The dentate gyrus (DG) is a critical entry point regulating function of the hippocampus. Integral to this role are the sparse, selective activation characteristics of the principal cells of the DG, dentate granule cells (DGCs). This sparse activation is important both in cognitive processing, and in regulation of pathological activity in disease states. Using a novel, combined dynamic imaging approach capable of resolving sequentially both synaptic potentials and action potential firing in large populations of DGCs, we characterized the postnatal development of firing properties of DG neurons in response to afferent activation in mouse hippocampal-entorhinal cortical slices. During postnatal development, there was a protracted, progressive sparsification of responses, accompanied by increased temporal precision of activation. Both of these phenomena were primarily mediated by changes in local circuit inhibition, and not by alterations in afferent innervation of DGCs, since GABAA antagonists normalized developmental differences. There was significant theta and gamma frequency-dependent synaptic recruitment of DGC activation in adult, but not developing, animals. Finally, we found that the decision to fire or not fire by individual DGCs was robust and repeatable at all stages of development. The protracted postnatal development of sparse, selective firing properties, increased temporal precision and frequency dependence of activation, and the fidelity with which the decision to fire is made are all fundamental circuit determinants of DGC excitation, critical in both normal and pathological function of the DG.
Calcium Imaging; Two-Photon Microscopy; Voltage Sensitive Dye; Confocal Microscopy; Hippocampus; Dentate; Granule Cells
Sodium bicarbonate (NaHCO3) is a common treatment for metabolic acidemia, however little definitive information exists regarding its treatment efficacy and cerebral hemodynamic effects. This pilot observational study quantifies relative changes in cerebral blood flow (rCBF) and oxy and deoxy-hemoglobin concentrations (ΔHbO2 and ΔHb) due to bolus administration of NaHCO3 in patients with mild base deficits.
Infants and children with hypoplastic left heart syndrome (HLHS) were recruited prior to cardiac surgery. NaHCO3 was given as needed for treatment of base deficit. Diffuse optical spectroscopies were employed for 15 minutes post-injection to non-invasively monitor ΔHb, ΔHbO2 and rCBF relative to baseline prior to NaHCO3 administration.
Twenty-two anesthetized and mechanically ventilated HLHS patients (1 day to 4 years old) received a median (interquartile range) dose of 1.1 (0.8, 1.8) mEq/kg NaHCO3 administered intravenously over 10–20 seconds to treat a base deficit of −4 (−6, −3) mEq/l. NaHCO3 caused significant dose-dependent increases in rCBF, however population averaged ΔHb or Δ4HbO2 compared to controls were not significant.
Dose-dependent increases in cerebral blood flow (CBF) caused by bolus NaHCO3 are an important consideration in vulnerable populations wherein risk of rapid CBF fluctuations does not outweigh the benefit of treating a base deficit.
Striatal-enriched tyrosine phosphatase (STEP) has been identified as a component of physiological and pathophysiological signaling pathways mediated by N-methyl-d-aspartate (NMDA) receptor/calcineurin/calpain activation. Activation of these pathways produces a subsequent change in STEP isoform expression or activation via dephosphorylation. In this study, we evaluated changes in STEP phosphorylation and proteolysis in dissociated cortical neurons after sublethal and lethal mechanical injury using an in vitro stretch injury device. Sublethal stretch injury produces minimal changes in STEP phosphorylation at early time points, and increased STEP phosphorylation at 24 h that is blocked by the NMDA-receptor antagonist APV, the calcineurin-inhibitor FK506, and the sodium channel blocker tetrodotoxin. Lethal stretch injury produces rapid STEP dephosphorylation via NR2B-containing NMDA receptors, but not calcineurin, and a subsequent biphasic phosphorylation pattern. STEP61 expression progressively increases after sublethal stretch with no change in calpain-mediated STEP33 formation, while lethal stretch injury results in STEP33 formation via a NR2B-containing NMDA receptor pathway within 1 h of injury. Blocking calpain activation in the initial 30 min after stretch injury increases the ratio of active STEP in cells and blocks STEP33 formation, suggesting that STEP is an early substrate of calpain after mechanical injury. There is a strong correlation between the amount of STEP33 formed and the degree of cell death observed after lethal stretch injury. In summary, these data demonstrate that previously characterized pathways of STEP regulation via the NMDA receptor are generally conserved in mechanical injury, and suggest that calpain-mediated cleavage of STEP33 should be further examined as an early marker of neuronal fate after stretch injury.
altered signal transduction; in vitro studies; neural injury; traumatic brain injury
Background and Objective
Photodynamic therapy (PDT) of thoracic malignancies involving the pleural surfaces is an active area of clinical investigation. The present report aims to characterize a model for PDT of disseminated non-small cell lung carcinoma grown orthotopically in nude mice, and to evaluate PDT effect on tumor and normal tissues.
H460 human non-small cell lung carcinoma (NSCLC) cells were injected percutaneously into the thoracic cavity of nude mice. HPPH-PDT (1 mg/kg, 24 h) was performed via the interstitial delivery (150 mW/cm) of 661 nm light to the thoracic cavity at fluences of 25-200 J/cm.
H460 tumors exhibited exponential growth within the thoracic cavity consisting of diffuse, gross nodular disease within 9 days after intrathoracic injection. Tumor volume, measured by magnetic resonance imaging (MRI), was highly correlated with the aggregate tumor mass extracted from the corresponding animal. Intrathoracic PDT at fluences of ≥ 50 J/cm produced significant decreases in tumor burden as compared to untreated controls, however mortality increased with rising fluence. Accordingly, 50 J/cm was selected for MRI studies to measure intra-animal PDT effects. Tumor distribution favored the ventral (vs. dorsal), caudal (vs. cranial), and right (vs. left) sides of the thoracic cavity by MRI; PDT did not change this spatial pattern despite an overall effect on tumor burden. Histopathology revealed edema and fibrin deposition within the pulmonary interstitium and alveoli of the PDT-treated thoracic cavity, as well as occasional evidence of vascular disruption. Prominent neutrophil infiltration with a concomitant decline in the lymphocyte compartment was also noted in the lung parenchyma within 24 hours after PDT.
HPPH-PDT of an orthotopic model of disseminated NSCLC is both feasible and effective using intracavitary light delivery. We establish this animal model, together with the treatment and monitoring approaches, as novel and valuable methods for the pre-clinical investigation of intrathoracic PDT of disseminated pleural malignancies.
HPPH; Photochlor®; 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a; interstitial illumination; magnetic resonance imaging; non-small cell lung carcinoma; photodynamic therapy; pleural malignancy
Polyalanine (poly-A) tracts exist in 494 annotated proteins; to date, expansions in these tracts have been associated with nine human diseases. The pathogenetic mechanism by which a poly-A tract results in these various human disorders remains uncertain. To understand the role of this mutation type, we investigated the change in functional properties of the transcription factor Arx when it has an expanded poly-A tract (ArxE), a mutation associated with infantile spasms and intellectual disabilities in humans. We found that although ArxE functions normally in the dorsal brain, its function in subpallial-derived populations of neurons is compromised. These contrasting functions are associated with the misregulation of Arx targets through the loss of the ability of ArxE to interact with the Arx cofactor Tle1. Our data demonstrate a novel mechanism for poly-A expansion diseases: the misregulation of a subset of target genes normally regulated by a transcription factor.
Early allograft dysfunction (EAD) occurring in the first week post-liver transplantation is associated with increased graft failure and mortality and is believed to be largely due to ischemia/reperfusion injury. We anticipated that the presence of EAD would be reflected by alterations in expression of serum proteins associated with an inflammatory response in the peri-operative period, and hypothesized that a specific pattern of expression might correlate with the development of EAD. The serum levels of 25 cytokines, chemokines, and immunoreceptors were measured by Luminex multiplex assays pre- and post-liver transplantation. Levels of each cytokine biomarker were compared in adult recipients with or without EAD at serial time points using samples collected pre-operatively and at 1, 7, 14, and 30 days post-transplant. EAD was defined according to standard criteria as maximum alanine transferase (ALT) or aspartate transferase (AST) levels on days 1–7 of >2000 U/ml, day 7 bilirubin level ≥10 mg/dl, or a day 7 international normalized ratio (INR) ≥1.7. Multivariable analyses showed that patients experiencing EAD had lower pre-operative IL-6 and higher IL-2R levels. Patients with EAD also showed higher MCP-1 (CCL2), IL-8 (CXCL8), and RANTES (CCL5) chemokine levels in the early post-operative period, suggesting up-regulation of the NF-κB pathway, in addition to higher levels of chemokines and cytokines associated with T cell immunity, including Mig (CXCL9), IP-10 (CXCL10) and IL-2R. These findings identify several possible biomarkers and pathways associated with EAD, that may guide future validation studies and investigation of specific cellular and molecular mechanisms of graft dysfunction. Furthermore, if validated, our findings may contribute to perioperative prediction of the occurrence of EAD and ultimately lead to identification of potential interventional therapies.
immune monitoring; multiplex analysis; chemokines; immunobiology
Computational studies have suggested that stochastic, deterministic, and mixed processes all could be possible determinants of spontaneous, synchronous network bursts. In the present study, utilizing multicellular calcium imaging coupled with fast confocal microscopy, we describe neuronal behavior underlying spontaneous network bursts in developing rat and mouse hippocampal area CA3 networks. Two primary burst types were studied: giant depolarizing potentials (GDPs) and spontaneous interictal bursts recorded in bicuculline, a GABAA receptor antagonist. Analysis of the simultaneous behavior of multiple CA3 neurons during synchronous GDPs revealed a repeatable activation order from burst to burst. This was validated using several statistical methods, including high Kendall’s W values for firing order during GDPs, high Pearson’s correlations of cellular activation times between burst pairs, and latent class analysis, which revealed a population of 5-6% of CA3 neurons reliably firing very early during GDPs. In contrast, neuronal firing order during interictal bursts appeared homogenous, with no particular cells repeatedly leading or lagging during these synchronous events. We conclude that GDPs activate via a deterministic mechanism, with distinct, repeatable roles for subsets of neurons during burst generation, while interictal bursts appear to be stochastic events with cells assuming interchangeable roles in the generation of these events.
Calcium Imaging; Fast Confocal Microscopy; Network Bursts; Hippocampus; CA3 Neurons; Latent Class Model Analysis
Leukocyte infiltration of adipose is a critical determinant of obesity-related metabolic diseases. Fractalkine (CX3CL1) and its receptor (CX3CR1) comprise a chemokine system involved in leukocyte recruitment and adhesion in atherosclerosis, but its role in adipose inflammation and type 2 diabetes is unknown.
RESEARCH DESIGN AND METHODS
CX3CL1 mRNA and protein were quantified in subcutaneous adipose and blood during experimental human endotoxemia and in lean and obese human adipose. CX3CL1 cellular source was probed in human adipocytes, monocytes, and macrophages, and CX3CL1-blocking antibodies were used to assess its role in monocyte-adipocyte adhesion. The association of genetic variation in CX3CR1 with metabolic traits was determined in a community-based sample. Finally, plasma CX3CL1 levels were measured in a case-control study of type 2 diabetes.
Endotoxemia induced adipose CX3CL1 mRNA (32.7-fold, P < 1 × 10−5) and protein (43-fold, P = 0.006). Obese subjects had higher CX3CL1 levels in subcutaneous adipose compared with lean (0.420 ± 0.387 vs. 0.228 ± 0.187 ng/mL, P = 0.04). CX3CL1 was expressed and secreted by human adipocytes and stromal vascular cells. Inflammatory cytokine induction of CX3CL1 in human adipocytes (27.5-fold mRNA and threefold protein) was completely attenuated by pretreatment with a peroxisome proliferator–activated receptor-γ agonist. A putative functional nonsynonymous single nucleotide polymorphism (rs3732378) in CX3CR1 was associated with adipose and metabolic traits, and plasma CX3CL1 levels were increased in patients with type 2 diabetes vs. nondiabetics (0.506 ± 0.262 vs. 0.422 ± 0.210 ng/mL, P < 0.0001).
CX3CL1-CX3CR1 is a novel inflammatory adipose chemokine system that modulates monocyte adhesion to adipocytes and is associated with obesity, insulin resistance, and type 2 diabetes. These data provide support for CX3CL1 as a diagnostic and therapeutic target in cardiometabolic disease.
Pathologic stresses induce heart failure in animal models through activation of multiple cardiac transcription factors (TFs) working cooperatively. However, interactions among TFs in human heart failure are less well understood. Here we use genomic data to examine the evidence that five candidate TF families co-regulate gene expression in human heart failure.
Methods and Results
RNA isolates from failing (n=86) and non-failing (n=16) human hearts were hybridized with Affymetrix HU133A arrays. For each gene on the array, we determined conserved MEF2, NFAT, NKX, GATA, and FOX binding motifs within the −1 kb promoter region using human-murine sequence alignments and the TRANSFAC database. Across 9,076 genes expressed in the heart, TF binding motifs tended to cluster together in nonrandom patterns within promoters of specific genes (P-values ranging from 10−2 to 10−21), suggesting co-regulation. We then modeled differential expression as a function of TF combinations present in promoter regions. Several combinations predicted increased odds of differential expression in the failing heart, with highest odds ratios noted for genes containing both MEF2 and NFAT bindings motifs together in the same promoter (peak OR 3.47, P=0.005).
These findings provide genomic evidence for co-regulation of myocardial gene expression by MEF2 and NFAT in human heart failure. In doing so, they extend the paradigm of combinatorial regulation of gene expression to the human heart and identify new target genes for mechanistic study. More broadly, we demonstrate how integrating diverse sources of genomic data yields novel insights into human cardiovascular disorders.
heart failure; hypertrophy; remodeling; genes; transcription factors
In a quantitative trait locus (QTL) study, it is usually not feasible to select families with offspring that simultaneously display variability in more than one phenotype. When multiple phenotypes are of interest, the sample will, with high probability, contain ‘non-segregating’ families, i.e. families with both parents homozygous at the QTL. These families potentially reduce the power of regression-based methods to detect linkage. Moreover, follow-up studies in individual families will be inefficient, and potentially even misleading, if non-segregating families are selected for the study. Our work extends Haseman-Elston regression using a latent class model to account for the mixture of segregating and non-segregating families. We provide theoretical motivation for the method using an additive genetic model with two distinct functions of the phenotypic outcome, squared difference (SqD) and mean-corrected product (MCP). A permutation procedure is developed to test for linkage; simulation shows that the test is valid for both phenotypic functions. For rare alleles, the method provides increased power compared to a ‘marginal’ approach that ignores the two types of families; for more common alleles, the marginal approach has better power. These results appear to reflect the ability of the algorithm to accurately assign families to the two classes and the relative weights of segregating and non-segregating families to the test of linkage. An application of Bayes rule is used to estimate the family-specific probability of segregating. High predictive value positive values for segregating families, particularly for MCP, suggest that the method has considerable value for identifying segregating families. The method is illustrated for gene expression phenotypes measured on 27 candidate genes previously demonstrated to show linkage in a sample of 14 families.
Linkage; Heterogeneity; Haseman-Elston regression; Latent class analysis; Gene expression
Background & Aims
Pernicious anemia (PA) is characterized by vitamin B12 deficiency and achlorhydria, both of which have a detrimental effect on bone strength. The principle aim of this study was to determine the risk of hip fracture in patients with PA.
This is a retrospective cohort study using the General Practice Research Database (GPRD) from the United Kingdom. GPRD data from May 1987 until April 2002 were utilized to identify patients between 40 and 90 years of age at the time of GPRD enrollment. The exposed group contained patients with a diagnosis of PA being treated with vitamin B12 therapy. We matched each patient having a diagnosis of PA with 4 randomly selected non-PA patients with respect to age (+/− 1 year) and sex. Cox regression analysis was used to determine the hazard ratio (HR) for hip fracture associated with PA.
9,506 patients with a diagnosis of PA receiving vitamin B12 injection therapy were identified and compared to 38,024 controls. Patients with PA had a greater risk of hip fracture than the controls (HR 1.74, 95% CI 1.45–2.08). The increase in hip fracture risk was even more pronounced among those patients newly diagnosed with PA during GPRD follow-up (HR 2.63, 95% CI 2.03–3.41).
Patients with a diagnosis of PA have an elevated risk of hip fracture. The increased hip fracture risk was persistent even years after vitamin B12 therapy. Chronic achlorhydria could be the mechanism contributing to the persistently elevated hip fracture risk.
Pernicious anemia; vitamin b12; osteoporosis; hip fracture
Neuregulin-1 (NRG-1) is a paracrine factor released by microvascular endothelial cells that has cardioprotective effects in animal models of heart failure. However, circulating NRG-1 has not been studied in human heart disease. We used a novel immunoassay to test whether circulating neuregulin-1β (NRG-1β) is associated with disease severity and clinical outcome in chronic heart failure.
Methods and Results
Serum NRG-1β was quantified in 899 outpatients in the Penn Heart Failure Study, a referral cohort representing a broad spectrum of systolic heart failure. Circulating NRG-1β was significantly elevated in patients with worse disease severity (NYHA Class IV median 6.2 versus 4.4ng/ml for Class I, p=0.002). In adjusted models, NRG-1β was independently associated with an increased risk of death or cardiac transplantation over a median follow-up of 2.4 years (adjusted HR 1.58 [95% CI 1.04–2.39, p=0.03] comparing 4th versus 1st NRG-1β quartile). Associations with outcome differed by heart failure etiology and symptom severity, with the strongest associations observed in patients with ischemic cardiomyopathy (interaction p=0.008) and NYHA Class III/IV symptoms (interaction p=0.01). These findings were all independent of BNP, and assessment of NRG-1β and BNP jointly provided better risk stratification than each biomarker individually in patients with ischemic or NYHA Class III/IV heart failure.
Circulating NRG-1β is independently associated with heart failure severity and risk of death or cardiac transplantation. These findings support a role for NRG-1/ErbB signaling in human heart failure and identify serum NRG-1β as a novel biomarker that may have clinical applications.
Neuregulin; Heart Failure; Cardiomyopathy
We have developed a novel parallel-plate diffuse optical tomography (DOT) system for three-dimensional in vivo imaging of human breast tumor based on large optical data sets. Images of oxy-, deoxy-, total-hemoglobin concentration, blood oxygen saturation, and tissue scattering were reconstructed. Tumor margins were derived using the optical data with guidance from radiology reports and Magnetic Resonance Imaging. Tumor-to-normal ratios of these endogenous physiological parameters and an optical index were computed for 51 biopsy-proven lesions from 47 subjects. Malignant cancers (N=41) showed statistically significant higher total hemoglobin, oxy-hemoglobin concentration, and scattering compared to normal tissue. Furthermore, malignant lesions exhibited a two-fold average increase in optical index. The influence of core biopsy on DOT results was also explored; the difference between the malignant group measured before core biopsy and the group measured more than one week after core biopsy was not significant. Benign tumors (N=10) did not exhibit statistical significance in the tumor-to-normal ratios of any parameter. Optical index and tumor-to-normal ratios of total hemoglobin, oxy-hemoglobin concentration, and scattering exhibited high area under the receiver operating characteristic curve values from 0.90 to 0.99, suggesting good discriminatory power. The data demonstrate that benign and malignant lesions can be distinguished by quantitative three-dimensional DOT.
Breast Cancer; Diffuse Optical Tomography; Near Infrared Light; Photon Migration; Optical Mammography
“Diffuse correlation spectroscopy” (DCS) is a technology for non-invasive transcranial measurement of cerebral blood flow (CBF) that can be hybridized with “near-infrared spectroscopy” (NIRS). Taken together these methods hold potential for monitoring hemodynamics in stroke patients. We explore the utility of DCS and NIRS to measure effects of head-of-bed (HOB) positioning at 30°, 15°, 0°, −5° and 0° angles in patients with acute ischemic stroke affecting frontal cortex and in controls. HOB positioning significantly altered CBF, oxy-hemoglobin (HbO2) and total-hemoglobin (THC) concentrations. Moreover, the presence of an ipsilateral infarct was a significant effect for all parameters. Results are consistent with the notion of impaired CBF autoregulation in the infarcted hemisphere.
Four very low birth weight, very premature infants were monitored during a 12° postural elevation using diffuse correlation spectroscopy (DCS) to measure microvascular cerebral blood flow (CBF) and transcranial Doppler ultrasound (TCD) to measure macrovascular blood flow velocity in the middle cerebral artery. DCS data correlated significantly with peak systolic, end diastolic, and mean velocities measured by TCD (pA =0.036, 0.036, 0.047). Moreover, population averaged TCD and DCS data yielded no significant hemodynamic response to this postural change (p>0.05). We thus demonstrate feasibility of DCS in this population, we show correlation between absolute measures of blood flow from DCS and blood flow velocity from TCD, and we do not detect significant changes in CBF associated with a small postural change (12°) in these patients.
Heterogeneity poses a challenge to linkage mapping. Here, we apply a latent class extension of Haseman-Elston regression to expression phenotypes with significant evidence of linkage to trans regulators in 14 large pedigrees. We test for linkage, accounting for heterogeneity, and classify individual families as "linked" and "unlinked" on the basis of their contribution to the overall evidence of linkage.
Fluctuations in tumor blood flow are common and attributed to factors such as vasomotion or local vascular structure, yet, because vessel structure and physiology are host-derived, animal strain of tumor propagation may further determine blood flow characteristics. In the present report, baseline and stress-altered tumor hemodynamics as a function of murine strain were studied using radiation-induced fibrosacomas (RIF) grown in C3H or nude mice. Fluctuations in tumor blood flow during one hour of baseline monitoring or during vascular stress induced by photodynamic therapy (PDT) were measured by diffuse correlation spectroscopy. Baseline monitoring revealed fluctuating tumor blood flow highly correlated with heart rate and with similar median periods (i.e., ∼9 and 14 min in C3H and nudes, respectively). However, tumor blood flow in C3H animals was more sensitive to physiologic or stress-induced perturbations. Specifically, PDT-induced vascular insults produced greater decreases in blood flow in the tumors of C3H versus nude mice; similarly, during baseline monitoring, fluctuations in blood flow were more regular and more prevalent within the tumors of C3H mice versus nude mice; finally, the vasoconstrictor L-NNA reduced tumor blood flow in C3H mice but did not affect tumor blood flow in nudes. Underlying differences in vascular structure, such as smaller tumor blood vessels in C3H versus nude animals, may contribute to strain-dependent variation in vascular function. These data thus identify clear effects of mouse strain on tumor hemodynamics with consequences to PDT and potentially other vascular-mediated therapies.