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1.  Influence of ATP-Binding Cassette Polymorphisms on Neurological Outcome After Traumatic Brain Injury 
Neurocritical care  2013;19(2):192-198.
As important mediators of solute transport at the blood–brain and blood–cerebrospinal fluid barriers, ATP-binding cassette (ABC) transporters (including ABCB1, ABCC1, and ABCC2), impact the bioavailability of drugs and endogenous substrates in the brain. While several ABCB1, ABCC1, and ABCC2 single nucleotide polymorphisms (SNPs) have been identified, their impact on outcome after traumatic brain injury (TBI) is unknown.
ABCB1, ABCC1, and ABCC2 SNPs are associated with Glasgow Outcome Scale (GOS) score after TBI.
DNA samples from 305 adult patients with severe TBI (Glasgow Coma Scale, GCS score ≤ 8) were genotyped for tagging SNPs of ABCB1 (rs1045642; rs1128503), ABCC1 (rs212093; rs35621; rs4148382), and ABCC2 (rs2273697). For each SNP, patients were dichotomized based on presence of variant allele for multivariate analysis to determine associations with GOS assigned at 6 months adjusting for GCS, Injury Severity score, age, and patient sex.
For ABCB1 rs1045642, patients homozygous for the T allele were less likely to be assigned poor outcome versus those possessing the C allele [CT/CC; odds of unfavorable GOS = 0.71(0.55−0.92)]. For ABCC1 rs4148382, patients homozygous for the G allele were less likely to be assigned poor outcome versus those possessing the A allele [AG/AA; odds of unfavorable GOS = 0.73(0.55−0.98)].
In this single-center study, patients homozygous for the T allele of ABCB1 rs1045642 or the G allele of ABCC1 rs4148382 were found to have better outcome after severe TBI. Further study is necessary to replicate these very preliminary findings and to determine whether these associations are due to central nervous system bioavailability of ABC transporter drug substrates commonly used in the management of TBI, brain efflux of endogenous solutes, or both.
PMCID: PMC4332629  PMID: 23896815
Blood-brain barrier; Head trauma; Membrane transporter; Multidrug resistance protein; Multidrug resistance-associated protein; P-Glycoprotein
2.  Polynitroxylated-pegylated hemoglobin attenuates fluid requirements and brain edema in combined traumatic brain injury plus hemorrhagic shock in mice 
Polynitroxylated-pegylated hemoglobin (PNPH), a bovine hemoglobin decorated with nitroxide and polyethylene glycol moieties, showed neuroprotection vs. lactated Ringer's (LR) in experimental traumatic brain injury plus hemorrhagic shock (TBI+HS). Hypothesis: Resuscitation with PNPH will reduce intracranial pressure (ICP) and brain edema and improve cerebral perfusion pressure (CPP) vs. LR in experimental TBI+HS. C57/BL6 mice (n=20) underwent controlled cortical impact followed by severe HS to mean arterial pressure (MAP) of 25 to 27 mm Hg for 35 minutes. Mice (n=10/group) were then resuscitated with a 20 mL/kg bolus of 4% PNPH or LR followed by 10 mL/kg boluses targeting MAP>70 mm Hg for 90 minutes. Shed blood was then reinfused. Intracranial pressure was monitored. Mice were killed and %brain water (%BW) was measured (wet/dry weight). Mice resuscitated with PNPH vs. LR required less fluid (26.0±0.0 vs. 167.0±10.7 mL/kg, P<0.001) and had a higher MAP (79.4±0.40 vs. 59.7±0.83 mm Hg, P<0.001). The PNPH-treated mice required only 20 mL/kg while LR-resuscitated mice required multiple boluses. The PNPH-treated mice had a lower peak ICP (14.5±0.97 vs. 19.7±1.12 mm Hg, P=0.002), higher CPP during resuscitation (69.2±0.46 vs. 45.5±0.68 mm Hg, P<0.001), and lower %BW vs. LR (80.3±0.12 vs. 80.9±0.12%, P=0.003). After TBI+HS, resuscitation with PNPH lowers fluid requirements, improves ICP and CPP, and reduces brain edema vs. LR, supporting its development.
PMCID: PMC3764379  PMID: 23801241
blood substitute; cerebral edema; hemoglobin blood oxygen carrier; intracranial pressure; nitroxide; resuscitation
3.  The role of chromogranin B in an animal model of multiple sclerosis 
Chromogranin B (CGB) is a high capacity, low affinity calcium binding protein in the endoplasmic reticulum (ER) that binds to the inositol 1,4,5 trisphosphate receptor (InsP3R) and amplifies calcium release from ER stores. Recently, it was discovered that levels of CGB-derived peptides are decreased in the cerebrospinal fluid of multiple sclerosis (MS) patients. One of the mechanisms by which neurodegeneration in MS is thought to occur is through increased levels of intra-axonal calcium. The combination of excess intracellular calcium and dysregulated levels of CGB in MS led us to hypothesize that CGB may be involved in MS pathophysiology. Here, we show in a mouse model of MS that CGB levels are elevated in neurons prior to onset of symptoms. Once symptoms develop, CGB protein levels increase with disease severity. Additionally, we show that elevated levels of CGB may have a role in the pathophysiology of MS and suggest that the initial elevation of CGB, prior to symptom onset, is due to inflammatory processes. Upon development of symptoms, CGB accumulation in neurons results from decreased ubiquitination and decreased secretion. Furthermore, we show that calpain activity is increased and levels of InsP3R are decreased. From these results, we suggest that the elevated levels of CGB and altered InsP3R levels may contribute to the axonal/neuronal damage and dysregulated calcium homeostasis observed in MS. Additionally, we propose that CGB can be a biomarker that predicts the onset and severity of disease in patients with MS.
PMCID: PMC3791207  PMID: 23624073
Chromogranin B (CgB); Inositol 1,4,5 trisphosphate receptor (InsP3R); Intracellular calcium signaling; Multiple Sclerosis (MS)
4.  Hypothermia Decreases Cerebrospinal Fluid Asymmetric Dimethylarginine Levels in Traumatic Brain Injury Children 
Pathological increases in asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, have been implicated in endothelial dysfunction and vascular diseases. Reduced NO early after traumatic brain injury (TBI) may contribute to hypoperfusion. Currently, methods to quantify ADMA in the cerebrospinal fluid (CSF) have not been fully explored. We aimed to develop and validate a method to determine ADMA in the CSF of a pediatric TBI population and to use this method to assess the effects of (i) TBI and (ii) therapeutic hypothermia (TH) on this mediator.
Design, Setting, Patients
An ancillary study to a prospective, phase II randomized clinical trial (RCT) of early hypothermia in a tertiary care pediatric intensive care unit for children with TBI admitted to Children's Hospital of Pittsburgh.
Measurements and Main Results
A UPLC-MS/MS method was developed and validated to quantitate ADMA. A total of 56 samples collected over 3 days starting with injury onset were analyzed from the CSF of consented therapeutic hypothermia (n=9) and normothermia (n=10) children. Children undergoing diagnostic lumbar puncture (n=5) were controls. ADMA was present at a quantifiable level in all samples. Mean ADMA levels were significantly increased in normothermic TBI children compared to control (0.19± 0.08 μmol/L and 0.11± 0.02μmol/L respectively, p=0.01), and hypothermic children had significantly reduced mean ADMA levels (0.11 ± 0.05 μmol/L) vs. normothermic (p=0.03) measured on day 3. Patient demographics including age, gender, and NO levels (measured as nitrite and nitrate using liquid chromatography coupled with Griess reaction) did not significantly differ between normothermia and hypothermia groups. Also, NO levels did not correlate with ADMA concentrations.
ADMA levels were significantly increased in the CSF of TBI children. Early hypothermia attenuated this increase. The implications of attenuated ADMA on NOS activity and regional cerebral blood flow after TBI by TH deserve future attention.
PMCID: PMC4134918  PMID: 23439461
traumatic brain injury; nitric oxide synthase; cerebrospinal fluid; asymmetric dimethyl arginine; therapeutic hypothermia; neuroprotection
5.  Phospholamban Knockout Breaks Arrhythmogenic Ca2+ Waves and Suppresses Catecholaminergic Polymorphic Ventricular Tachycardia in Mice 
Circulation research  2013;113(5):10.1161/CIRCRESAHA.113.301678.
Phospholamban (PLN) is an inhibitor of cardiac sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a). PLN knockout (PLN-KO) enhances sarcoplasmic reticulum (SR) Ca2+ load and Ca2+ leak. Conversely, PLN-KO accelerates Ca2+ sequestration and aborts arrhythmogenic spontaneous Ca2+ waves (SCWs). An important question is whether these seemingly paradoxical effects of PLN-KO exacerbate or protect against Ca2+-triggered arrhythmias.
We investigate the impact of PLN-KO on SCWs, triggered activities, and stress-induced ventricular tachyarrhythmias (VTs) in a mouse model of cardiac ryanodine receptor (RyR2)-linked catecholaminergic polymorphic ventricular tachycardia (CPVT).
Methods and Results
We generated a PLN-deficient, RyR2 mutant mouse model (PLN−/−/RyR2-R4496C+/−) by crossbreeding PLN-KO mice with CPVT-associated RyR2-R4496C mutant mice. Ca2+ imaging and patch-clamp recording revealed cell-wide propagating SCWs and triggered activities in RyR2-R4496C+/− ventricular myocytes during SR Ca2+ overload. PLN-KO fragmented these cell-wide SCWs into mini-waves and Ca2+ sparks, and suppressed triggered activities evoked by SR Ca2+ overload. Importantly, these effects of PLN-KO were reverted by partially inhibiting SERCA2a with 2,5-Di-tert-butylhydroquinone (tBHQ). However, Bay K, caffeine, or Li+ failed to convert mini-waves to cell-wide SCWs in PLN−/−/RyR2-R4496C+/− ventricular myocytes. Furthermore, ECG analysis showed that PLN-KO mice are not susceptible to stress-induced VTs. On the contrary, PLN-KO protected RyR2-R4496C mutant mice from stress-induced VTs.
Our results demonstrate that despite severe SR Ca2+ leak, PLN-KO suppresses triggered activities and stress-induced VTs in a mouse model of CPVT. These data suggest that breaking up cell-wide propagating SCWs by enhancing Ca2+ sequestration represents an effective approach for suppressing Ca2+-triggered arrhythmias.
PMCID: PMC3864692  PMID: 23856523
Sarcoplasmic reticulum; ryanodine receptor; phospholamban; Ca2+ waves; Ca2+ leak; Ca2+-triggered arrhythmias
6.  Na+ current expression in human atrial myofibroblasts: identity and functional roles 
In the mammalian heart fibroblasts have important functional roles in both healthy conditions and diseased states. During pathophysiological challenges, a closely related myofibroblast cell population emerges, and can have distinct, significant roles. Recently, it has been reported that human atrial myofibroblasts can express a Na+ current, INa. Some of the biophysical properties and molecular features suggest that this INa is due to expression of Nav 1.5, the same Na+ channel α subunit that generates the predominant INa in myocytes from adult mammalian heart. In principle, expression of Nav 1.5 could give rise to regenerative action potentials in the fibroblasts/myofibroblasts. This would suggest an active as opposed to passive role for fibroblasts/myofibroblasts in both the “trigger” and the “substrate” components of cardiac rhythm disturbances. Our goals in this preliminary study were: (i) to confirm and extend the electrophysiological characterization of INa in a human atrial fibroblast/myofibroblast cell population maintained in conventional 2-D tissue culture; (ii) to identify key molecular properties of the α and β subunits of these Na+ channel(s); (iii) to define the biophysical and pharmacological properties of this INa; (iv) to integrate the available multi-disciplinary data, and attempt to illustrate its functional consequences, using a mathematical model in which the human atrial myocyte is coupled via connexins to fixed numbers of fibroblasts/myofibroblasts in a syncytial arrangement. Our experimental findings confirm that a significant fraction (approximately 40–50%) of these human atrial myofibroblasts can express INa. However, our data suggest that INa may be generated by a combination of Nav 1.9, Nav 1.2, and Nav 1.5. Our results, when complemented with mathematical modeling, provide a background for re-evaluating pharmacological management of supraventricular rhythm disorders, e.g., persistent atrial fibrillation.
PMCID: PMC4124488  PMID: 25147525
Na+ current; atrial arrhythmias; mathematical models; fibroblast; myofibroblast
7.  Brain tissue oxygen monitoring identifies cortical hypoxia and thalamic hyperoxia after experimental pediatric cardiac arrest 
Pediatric research  2013;75(2):295-301.
Optimization of cerebral oxygenation after pediatric cardiac arrest (CA) may reduce neurological damage associated with the post-CA syndrome. We hypothesized that important alterations in regional partial pressure of brain tissue oxygen (PbO2) occur after resuscitation from CA and that clinically relevant interventions such as hyperoxia and blood pressure augmentation would influence PbO2.
Cortical and thalamic PbO2 were monitored in immature rats subjected to asphyxial CA (9 or 12 min asphyxia) and sham-operated rats using oxygen sensors.
Thalamus and cortex showed similar baseline PbO2. Post-resuscitation there was early and sustained cortical hypoxia in an insult-duration fashion. In contrast, thalamic PbO2 initially increased four-fold, and afterwards returned to baseline values. PbO2 was FiO2-dependent, and the response to oxygen was more pronounced after a 9 min vs. 12 min CA. After a 12 min CA, PbO2 was modestly affected by blood pressure augmentation using epinephrine in the thalamus but not cortex.
After asphyxial pediatric CA, there is marked regional variability of cerebral oxygenation. Cortical hypoxia is pronounced and appears early, while thalamic hyperoxia is followed by normoxia. Compromised PbO2 in the cortex may represent a relevant and clinically measurable therapeutic target aimed at improving neurological outcome after pediatric CA.
PMCID: PMC3970819  PMID: 24226633
8.  Advanced oxidation protein products induce cardiomyocyte death via Nox2/Rac1/superoxide-dependent TRAF3IP2/JNK signaling 
Advanced oxidation protein products (AOPPs) are formed during chronic oxidative stress as a result of reactions between plasma proteins and chlorinated oxidants. Their levels are elevated during various cardiovascular diseases. Since elevated AOPPs serve as independent risk factors for ischemic heart disease, and cardiomyocyte death is a hallmark of ischemic heart disease, we hypothesized that AOPPs will induce cardiomyocyte death. AOPPs-modified mouse serum albumin (AOPPs-MSA) induced significant death of neonatal mouse cardiomyocytes that was attenuated by Receptor for Advanced Glycation End Products (RAGE), but not CD36 knockdown. Notably, TRAF3 interacting protein 2 (TRAF3IP2; also known as CIKS or Act1) knockdown blunted AOPPs-induced apoptosis. AOPPs-MSA stimulated Nox2/Rac1-dependent superoxide generation, TRAF3IP2 expression, and TRAF3IP2-dependent JNK activation. The superoxide anion generating xanthine-xanthine oxidase system and hydrogen peroxide both induced TRAF3IP2 expression. Further, AOPPs-MSA induced mitochondrial Bax translocation and release of cytochrome c into cytoplasm. Moreover, AOPPs-MSA suppressed anti-apoptotic Bcl-2 and Bcl-xL expression. These effects were reversed by TRAF3IP2 knockdown or forced expression of mutant JNK. Similar to its effects in neonatal cardiomyocytes, AOPPs-MSA induced adult cardiomyocyte death in part via TRAF3IP2. These results demonstrate for the first time that AOPPs induce cardiomyocyte death via Nox2/Rac1/superoxide-dependent TRAF3IP2/JNK activation in vitro, and suggest that AOPPs may contribute to myocardial injury in vivo. Thus TRAF3IP2 may represent a potential therapeutic target in ischemic heart disease.
PMCID: PMC3714806  PMID: 23453926
AOPPs; TRAF3IP2; oxidative stress; cell death; myocardial injury
9.  Using beta binomials to estimate classification uncertainty for ensemble models 
Quantitative structure-activity (QSAR) models have enormous potential for reducing drug discovery and development costs as well as the need for animal testing. Great strides have been made in estimating their overall reliability, but to fully realize that potential, researchers and regulators need to know how confident they can be in individual predictions.
Submodels in an ensemble model which have been trained on different subsets of a shared training pool represent multiple samples of the model space, and the degree of agreement among them contains information on the reliability of ensemble predictions. For artificial neural network ensembles (ANNEs) using two different methods for determining ensemble classification – one using vote tallies and the other averaging individual network outputs – we have found that the distribution of predictions across positive vote tallies can be reasonably well-modeled as a beta binomial distribution, as can the distribution of errors. Together, these two distributions can be used to estimate the probability that a given predictive classification will be in error. Large data sets comprised of logP, Ames mutagenicity, and CYP2D6 inhibition data are used to illustrate and validate the method. The distributions of predictions and errors for the training pool accurately predicted the distribution of predictions and errors for large external validation sets, even when the number of positive and negative examples in the training pool were not balanced. Moreover, the likelihood of a given compound being prospectively misclassified as a function of the degree of consensus between networks in the ensemble could in most cases be estimated accurately from the fitted beta binomial distributions for the training pool.
Confidence in an individual predictive classification by an ensemble model can be accurately assessed by examining the distributions of predictions and errors as a function of the degree of agreement among the constituent submodels. Further, ensemble uncertainty estimation can often be improved by adjusting the voting or classification threshold based on the parameters of the error distribution. Finally, the profiles for models whose predictive uncertainty estimates are not reliable provide clues to that effect without the need for comparison to an external test set.
PMCID: PMC4076254  PMID: 24987464
Artificial neural network ensemble; ANNE; Classification; Confidence; Error estimation; Predictive value; QSAR; Uncertainty
10.  Assessing the Stages of Change Among African American Women in a Weight Management Program 
The Journal of black psychology  2011;38(1):81-103.
The objective of this study was to assess the relationship between stage of change (SOC) and behavioral outcomes among African American women entering obesity treatment in two settings. Fifty-five overweight/obese (body mass index = 26.50–48.13), but otherwise healthy African American women, 23 to 56 years old, attended a 13-week weight loss–treatment program that took place at churches (n = 36) or a university (n = 19). Participants were weighed, completed SOC measures, and had a physical fitness test at pre- and posttreatment. Pretreatment measures of SOC placed 47% of the participants as actors, 31% as contemplators, and 22% as maintainers. Of the 45 women who reported posttreatment SOC, 7% regressed, 44% did not change, and 31% progressed in SOC. Pretreatment SOC predicted posttreatment weight loss in the church setting but not in the university setting. At churches, contemplators lost more weight than actors and maintainers. The church may be a more conducive setting for weight change behaviors for African American women who are categorized as contemplators in the SOC model.
PMCID: PMC3968858  PMID: 24683280
stages of change; African American women; obesity; weight loss
11.  Serine Lipids of Porphyromonas gingivalis Are Human and Mouse Toll-Like Receptor 2 Ligands 
Infection and Immunity  2013;81(9):3479-3489.
The total cellular lipids of Porphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. In vitro, both lipid 654 and lipid 430 activated TLR2-expressing HEK cells, and this activation was inhibited by anti-TLR2 antibody. In contrast, TLR4-expressing HEK cells failed to be activated by either lipid 654 or lipid 430. Wild-type (WT) or TLR2-deficient (TLR2−/−) mice were injected with either lipid 654 or lipid 430, and the effects on serum levels of the chemokine CCL2 were measured 4 h later. Administration of either lipid 654 or lipid 430 to WT mice resulted in a significant increase in serum CCL2 levels; in contrast, the administration of lipid 654 or lipid 430 to TLR2−/− mice resulted in no increase in serum CCL2. These results thus identify a new class of TLR2 ligands that are produced by P. gingivalis that likely play a significant role in mediating inflammatory responses both at periodontal sites and, potentially, in other tissues where these lipids might accumulate.
PMCID: PMC3754206  PMID: 23836823
12.  Bone marrow-derived microglia-based neurturin delivery protects against dopaminergic neurodegeneration in a mouse model of Parkinson’s disease 
Neuroscience letters  2013;535:24-29.
Although neurotrophic factors have long been recognized as potent agents for protecting against neuronal degeneration, clinical success in treating Parkinson’s disease and other neurodegenerative disorders has been hindered by difficulties in delivery of trophic factors across the blood brain barrier (BBB). Bone marrow hematopoietic stem cell-based gene therapy is emerging as a promising tool for overcoming drug delivery problems, as myeloid cells can cross the BBB and are recruited in large numbers to sites of neurodegeneration, where they become activated microglia that can secrete trophic factors. We tested the efficacy of bone marrow-derived microglial delivery of neurturin (NTN) in protecting dopaminergic neurons against neurotoxin-induced death in mice. Bone marrow cells were transduced ex vivo with lentivirus expressing the NTN gene driven by a synthetic macrophage-specific promoter. Infected bone marrow cells were then collected and transplanted into recipient animals. Eight weeks after transplantation, the mice were injected with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropuridine (MPTP) for seven days to induce dopaminergic neurodegeneration. Microglia-mediated NTN delivery dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and their terminals in the striatum. Microglia-mediated NTN delivery also induced significant recovery of synaptic marker staining in the striatum of MPTP-treated animals. Functionally, NTN treatment restored MPTP-induced decline in general activity, rearing behavior, and food intake. Thus, bone marrow-derived microglia can serve as cellular vehicles for sustained delivery of neurotrophic factors capable of mitigating dopaminergic injury.
PMCID: PMC3645298  PMID: 23295906
Parkinson’s disease; Gene therapy; Neurodegenerative disease; Neurotrophic factors; Dopamine
13.  Inhibition of PKC disrupts addiction-related memory 
The atypical PKC isoforms, PKMζ and PKCλ have been proposed as integral substrates of long-term memory (LTM). Inhibition of these isoforms has recently been demonstrated to be sufficient for impairing the expression and maintenance of long-term potentiation. Additionally, the pseudosubstrate inhibitor, zeta inhibitory peptide (ZIP), which effectively blocks PKMζ and PKCλ, has previously been shown to disrupt associative memory; very little is known about its effects on pathological nonassociative forms of memory related to addiction. The neural and molecular substrates of memory and addiction have recently been argued to overlap. Here, we used ZIP to disrupt PKMζ and PKCλ activity to examine their role in cocaine sensitization, a nonassociative, addiction-related memory argued to underlie the transition from casual to pathological drug use. We examined the effects of both continuous and acute administration of ZIP. Even a single application of ZIP blocked the development of sensitization; sustained inhibition using osmotic pumps produced an almost complete blockade of sensitization. Further, a single application of ZIP was shown to reduce membrane-bound AMPAR expression. These results demonstrate a novel, critical role for the atypical PKC isoforms in nonassociative memory and cocaine addiction.
PMCID: PMC3945752  PMID: 24639635
PKC; cocaine; sensitization; memory; nonassociative; ZIP
14.  Developing the design of a continuous national health survey for New Zealand 
A continuously operating survey can yield advantages in survey management, field operations, and the provision of timely information for policymakers and researchers. We describe the key features of the sample design of the New Zealand (NZ) Health Survey, which has been conducted on a continuous basis since mid-2011, and compare to a number of other national population health surveys.
A number of strategies to improve the NZ Health Survey are described: implementation of a targeted dual-frame sample design for better Māori, Pacific, and Asian statistics; movement from periodic to continuous operation; use of core questions with rotating topic modules to improve flexibility in survey content; and opportunities for ongoing improvements and efficiencies, including linkage to administrative datasets.
Results and discussion
The use of disproportionate area sampling and a dual frame design resulted in reductions of approximately 19%, 26%, and 4% to variances of Māori, Pacific and Asian statistics respectively, but at the cost of a 17% increase to all-ethnicity variances. These were broadly in line with the survey’s priorities. Respondents provided a high degree of cooperation in the first year, with an adult response rate of 79% and consent rates for data linkage above 90%.
A combination of strategies tailored to local conditions gives the best results for national health surveys. In the NZ context, data from the NZ Census of Population and Dwellings and the Electoral Roll can be used to improve the sample design. A continuously operating survey provides both administrative and statistical advantages.
PMCID: PMC3880001  PMID: 24364838
Health surveys; Indigenous populations; Sample design; Sampling rare populations; Survey planning
15.  Increased cytochrome c in rat cerebrospinal fluid after cardiac arrest and its effects on hypoxic neuronal survival 
Resuscitation  2012;83(12):1491-1496.
Cerebrospinal fluid (CSF) proteins may be useful biomarkers of neuronal death and ultimate prognosis after hypoxic-ischemic brain injury. Cytochrome c has been identified in the CSF of children following traumatic brain injury. Cytochrome c is required for cellular respiration but it is also a central component of the intrinsic pathway of apoptosis. Thus, in addition to serving as a biomarker, cytochrome c release into CSF may have an effect upon survival of adjacent neurons. In this study, we use Western blot and ELISA to show that cytochrome c is elevated in CSF obtained from pediatric rats following resuscitation from cardiac arrest. Using biotinylated human cytochrome c in culture media we show that cytochrome c crosses the cell membrane and is incorporated into mitochondria of neurons exposed to anoxia. Lastly, we show that addition of human cytochrome c to primary neuronal culture exposed to anoxia improves survival. To our knowledge, this is the first study to show cytochrome c is elevated in CSF following hypoxic ischemic brain injury. Results from primary neuronal culture suggest that extracellular cytochrome c is able to cross the cell membrane of injured neurons, incorporate into mitochondria, and promote survival following anoxia.
PMCID: PMC3445744  PMID: 22554683
cerebrospinal fluid; brain ischemia; cardiac arrest; cytochrome c
16.  Bacterial lipodipeptide, Lipid 654, is a microbiome-associated biomarker for multiple sclerosis 
Multiple sclerosis (MS) is an autoimmune disease of unknown etiology. Infectious agents have been suggested to have a role as environmental factors in MS, but this concept remains controversial. Recently, gastrointestinal commensal bacteria have been implicated in the pathogenesis of autoimmune diseases, but mechanisms underlying the relationship of human systemic autoimmunity with the commensal microbiome have yet to be identified. Consistent with the lack of understanding of pathogenic mechanisms and relevant environmental factors in MS, no blood biomarkers have been identified that distinguish MS patients from healthy individuals. We recently identified a unique gastrointestinal and oral bacteria-derived lipodipeptide, Lipid 654, which is produced by commensal bacteria and functions as a human and mouse Toll-like receptor 2 ligand. Using multiple-reaction-monitoring mass spectrometry, a critical approach in targeted lipidomics, we now report that Lipid 654 can be recovered in the serum of healthy individuals. Most interestingly, we find that Lipid 654 is expressed at significantly lower levels in the serum of patients with MS compared with both healthy individuals and patients with Alzheimer's disease. These results thus identify for the first time a potential mechanism relating the gastrointestinal and oral commensal microbiome to a human systemic autoimmune disease. In addition, these results also identify a potential etiologic environmental factor and novel clinically relevant serum biomarker for MS.
PMCID: PMC4232052  PMID: 25505950
autoimmunity; biomarker; commensal bacteria; microbiome; multiple sclerosis; TLR2
17.  Severe Brief Pressure-Controlled Hemorrhagic Shock after Traumatic Brain Injury Exacerbates Functional Deficits and Long-Term Neuropathological Damage in Mice 
Journal of Neurotrauma  2012;29(12):2192-2208.
Hypotension after traumatic brain injury (TBI) worsens outcome. We published the first report of TBI plus hemorrhagic shock (HS) in mice using a volume-controlled approach and noted increased neuronal death. To rigorously control blood pressure during HS, a pressure-controlled HS model is required. Our hypothesis was that a brief, severe period of pressure-controlled HS after TBI in mice will exacerbate functional deficits and neuropathology versus TBI or HS alone. C57BL6 male mice were randomized into four groups (n=10/group): sham, HS, controlled cortical impact (CCI), and CCI+HS. We used a pressure-controlled shock phase (mean arterial pressure [MAP]=25–27 mm Hg for 35 min) and its treatment after mild to moderate CCI including, a 90 min pre-hospital phase, during which lactated Ringer's solution was given to maintain MAP >70 mm Hg, and a hospital phase, when the shed blood was re-infused. On days 14–20, the mice were evaluated in the Morris water maze (MWM, hidden platform paradigm). On day 21, the lesion and hemispheric volumes were quantified. Neuropathology and hippocampal neuron counts (hematoxylin and eosin [H&E], Fluoro-Jade B, and NeuN) were evaluated in the mice (n=60) at 24 h, 7 days, or 21 days (n=5/group/time point). HS reduced MAP during the shock phase in the HS and CCI+HS groups (p<0.05). Fluid requirements during the pre-hospital phase were greatest in the CCI+HS group (p<0.05), and were increased in HS versus sham and CCI animals (p<0.05). MWM latency was increased on days 14 and 15 after CCI+HS (p<0.05). Swim speed and visible platform latency were impaired in the CCI+HS group (p<0.05). CCI+HS animals had increased contusion volume versus the CCI group (p<0.05). Hemispheric volume loss was increased 33.3% in the CCI+HS versus CCI group (p<0.05). CA1 cell loss was seen in CCI+HS and CCI animals at 24 h and 7 days (p<0.05). CA3 cell loss was seen after CCI+HS (p<0.05 at 24 h and 7 days). CA1 cell loss at 21 days was seen only in CCI+HS animals (p<0.05). Brief, severe, pressure-controlled HS after CCI produces robust functional deficits and exacerbates neuropathology versus CCI or HS alone.
PMCID: PMC3472682  PMID: 22738159
blast injury; controlled cortical impact; head injury; head trauma; Morris water maze; polytrauma; secondary insult
18.  Cerebrospinal Fluid Levels of High-Mobility Group Box 1 and Cytochrome C Predict Outcome after Pediatric Traumatic Brain Injury 
Journal of Neurotrauma  2012;29(11):2013-2021.
High-mobility group box 1 (HMGB1) is a ubiquitous nuclear protein that is passively released from damaged and necrotic cells, and actively released from immune cells. In contrast, cytochrome c is released from mitochondria in apoptotic cells, and is considered a reliable biomarker of apoptosis. Thus, HMGB1 and cytochrome c may in part reflect the degree of necrosis and apoptosis present after traumatic brain injury (TBI), where both are felt to contribute to cell death and neurological morbidity. Ventricular cerebrospinal fluid (CSF) was obtained from children admitted to the intensive care unit (ICU) after TBI (n=37). CSF levels of HMGB1 and cytochrome c were determined at four time intervals (0–24 h, 25–48 h, 49–72 h, and>72 h after injury) using enzyme-linked immunosorbent assay (ELISA). Lumbar CSF from children without TBI served as controls (n=12). CSF HMGB1 levels were: control=1.78±0.29, 0–24 h=5.73±1.45, 25–48 h=5.16±1.73, 49–72 h=4.13±0.75,>72 h=3.80±0.90 ng/mL (mean±SEM). Peak HMGB1 levels were inversely and independently associated with favorable Glasgow Outcome Scale (GOS) scores at 6 mo (0.49 [0.24–0.97]; OR [5–95% CI]). CSF cytochrome c levels were: control=0.37±0.10, 0–24 h=0.69±0.15, 25–48 h=0.82±0.48, 49–72 h=1.52±1.08,>72 h=1.38±1.02 ng/mL (mean±SEM). Peak cytochrome c levels were independently associated with abusive head trauma (AHT; 24.29 [1.77–334.03]) and inversely and independently associated with favorable GOS scores (0.42 [0.18–0.99]). In conclusion, increased CSF levels of HMGB1 and cytochrome c were associated with poor outcome after TBI in infants and children. These data are also consistent with the designation of HMGB1 as a “danger signal.” Distinctly increased CSF cytochrome c levels in infants and children with AHT and poor outcome suggests that apoptosis may play an important role in this unique patient population.
PMCID: PMC3408241  PMID: 22540160
abusive head trauma; child abuse; cytochrome c; high mobility group box 1
19.  Enhanced CD4+ T-Cell Recovery with Earlier HIV-1 Antiretroviral Therapy 
The New England journal of medicine  2013;368(3):218-230.
The relationship between the timing of the initiation of antiretroviral therapy (ART) after infection with human immunodeficiency virus type 1 (HIV-1) and the recovery of CD4+ T-cell counts is unknown.
In a prospective, observational cohort of persons with acute or early HIV-1 infection, we determined the trajectory of CD4+ counts over a 48-month period in partially overlapping study sets: study set 1 included 384 participants during the time window in which they were not receiving ART and study set 2 included 213 participants who received ART soon after study entry or sometime thereafter and had a suppressed plasma HIV viral load. We investigated the likelihood and rate of CD4+ T-cell recovery to 900 or more cells per cubic millimeter within 48 months while the participants were receiving viral-load–suppressive ART.
Among the participants who were not receiving ART, CD4+ counts increased spontaneously, soon after HIV-1 infection, from the level at study entry (median, 495 cells per cubic millimeter; interquartile range, 383 to 622), reached a peak value (median, 763 cells per cubic millimeter; interquartile range, 573 to 987) within approximately 4 months after the estimated date of infection, and declined progressively thereafter. Recovery of CD4+ counts to 900 or more cells per cubic millimeter was seen in approximately 64% of the participants who initiated ART earlier (≤4 months after the estimated date of HIV infection) as compared with approximately 34% of participants who initiated ART later (>4 months) (P<0.001). After adjustment for whether ART was initiated when the CD4+ count was 500 or more cells per cubic millimeter or less than 500 cells per cubic millimeter, the likelihood that the count would increase to 900 or more cells per cubic millimeter was lower by 65% (odds ratio, 0.35), and the rate of recovery was slower by 56% (rate ratio, 0.44), if ART was initiated later rather than earlier. There was no association between the plasma HIV RNA level at the time of initiation of ART and CD4+ T-cell recovery.
A transient, spontaneous restoration of CD4+ T-cell counts occurs in the 4-month time window after HIV-1 infection. Initiation of ART during this period is associated with an enhanced likelihood of recovery of CD4+ counts. (Funded by the National Institute of Allergy and Infectious Diseases and others.)
PMCID: PMC3657555  PMID: 23323898
20.  Increases in cerebrospinal fluid caffeine concentration are associated with favorable outcome after severe traumatic brain injury in humans 
Caffeine, the most widely consumed psychoactive drug and a weak adenosine receptor antagonist, can be neuroprotective or neurotoxic depending on the experimental model or neurologic disorder. However, its contribution to pathophysiology and outcome in traumatic brain injury (TBI) in humans is undefined. We assessed serial cerebrospinal fluid (CSF) concentrations of caffeine and its metabolites (theobromine, paraxanthine, and theophylline) by high-pressure liquid chromatography/ultraviolet in 97 ventricular CSF samples from an established bank, from 30 adults with severe TBI. We prospectively selected a threshold caffeine level of ≥1 μmol/L (194 ng/mL) as clinically significant. Demographics, Glasgow Coma Scale (GCS) score, admission blood alcohol level, and 6-month dichotomized Glasgow Outcome Scale (GOS) score were assessed. Mean time from injury to initial CSF sampling was 10.77±3.13 h. On initial sampling, caffeine was detected in 24 of 30 patients, and the threshold was achieved in 9 patients. Favorable GOS was seen more often in patients with CSF caffeine concentration ≥ versus < the threshold (55.6 versus 11.8%, P = 0.028). Gender, age, admission CGS score, admission blood alcohol level, and admission systolic arterial blood pressure did not differ between patients with CSF caffeine concentration ≥ versus < the threshold. Increases in CSF concentrations of the caffeine metabolites theobromine and paraxanthine were also associated with favorable outcome (P = 0.018 and 0.056, respectively). Caffeine and its metabolites are commonly detected in CSF in patients with severe TBI and in an exploratory assessment are associated with favorable outcome. We speculate that caffeine may be neuroprotective by long-term upregulation of adenosine A1 receptors or acute inhibition of A2a receptors.
PMCID: PMC3714395  PMID: 17684518
adenosine; alcohol; coffee; head injury; head trauma; theobromine
21.  CIKS (Act1 or TRAF3IP2) mediates Angiotensin-II-induced Interleukin-18 Expression, and Nox2-dependent Cardiomyocyte Hypertrophy 
Chronic elevation of angiotensin (Ang)-II can lead to myocardial inflammation, hypertrophy and cardiac failure. The adaptor molecule CIKS (connection to IKK and SAPK/JNK) activates the IκB kinase/nuclear factor (NF)-κB and JNK/activator protein (AP)-1 pathways in autoimmune and inflammatory diseases. Since Ang-II is a potent activator of NF-κB and AP-1, we investigated whether CIKS is critical in Ang-II-mediated cardiac hypertrophy. Here we report that Ang-II induced CIKS mRNA and protein expression, CIKS binding to IKK and JNK perhaps functioning as a scaffold protein, CIKS-dependent IKK/NF-κB and JNK/AP-1 activation, p65 and c-Jun phosphorylation and nuclear translocation, NF-κB- and AP-1-dependent IL-18 and MMP-9 induction, and hypertrophy of adult cardiomyocytes isolated from WT, but not CIKS-null mice. These results were recapitulated in WT-cardiomyocytes following CIKS knockdown. Infusion of Ang-II for 7 days induced cardiac hypertrophy, increased collagen content, and upregulated CIKS mRNA and protein expression in WT mice, whereas cardiac hypertrophy and collagen deposition were markedly attenuated in the CIKS-null mice, despite a similar increase in systolic blood pressure and DPI-inhibitable superoxide generation in both types of animals. Further, Ang-II-induced IKK/p65 and JNK/c-Jun phosphorylation, NF-κB and AP-1 activation, and IL-18 and MMP-9 expression were also markedly attenuated in CIKS-null mice. These results demonstrate that CIKS is critical in Ang-II-induced cardiomyocyte hypertrophy and fibrosis, and that CIKS is an important intermediate in Ang-II induced redox signaling. CIKS is a potential therapeutic target in cardiac hypertrophy, fibrosis, and congestive heart failure.
PMCID: PMC3477707  PMID: 22575763
RAAS; NADPH oxidase; Act1; TRAF3IP2; fibrosis; cardiac hypertrophy
22.  Global lipidomics identifies cardiolipin oxidation as a mitochondrial target for redox therapy of acute brain injury 
Nature neuroscience  2012;15(10):1407-1413.
Brain contains a highly diversified complement of molecular species of a mitochondria-specific phospholipid, cardiolipin (CL), which - due to its polyunsaturation - can readily undergo oxygenation. Here, we used global lipidomics analysis in experimental traumatic brain injury (TBI) and showed that TBI was accompanied by oxidative consumption of polyunsaturated CL and accumulation of more than 150 new oxygenated molecular species in CL. RNAi-based manipulations of CL-synthase and CL levels conferred resistance of primary rat cortical neurons to mechanical stretch - an in vitro model of traumatic neuronal injury. By applying the novel brain permeable mitochondria-targeted electron-scavenger, we prevented CL oxygenation in the brain, achieved a substantial reduction in neuronal death both in vitro and in vivo, and markedly reduced behavioral deficits and cortical lesion volume. We conclude that CL oxygenation generates neuronal death signals and that its prevention by mitochondria-targeted small molecule inhibitors represents a new target for neuro-drug discovery.
PMCID: PMC3697869  PMID: 22922784
23.  Brain Resuscitation in the Drowning Victim 
Neurocritical care  2012;17(3):441-467.
Drowning is a leading cause of accidental death. Survivors may sustain severe neurologic morbidity. There is negligible research specific to brain injury in drowning making current clinical management non-specific to this disorder. This review represents an evidence-based consensus effort to provide recommendations for management and investigation of the drowning victim. Epidemiology, brain-oriented prehospital and intensive care, therapeutic hypothermia, neuroimaging/monitoring, biomarkers, and neuroresuscitative pharmacology are addressed. When cardiac arrest is present, chest compressions with rescue breathing are recommended due to the asphyxial insult. In the comatose patient with restoration of spontaneous circulation, hypoxemia and hyperoxemia should be avoided, hyperthermia treated, and induced hypothermia (32–34 °C) considered. Arterial hypotension/hypertension should be recognized and treated. Prevent hypoglycemia and treat hyperglycemia. Treat clinical seizures and consider treating non-convulsive status epilepticus. Serial neurologic examinations should be provided. Brain imaging and serial biomarker measurement may aid prognostication. Continuous electroencephalography and N20 somatosensory evoked potential monitoring may be considered. Serial biomarker measurement (e.g., neuron specific enolase) may aid prognostication. There is insufficient evidence to recommend use of any specific brain-oriented neuroresuscitative pharmacologic therapy other than that required to restore and maintain normal physiology. Following initial stabilization, victims should be transferred to centers with expertise in age-specific post-resuscitation neurocritical care. Care should be documented, reviewed, and quality improvement assessment performed. Preclinical research should focus on models of asphyxial cardiac arrest. Clinical research should focus on improved cardiopulmonary resuscitation, re-oxygenation/reperfusion strategies, therapeutic hypothermia, neuroprotection, neurorehabilitation, and consideration of drowning in advances made in treatment of other central nervous system disorders.
PMCID: PMC3677166  PMID: 22956050
Drowning; Brain; Asphyxia; Cardiac arrest
24.  Relationship Between Hyperglycemia and Outcome in Children with Severe Traumatic Brain Injury 
To determine the relationship between hyperglycemia and outcome in infants and children after severe traumatic brain injury (TBI)
Retrospective review of a prospectively-collected Pediatric Neurotrauma Registry
Setting and Patients
Children admitted after severe TBI (post-resuscitation GCS ≤ 8) were studied (1999 – 2004). A subset of children (n = 28) were concurrently enrolled in a randomized, controlled clinical trial of early hypothermia for neuroprotection
Demographic data, serum glucose concentrations and outcome assessments were collected
Methods and Main Results
Children (n = 57) were treated with a standard TBI protocol. Exogenous glucose was withheld for 48 h after injury unless hypoglycemia was observed (blood glucose < 70 mg/dl). Early (first 48 h) and Late (49 - 168 h) time periods were defined and mean blood glucose concentrations were calculated. Additionally, children were categorized based on peak blood glucose concentrations during each time period (normal (NG) – blood glucose < 150 mg/dl; mild hyperglycemia (MHG) – blood glucose ≤ 200 mg/dl; severe hyperglycemia (SHG) – blood glucose > 200 mg/dl). In the Late period, an association between elevated mean serum glucose concentration and outcome was observed (133.5 ± 5.6 mg/dl in the unfavorable group vs. 115.4 ± 4.1 mg/dl in favorable group, p = 0.02). This association continued to be significant after correcting for injury severity, age, and exposure to insulin (p = 0.03). Similarly, in the Late period, children within the SHG had decreased incidence of good outcome compared to children within the other glycemic groups (% good outcome: NG – 61.9%, MHG – 73.7%, SHG – 33.3%; p = 0.05). However, when adjusted for exposure to insulin, this relationship was no longer statistically significant.
In children with severe TBI, hyperglycemia beyond the initial 48 hours is associated with poor outcome. This relationship was observed in both our analysis of mean blood glucose concentrations as well as amongst the patients with episodic severe hyperglycemia. This observation suggests a relationship between hyperglycemia and outcome from TBI. However, only a prospective study can answer the important question, whether manipulating serum glucose concentration can improve outcome after TBI in children
PMCID: PMC3677026  PMID: 21499170
To compare the correlation of intracranial pressure (ICP) measurement and time to detection of ICP crises (defined as ICP ≥ 20 mm Hg for ≥ 5 min) between an intraparenchymal (IP) monitor and external ventricular drain (EVD) in children where continuous cerebrospinal fluid (CSF) diversion was used as a therapy for severe traumatic brain injury (TBI).
Academic, pediatric intensive care unit.
Retrospective review of a prospectively-collected Pediatric Neurotrauma database.
Children with severe TBI (GCS ≤ 8) who underwent ICP monitoring with both IP and EVD techniques were studied. In Cohort 1 (n = 58), hourly ICP measurements were extracted from the medical record. In Cohort 2 (n = 4), ICP measurements were collected every minute by an automated data collection system.
Measurements and Main Results
The mean absolute difference in ICP (|ICP|) and intraclass correlation coefficients (ICC) were calculated. Timing to detection of ICP crises was analyzed. Data expressed as mean ± SEM. In cohort 1, 7,387 hours of data were analyzed and 399 hours (23,940 min) were analyzed in Cohort 2. In Cohort 1, |ICP| = 3.10 ± 0.04 mm Hg (ICC = 0.98, p < 0.001). |ICP| in Cohort 2 was 3.30 ± 0.05 mm Hg (ICC = 0.98, p < 0.001). In Cohort 2, a total of 75 ICP crises were observed. Fifty-five (73%) were detected first by the IP monitor, of which 35 were not identified by the EVD monitor. Time between IP and EVD detection of a crisis was 12.60 ± 2.34 min.
EVD and IP measurements of ICP were highly correlated, although intermittent EVD ICP measurements may fail to identify ICP events when continuously draining CSF. In institutions using continuous CSF diversion as a therapy, a two-monitor system may be valuable for accomplishing monitoring and therapeutic goals.
PMCID: PMC3670608  PMID: 20625341

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