lesser effect and neither IFNgamma nor IL1beta was active when applied singly. The TNFalpha stimulus was potently magnified in the presence of IL6 or IFNgamma. When applied in combination at very low concentrations, IFNgamma co-synergized with IL6 to produce a multiplicative increase in NO(2)(-) after stimulation with TNFalpha. Taken together, these data suggest that modest increases in multiple synergistic cytokines could produce a disproportionately severe activation of microglia within the degenerating spinal cord. Our data support a model wherein TNFalpha acts as a principal driver for neuroinflammation, while several co-stimulating cytokines and chemokines act to potentiate the TNFalpha effects.

Temporal patterns of cytokine and apoptosis-related gene expression in spinal cords of the G93A-SOD1 mouse model of amyotrophic lateral sclerosis.

Familial amyotrophic lateral sclerosis (FALS) is often caused by gain-of-function mutations in Cu,Zn-superoxide dismutase (SOD1). Multiprobe ribonuclease protection assays (RPAs) were used to investigate expression of 36 different cytokines and apoptosis-related genes in spinal cords of mice that ubiquitously express human SOD1 bearing a glycine (r) alanine substitution at residue 93 (G93A-SOD1). Mice were studied at late presymptomatic stage (80 days), and at 120 days when the animals experience severe hindlimb paralysis and accumulation of oxidatively modified proteins. Spinal cord tissue from G93A-SOD1 mice expressed a selective subset of macrophage-typical cytokines (monokines) including interleukin (IL)1alpha, IL1beta and IL1RA at 80 days increasing by 120 days. Contrastingly, T-cell derived cytokines (lymphokines) including IL2, IL3 and IL4 were detected at low levels in non-transgenic mice but these were not elevated in G93A-SOD1 mice even at 120 days. Apoptosis-related genes were generally unaffected at 80 days but multiple caspases and death receptor components were up-regulated at 120 days; the only exceptions being FADD and the tumor necrosis factor (TNF)alpha receptor p55 which was up-regulated at 80 days and increased further at 120 days. These data indicate that in the G93A-SOD1 mouse: (i) cytokine expression changes precede bulk protein oxidation and apoptosis gene expression; (ii) lymphocyte contributions to cytokine expression in FALS are likely minor; and (iii) TNFalpha and its receptors may link inflammation to apoptosis in ALS.