Metals such as copper disrupt olfactory function in fish. Unfortunately, little is understood of the molecular consequences of copper olfactory impairment, thus hindering the development of relevant diagnostic tools of olfactory injury. To address this critical data gap, we analyzed gene expression within olfactory tissues of adult zebrafish exposed to CuCl2 (6, 16, 40 ppb) for 24 hrs. Transcriptional markers of copper impairment within the entire olfactory system were identified and specific genes of interest (e.g. S100a, parvalbumin 8, olfactory marker protein, and calbindin 2-like protein) were confirmed with quantitative real-time PCR. In addition, we performed gene set analysis (GSA) using both a-priori and custom pathways of gene sets specifically targeting the olfactory signal transduction (OST) pathway. These analyses revealed down-regulated gene sets related to calcium channels and ion transport, g-proteins, and olfactory receptors. Collectively, these data demonstrate that copper causes a depression of transcription of key genes within the OST pathway and elsewhere within olfactory tissues, likely resulting in an olfactory system less responsive to odorants. Further, these data provide a mechanistic explanation in support of earlier studies of functional olfactory impairment in fish following copper exposure.
Two screening methods for urine microbiology are proposed: one in which the Gram-stained smear is used to detect significant bacteriuria, and another in which Autobac antibiotic susceptibility tests are performed directly on positive urine samples. Results on 1,350 specimens indicated that an average of 18 bacteria per oil immersion field were observed in the urine of patients with significant bacteriuria, and an average of less than 1 bacterium per oil immersion field was found in the urine of patients without significant bacteriuria. Direct susceptibility testing by Autobac proved to be rapid (3 h versus 24 h) and reliable (0.5 to 1.2% discrepancies).
Trimethyltin chloride (TMT) is an organotin contaminant, widely detected in aqueous environments, posing potential human and environmental risks. In this study, we utilized the zebrafish model to investigate the impact of transient TMT exposure on developmental progression, angiogenesis, and cardiovascular development. Embryos were waterborne exposed to a wide TMT concentration range from 8 to 96 hours post fertilization (hpf). The TMT concentration that led to mortality in 50% of the embryos (LC50) at 96 hpf was 8.2 μM; malformations in 50% of the embryos (EC50) was 2.8 μM. The predominant response observed in surviving embryos was pericardial edema. Additionally, using the Tg (fli1a: EGFP) y1 transgenic zebrafish line to non-invasively monitor vascular development, TMT exposure led to distinct disarrangements in the vascular system. The most susceptible developmental stage to TMT exposure was between 48–72 hpf. High density whole genome microarrays were used to identify the early transcriptional changes following TMT exposure from 48 to 60 hpf or 72 hpf. In total, 459 transcripts were differentially expressed at least 2-fold (P < 0.05) by TMT compared to control. Using Ingenuity Pathway Analysis (IPA) tools, it was revealed that the transcripts misregulated by TMT exposure were clustered in numerous categories including metabolic and cardiovascular disease, cellular function, cell death, molecular transport, and physiological development. In situ localization of highly elevated transcripts revealed intense staining of ADP-ribosylation factors arf3 and arf5 in the head, trunk, and tail regions. When arf5 expression was blocked by morpholinos, the zebrafish did not display the prototypical TMT-induced vascular deficits, indicating that the induction of arf5 was necessary for TMT-induced vascular toxicity.
zebrafish; trimethyltin chloride; vascular; ADP-ribosylation factors (arf); gene expression
Small-molecule CCR5 antagonists, such as maraviroc (MVC), likely block HIV-1 through an allosteric, noncompetitive inhibition mechanism, whereas inhibition by agonists such as PSC-RANTES is less defined and may involve receptor removal by cell surface downregulation, competitive inhibition by occluding the HIV-1 envelope binding, and/or allosteric effects by altering CCR5 conformation. We explored the inhibitory mechanisms of maraviroc and PSC-RANTES by employing pairs of virus clones with differential sensitivities to these inhibitors. Intrinsic PSC-RANTES-resistant virus (YA versus RT) or those selected in PSC-RANTES treated macaques (M584 versus P3-4) only displayed resistance in multiple-cycle assays or with a CCR5 mutant that cannot be downregulated. In single-cycle assays, these HIV-1 clones displayed equal sensitivity to PSC-RANTES inhibition, suggesting effective receptor downregulation. Prolonged PSC-RANTES exposure resulted in desensitization of the receptor to internalization such that increasing virus concentration (substrate) could saturate the receptors and overcome PSC-RANTES inhibition. In contrast, resistance to MVC was observed with the MVC-resistant HIV-1 (R3 versus S2) in both multiple- and single-cycle assays and with altered virus concentrations, which is indicative of allosteric inhibition. MVC could also mediate inhibition and possibly resistance through competitive mechanisms.
The polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), was compared to dibenzo[def,p]chrysene (DBC) and combinations of three environmental PAH mixtures (coal tar, diesel particulate and cigarette smoke condensate) using a two stage, FVB/N mouse skin tumor model. DBC (4 nmol) was most potent, reaching 100% tumor incidence with a shorter latency to tumor formation, less than 20 weeks of 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion compared to all other treatments. Multiplicity was 4 times greater than BaP (400 nmol). Both PAHs produced primarily papillomas followed by squamous cell carcinoma and carcinoma in situ. Diesel particulate extract (1 mg SRM 1650b; mix 1) did not differ from toluene controls and failed to elicit a carcinogenic response. Addition of coal tar extract (1 mg SRM 1597a; mix 2) produced a response similar to BaP. Further addition of 2 mg of cigarette smoke condensate (mix 3) did not alter the response with mix 2. PAH-DNA adducts measured in epidermis 12 h post initiation and analyzed by 32P post- labeling, did not correlate with tumor incidence. PAH- dependent alteration in transcriptome of skin 12 h post initiation was assessed by microarray. Principal component analysis (sum of all treatments) of the 922 significantly altered genes (p<0.05), showed DBC and BaP to cluster distinct from PAH mixtures and each other. BaP and mixtures up-regulated phase 1 and 2 metabolizing enzymes while DBC did not. The carcinogenicity with DBC and two of the mixtures was much greater than would be predicted based on published Relative Potency Factors (RPFs).
PAHs; Cyp1a1; Cyp1b1; adducts; relative potency factor; skin1
We investigated the phenomenon of incomplete wetting of a high energy liquid subphase by drops of pure amphiphilic molecules as well as drops of amphiphile solutions which are immiscible with the subphase. We show that amphiphiles escape across the contact line of the drop, move on the subphase/vapor interface and form a submonolayer or full monolayer external to the drop. If this monolayer is sufficiently dense, it can reduce the surface tension of the subphase, raise the contact angle of the drop and prevent the drop from fully wetting the subphase. This phenomenon is called autophobing and has been extensively studied on solid substrates. For the liquid subphase studied here, we measure the surface tensions of the three relevant interfaces before and after the drop is deposited. The measured surface tension external to the drop shows that amphiphiles can move across the contact line and form a monolayer outside of the drop. In some cases, at equilibrium, the monolayer is in a sufficiently packed state to create the nonwetting condition. In other cases, at equilibrium the monolayer density is insufficient to lower the surface tension enough to achieve the nonwetting condition. Unlike on solid substrates where the formation of the monolayer external to the drop is kinetically hindered, the amphiphiles can move rapidly across the liquid subphase by Marangoni driven surface transport and local equilbirum is achieved. However, because the amphiphile inventory and subphase area are limited, the achievement of autophobing on a liquid subphase depends not only on the instrinsic subphase/amphiphile interaction but also on the total amphiphile inventory and area of the liquid subphase.
The severe acute respiratory syndrome coronavirus accessory protein ORF6 antagonizes interferon signaling by blocking karyopherin-mediated nuclear import processes. Viral nuclear import antagonists, expressed by several highly pathogenic RNA viruses, likely mediate pleiotropic effects on host gene expression, presumably interfering with transcription factors, cytokines, hormones, and/or signaling cascades that occur in response to infection. By bioinformatic and systems biology approaches, we evaluated the impact of nuclear import antagonism on host expression networks by using human lung epithelial cells infected with either wild-type virus or a mutant that does not express ORF6 protein. Microarray analysis revealed significant changes in differential gene expression, with approximately twice as many upregulated genes in the mutant virus samples by 48 h postinfection, despite identical viral titers. Our data demonstrated that ORF6 protein expression attenuates the activity of numerous karyopherin-dependent host transcription factors (VDR, CREB1, SMAD4, p53, EpasI, and Oct3/4) that are critical for establishing antiviral responses and regulating key host responses during virus infection. Results were confirmed by proteomic and chromatin immunoprecipitation assay analyses and in parallel microarray studies using infected primary human airway epithelial cell cultures. The data strongly support the hypothesis that viral antagonists of nuclear import actively manipulate host responses in specific hierarchical patterns, contributing to the viral pathogenic potential in vivo. Importantly, these studies and modeling approaches not only provide templates for evaluating virus antagonism of nuclear import processes but also can reveal candidate cellular genes and pathways that may significantly influence disease outcomes following severe acute respiratory syndrome coronavirus infection in vivo.
Aerodynamic forces provide the primary means of distributing aerosol medications within the lungs. Partial airway obstructions can limit both air flow and aerosol penetration into diseased zones. We hypothesize that low surface tension additives may help to disperse aerosol medications after deposition in the airways, improving dose uniformity and drug delivery to underventilated regions. To test this, we performed a pilot scintigraphy study of surfactant and saline deposition and postdeposition dispersion.
Because inhaled antibiotics for cystic fibrosis provide an example of where self-dispersing medications may be useful, we administered calfactant and saline aerosols with added Technetium 99m sulfur colloid (Tc-SC; 100 nm filtered) on different days in randomized order to eight cystic fibrosis (CF) subjects (average FEV1%, p=85±12%). Nebulized delivery was matched (similar aerosol sizes and volume delivery rates, fixed breathing patterns). Tc-SC distribution in the lungs was imaged continuously for 30 min after delivery.
Both aerosols were well tolerated. Aerosol distribution was mostly peripheral (58/42%) and initially similar for saline and surfactant. Changes in whole lung counts after 30 min were also similar. Peripheral lung activity decreased more rapidly on average with calfactant though the difference versus saline was not statistically significant. Central to peripheral count ratio decreased with saline and increased with calfactant and c/p changes approached significance (−0.05±0.16 vs. 0.10±0.10; p=0.07 Wilcoxon).
Our results lack statistical significance, but suggest that inhaled calfactant increased peripheral clearance, due to either surfactant-based dispersion or mucociliary effects. Further studies are needed to define the potential for low surface tension carriers to improve drug delivery.
calfactant; cystic fibrosis; aerosol deposition; scintigraphy; surface tension
Like many diseases, diabetic nephropathy is defined in a histopathological context and studied using reductionist approaches that attempt to ameliorate structural changes. Novel technologies in mass spectrometry-based proteomics have the ability to provide a deeper understanding of the disease beyond classical histopathology, redefine the characteristics of the disease state, and identify novel approaches to reduce renal failure. The goal is to translate these new definitions into improved patient outcomes through diagnostic, prognostic and therapeutic tools. Here, we review progress made in studying the proteomics of diabetic nephropathy and provide an introduction to the informatics tools used in the analysis of systems biology data, while pointing out statistical issues for consideration. Novel bioinformatics methods may increase biomarker identification, and other tools, including selective reaction monitoring, may hasten clinical validation.
bioinformatics; diabetes; diabetic nephropathy; mass spectrometry; proteomics; sytems biology
Influenza infection causes respiratory disease that can lead to death. The complex interplay between virus-encoded and host-specific pathogenicity regulators – and the relative contributions of each toward viral pathogenicity – is not well-understood.
By analyzing a collection of lung samples from mice infected by A/Vietnam/1203/2004 (H5N1; VN1203), we characterized a signature of transcripts and proteins associated with the kinetics of the host response. Using a new geometrical representation method and two criteria, we show that inoculation concentrations and four specific mutations in VN1203 mainly impact the magnitude and velocity of the host response kinetics, rather than specific sets of up- and down- regulated genes. We observed analogous kinetic effects using lung samples from mice infected with A/California/04/2009 (H1N1), and we show that these effects correlate with morbidity and viral titer.
We have demonstrated the importance of the kinetics of the host response to H5N1 pathogenesis and its relationship with clinical disease severity and virus replication. These kinetic properties imply that time-matched comparisons of ‘omics profiles to viral infections give limited views to differentiate host-responses. Moreover, these results demonstrate that a fast activation of the host-response at the earliest time points post-infection is critical for protective mechanisms against fast replicating viruses.
Influenza; Host; Response; Kinetics; Magnitude; Velocity; Transcriptomics; Proteomics; Multidimensional; Scaling
Respiratory infections stemming from influenza viruses and the Severe Acute Respiratory Syndrome corona virus (SARS-CoV) represent a serious public health threat as emerging pandemics. Despite efforts to identify the critical interactions of these viruses with host machinery, the key regulatory events that lead to disease pathology remain poorly targeted with therapeutics. Here we implement an integrated network interrogation approach, in which proteome and transcriptome datasets from infection of both viruses in human lung epithelial cells are utilized to predict regulatory genes involved in the host response. We take advantage of a novel “crowd-based” approach to identify and combine ranking metrics that isolate genes/proteins likely related to the pathogenicity of SARS-CoV and influenza virus. Subsequently, a multivariate regression model is used to compare predicted lung epithelial regulatory influences with data derived from other respiratory virus infection models. We predicted a small set of regulatory factors with conserved behavior for consideration as important components of viral pathogenesis that might also serve as therapeutic targets for intervention. Our results demonstrate the utility of integrating diverse ‘omic datasets to predict and prioritize regulatory features conserved across multiple pathogen infection models.
MicroRNAs (miRNAs) are noncoding RNAs that direct post-transcriptional regulation of protein coding genes. Recent studies have shown miRNAs are important for controlling many biological processes, including nervous system development, and are highly conserved across species. Given their importance, computational tools are necessary for analysis, interpretation and integration of high-throughput (HTP) miRNA data in an increasing number of model species. The Bioinformatics Resource Manager (BRM) v2.3 is a software environment for data management, mining, integration and functional annotation of HTP biological data. In this study, we report recent updates to BRM for miRNA data analysis and cross-species comparisons across datasets.
BRM v2.3 has the capability to query predicted miRNA targets from multiple databases, retrieve potential regulatory miRNAs for known genes, integrate experimentally derived miRNA and mRNA datasets, perform ortholog mapping across species, and retrieve annotation and cross-reference identifiers for an expanded number of species. Here we use BRM to show that developmental exposure of zebrafish to 30 uM nicotine from 6–48 hours post fertilization (hpf) results in behavioral hyperactivity in larval zebrafish and alteration of putative miRNA gene targets in whole embryos at developmental stages that encompass early neurogenesis. We show typical workflows for using BRM to integrate experimental zebrafish miRNA and mRNA microarray datasets with example retrievals for zebrafish, including pathway annotation and mapping to human ortholog. Functional analysis of differentially regulated (p<0.05) gene targets in BRM indicates that nicotine exposure disrupts genes involved in neurogenesis, possibly through misregulation of nicotine-sensitive miRNAs.
BRM provides the ability to mine complex data for identification of candidate miRNAs or pathways that drive phenotypic outcome and, therefore, is a useful hypothesis generation tool for systems biology. The miRNA workflow in BRM allows for efficient processing of multiple miRNA and mRNA datasets in a single software environment with the added capability to interact with public data sources and visual analytic tools for HTP data analysis at a systems level. BRM is developed using Java™ and other open-source technologies for free distribution (http://www.sysbio.org/dataresources/brm.stm).
Systems biology; Genomics; MicroRNA; Bioinformatics; Zebrafish
Given the increasing prevalence of diabetes and the lack of patients reaching recommended therapeutic goals, novel models of team-based care are emerging. These teams typically include a combination of physicians, nurses, case managers, pharmacists, and community-based peer health promoters (HPs). Recent evidence supports the role of pharmacists in diabetes management to improve glycemic control, as they offer expertise in medication management with the ability to collaboratively intensify therapy. However, few studies of pharmacy-based models of care have focused on low income, minority populations that are most in need of intervention. Alternatively, HP interventions have focused largely upon low income minority groups, addressing their unique psychosocial and environmental challenges in diabetes self-care. This study will evaluate the impact of HPs as a complement to pharmacist management in a randomized controlled trial.
The primary aim of this randomized trial is to evaluate the effectiveness of clinical pharmacists and HPs on diabetes behaviors (including healthy eating, physical activity, and medication adherence), hemoglobin A1c, blood pressure, and LDL-cholesterol levels. A total of 300 minority patients with uncontrolled diabetes from the University of Illinois Medical Center ambulatory network in Chicago will be randomized to either pharmacist management alone, or pharmacist management plus HP support. After one year, the pharmacist-only group will be intensified by the addition of HP support and maintenance will be assessed by phasing out HP support from the pharmacist plus HP group (crossover design). Outcomes will be evaluated at baseline, 6, 12, and 24 months. In addition, program and healthcare utilization data will be incorporated into cost and cost-effectiveness evaluations of pharmacist management with and without HP support.
The study will evaluate an innovative, integrated approach to chronic disease management in minorities with poorly controlled diabetes. The approach is comprised of clinic-based pharmacists and community-based health promoters collaborating together. They will target patient-level factors (e.g., lack of adherence to lifestyle modification and medications) and provider-level factors (e.g., clinical inertia) that contribute to poor clinical outcomes in diabetes. Importantly, the study design and analytic approach will help determine the differential and combined impact of adherence to lifestyle changes, medication, and intensification on clinical outcomes.
ClinicalTrials.gov identifier: NCT01498159
(3–10): Diabetes mellitus/drug therapy; Patient compliance; Patient education; Pharmacists; Community health workers
Diabetes and smoking are known risk factors for cataract development. In this study, we evaluated the effect of nicotine on the progression of cataracts in a type 1 diabetic rat model. Diabetes was induced in Sprague-Dawley rats by a single injection of 65 mg/kg streptozotocin. Daily nicotine injections were administered subcutaneously. Forty-five rats were divided into groups of diabetics with and without nicotine treatment and controls with and without nicotine treatment. Progression of lens opacity was monitored using a slit lamp biomicroscope and scores were assigned. To assess whether systemic inflammation played a role in mediating cataractogenesis, we studied serum levels of eotaxin, IL-6, and IL-4. The levels of the measured cytokines increased significantly in nicotine-treated and untreated diabetic animals versus controls and demonstrated a positive trend in the nicotine-treated diabetic rats. Our data suggest the presence of a synergistic relationship between nicotine and diabetes that accelerated cataract formation via inflammatory mediators.
Klotho is an antiaging hormone present in the kidney that extends the lifespan, regulates kidney function, and modulates cellular responses to oxidative stress. We investigated whether Klotho levels and signaling modulate inflammation in diabetic kidneys.
RESEARCH DESIGN AND METHODS
Renal Klotho expression was determined by quantitative real-time PCR and immunoblot analysis. Primary mouse tubular epithelial cells were treated with methylglyoxalated albumin, and Klotho expression and inflammatory cytokines were measured. Nuclear factor (NF)-κB activation was assessed by treating human embryonic kidney (HEK) 293 and HK-2 cells with tumor necrosis factor (TNF)-α in the presence or absence of Klotho, followed by immunoblot analysis to evaluate inhibitor of κB (IκB)α degradation, IκB kinase (IKK) and p38 activation, RelA nuclear translocation, and phosphorylation. A chromatin immunoprecipitation assay was performed to analyze the effects of Klotho signaling on interleukin-8 and monocyte chemoattractant protein-1 promoter recruitment of RelA and RelA serine (Ser)536.
Renal Klotho mRNA and protein were significantly decreased in db/db mice, and a similar decline was observed in the primary cultures of mouse tubule epithelial cells treated with methylglyoxal-modified albumin. The exogenous addition of soluble Klotho or overexpression of membranous Klotho in tissue culture suppressed NF-κB activation and subsequent production of inflammatory cytokines in response to TNF-α stimulation. Klotho specifically inhibited RelA Ser536 phosphorylation as well as promoter DNA binding of this phosphorylated form of RelA without affecting IKK-mediated IκBα degradation, total RelA nuclear translocation, and total RelA DNA binding.
These findings suggest that Klotho serves as an anti-inflammatory modulator, negatively regulating the production of NF-κB–linked inflammatory proteins via a mechanism that involves phosphorylation of Ser536 in the transactivation domain of RelA.
Reactive oxygen species (ROS) are generated as by-products of many cellular processes and can modulate cellular signaling pathways. However, high ROS levels are toxic; thus, intracellular ROS need to be tightly controlled. Therefore, cells use a group of antioxidant molecules and detoxifying enzymes that remove or detoxify reactive species. We found that the level of the antioxidant glutathione is greatly increased in human cytomegalovirus (HCMV)-infected cells due to activation of glutathione synthetic enzymes. In addition, our data suggest that virus-specific mechanisms are used to induce the expression of target antioxidant and detoxifying enzymes critical for the success of the infection. As a result of this virus-induced anti-ROS environment, key signaling kinases, such as the mammalian target of rapamycin (mTOR) kinase in mTOR complex 1 (mTORC1), are protected from inhibition by exogenous hydrogen peroxide (H2O2). In this regard, we found that phosphorylation of mTOR kinase at serine 2448 (suggested to be activating) was maintained during infection even under ROS stress conditions that inhibited it in uninfected cells. We also show that AMP-dependent kinase (AMPK)-mediated phosphorylation of serine 792 of raptor, the specificity subunit of mTORC1, increases in infected cells after H2O2 treatment. This phosphorylation is normally inhibitory for mTORC1. However, in infected cells this did not result in inhibition of mTORC1 activity, suggesting that inhibitory effects of raptor phosphorylation are circumvented. Overall, our data suggest that HCMV utilizes virus-specific mechanisms to activate a variety of means to protect the cell and mTORC1 from the effects of ROS.
Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4β7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4β7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4β7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4β7, CD4 or CCR5 more efficiently.
Most new HIV-1 infections worldwide are caused by the sexual transmission of subtype C viruses, which are prevalent in Asia and southern Africa. While chronically infected individuals harbor a genetically diverse set of viruses, most new infections are established by single variants, termed transmitted/founder (T/F) viruses. This raises the question whether certain viral variants have particular properties allowing them to more efficiently overcome the transmission bottleneck. Preferential binding of the viral envelope (Env) to the integrin α4β7 has been hypothesized as one important feature of transmitted viruses. Here, we compared Envs from subtype C viruses that were transmitted to those that were prevalent in chronic infections for efficiency in utilizing α4β7, CD4 and CCR5 for cell entry and replication. We found that transmitted and chronic Envs engaged CD4 and CCR5 with equal efficiency, and that blocking the interaction between Env and α4β7 failed to inhibit replication of T/F as well as control viruses. While the search for determinants of transmission fitness remains an important goal, preferential CD4, CCR5 or α4β7 interactions do not appear to represent distinguishing features of T/F viruses.
Hyperglycemia and inflammation are hallmarks of burn injury. In this study, we used a rat model of hyperglycemia and burn injury to investigate the effects of hyperglycemia on inflammatory responses in the liver. Hyperglycemia was induced in male Sprague-Dawley rats with streptozotocin (STZ) (35–40 mg/kg), followed by a 60% third-degree scald burn injury. Cytokine levels (by multiplex, in cytosolic liver extracts), hormones (by enzyme-linked immunosorbent assay [ELISA], in serum), nuclear factor (NF)-κB protein deoxyribonucleic acid (DNA) binding (by ELISA, in nuclear liver extracts) and liver functional panel (using VetScan, in serum) were measured at different time points up to 7 d after burn injury. Blood glucose significantly increased after burn injury in both groups with different temporal patterns. Hyperglycemic rats were capable of endogenous insulin secretion, which was enhanced significantly versus controls 12 h after burn injury. DNA binding data of liver nuclear extracts showed a robust and significant activation of the noncanonical NF-κB pathway in the hyperglycemic versus control burn animals, including increased NF-κB–inducing kinase expression (p < 0.05). Liver acute-phase proteins and cytokine expression were increased, whereas secretion of constitutive proteins was decreased after burn injury in hyperglycemic versus control animals (p < 0.05). These results indicate that burn injury to the skin rapidly activated canonical and noncanonical NF-κB pathways in the liver. Robust activation of the NF-κB noncanonical pathway was associated with increased expression of inflammatory markers and acute-phase proteins, and impaired glucose metabolism. Hyperglycemia is detrimental to burn outcome by augmenting inflammation mediated by hepatic noncanonical NF-κB pathway activation.
Surface tension gradient driven, or “Marangoni,” flow can be used to move exogenous fluid, either surfactant dispersions or drug carrying formulations, through the lung. In this paper, we investigate the spreading of aqueous solutions of water-soluble surfactants over entangled, aqueous mucin solutions that mimic the airway surface liquid of the lung. We measure the movement of the formulation by incorporating dyes into the formulation while we measure surface flows of the mucin solution subphase using tracer particles. Surface tension forces and/or Marangoni stresses initiate a convective spreading flow over this rheologically complex subphase. As expected, when the concentration of surfactant is reduced until its surface tension is above that of the mucin solution, the convective spreading does not occur. The convective spreading front moves ahead of the drop containing the formulation. Convective spreading ends with the solution confined to a well-defined static area which must be governed by a surface tension balance. Further motion of the spread solution progresses by much slower diffusive processes. Spreading behaviors are qualitatively similar for formulations based on anionic, cationic, or nonionic surfactants, containing either hydrophilic or hydrophobic dyes, on mucin as well as on other entangled aqueous polymer solution subphases. This independence of qualitative spreading behaviors from the chemistry of the surfactant and subphase indicates that there is little chemical interaction between the formulation and the subphase during the spreading process. The spreading and final solution distributions are controlled by capillary and hydrodynamic phenomena and not by specific chemical interactions among the components of the system. It is suggested that capillary forces and Marangoni flows driven by soluble surfactants may thereby enhance the uniformity of drug delivery to diseased lungs.
Marangoni; soluble surfactant spreading; mucin; aerosol drug delivery; surfactant transport; pulmonary drug delivery
The role of adventitial fibroblasts in the vascular inflammation observed in the adventitia of large vessels in numerous cardiovascular diseases remains unclear. Our objective was to explore the contribution of these cells to angiotensin II (Ang II)-induced aortic inflammation and adventitial expansion.
Cytokine production by primary human aortic adventitial fibroblasts (AoAF) in tissue culture was detected using multiplex ELISA, and increases in cytokine mRNA following Ang II stimulation were quantitated by real-time PCR. The ability of AoAF-derived MCP-1 to attract monocytes was studied in vitro using Boyden assays, and the resulting effect of the monocyte-AoAF interaction on fibroblast proliferation was measured in vitro using proliferation and 3H-thymidine incorporation assays. Ang II-induced fibroblast proliferation was measured in vivo using aortic digestion of single cells followed by flow cytometric quantification of fibroblast numbers as well as fibroblast and PCNA immunostaining. The ability of monocytes to induce AoAF proliferation was demonstrated in vivo using CCR2+/+ wild-type monocyte adoptive transfer into Ang II-stimulated CCR2-null mice which can produce MCP-1 but have cells lacking the MCP-1 receptor – CCR2.
AoAF constitutively secreted numerous proinflammatory cytokines, particularly IL-6 and MCP-1, whose gene expressions were further upregulated in response to Ang II stimulation. AoAF-derived MCP-1 was potent in recruiting THP-1 monocytes in vitro, and these monocytes stimulated AoAF proliferation based on a flow cytometric assessment of cell number and 3H-thymidine incorporation in tissue culture. In vivo, Ang II induced fibroblast proliferation, increased fibroblast and PCNA adventitial staining, and blunted inflammatory responses in the CCR2−/− background. Injection of CCR2+/+ monocytes into Ang II-treated CCR2−/− mice restored adventitial thickening which resulted in increased fibrosis secondary to adventitial fibroblast proliferation.
Our results suggest that Ang II-stimulates AoAF to recruit monocytes via fibroblast-derived MCP-1, and the recruited monocytes further activate fibroblast proliferation, adventitial thickening, and additional cytokine production. This fibroblast-monocyte amplification loop may critically mediate hallmarks of adventitial inflammation common to many cardiovascular diseases.
Aorta; Aortic adventitial fibroblasts; Macrophages; Angiotensin II; Interleukin-6; MCP-1; CCR2
The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.
Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across mucosal barriers is responsible for the vast majority of new infections. This relatively inefficient process results in the transmission of a single transmitted/founder (T/F) virus, from a diverse viral swarm in the donor, in approximately 80% of cases. Here we compared the biological activities of 24 clade B T/F envelopes (Envs) with those from 17 chronic controls to determine whether the genetic bottleneck that occurs during transmission is linked to a particular Env phenotype. To maximize the likelihood of an intact mucosal barrier in the recipients and to enhance the sensitivity of detecting phenotypic differences, only T/F Envs from individuals infected with a single T/F variant were selected. Using pseudotyping to assess Env function in single-round infectivity assays, we compared coreceptor tropism, CCR5 utilization efficiencies, primary CD4+ T cell subset tropism, dendritic cell trans-infections, fusion kinetics, and neutralization sensitivities. T/F and chronic Envs were phenotypically equivalent in most assays; however, T/F Envs were modestly more sensitive to CD4 binding site antibodies b12 and VRC01, as well as pooled human HIV Ig. This finding was independently validated with a panel of 14 additional chronic HIV-1 Env controls. Moreover, the enhanced neutralization sensitivity was associated with more efficient binding of b12 and VRC01 to T/F Env trimers. These data suggest that there are subtle but significant structural differences between T/F and chronic clade B Envs that may have implications for HIV-1 transmission and the design of effective vaccines.
Dithiocarbamates (DTCs) are sulfhydryls (thiol)-containing compounds, often associated with metals, and have both antioxidant and pro-oxidant abilities depending on the compound, experimental system and condition. In this study we investigated whether cell death plays a role in the manifestation of DTC-induced notochord distortions in the developing zebrafish and if thiol-containing compounds or antioxidants could modify this developmental toxicity. Sodium metam (NaM) induced notochord distortions could not be protected with the antioxidants ascorbic acid, trolox (synthetic vitamin E) or lipoic acid. However, NaM-induced distortions could be protected with co-exposure to glutathione or N-Acetyl Cysteine. Staggering the NaM and glutathione exposures in consecutive 10 h developmental windows also resulted in protection. There were no discernable changes in TUNEL positive cells, a marker of apoptotic cells, at 24 h post-fertilization (hpf) in NaM, dimethyl-dithiocarbamate, carbon disulfide, or neocuproine exposed embryos. Live NaM-exposed embryos incubated with acridine orange, a general stain for cell death, for 1 h beginning at 11, 18 and 24 hpf showed clusters of stained nuclei near the somitogenic front but not in the cells making up the notochord. Overall, induction of apoptotic pathways and widespread cell death are not involved in the manifestation of the adverse developmental outcomes following NaM exposure. However, cellular thiol status or critical sulfhydryl moieties are important considerations in the mechanisms of DTC developmental toxicity.
Zebrafish; Developmental toxicity; Pesticides
Pesticides such as chlorpyrifos (CPF) and metals such as copper can impair swimming behavior in fish. However, the impact to swimming behavior from exposure to mixtures of neurotoxicants has received little attention. In the current study, we analyzed spontaneous swimming rates of adult zebrafish (Danio rerio) to investigate in vivo mixture interactions involving two chemical classes. Zebrafish were exposed to the neurotoxicants copper chloride (CuCl, 0.1 μM, 0.25 μM, 0.6 μM, or 6.3, 16, 40 ppb), chlorpyrifos (CPF, 0.1 μM, 0.25 μM, 0.6 μM, or 35, 88, 220 ppb) and binary mixtures for 24 hr to better understand the effects of Cu on CPF neurotoxicity. Exposure to CPF increased the number of animals undergoing freeze responses (an anti-predator behavior) and, at the highest CPF dose (0.6 μM), elicited a decrease in zebrafish swimming rates. Interestingly, the addition of Cu caused a reduction in the number of zebrafish in the CPF-exposure groups undergoing freeze responses. There was no evidence of additive or synergistic toxicity between Cu and CPF. Although muscle AChE activity was significantly reduced by CPF, there was a relatively poor relationship among muscle AChE concentrations and swimming behavior, suggesting non-muscle AChE mechanisms in the loss of swimming behavior. In summary, we have observed a modulating effect of Cu on CPF swimming impairment that appears to involve both AChE and non-AChE mechanisms. Our study supports the utility of zebrafish in understanding chemical mixture interactions and neurobehavioral injury.
mixtures; metals; organophosphates; behavior; acetylcholinesterase
Objective: To review the current status and recent trends in the American Board of Psychiatry and Neurology (ABPN) specialties and neurologic subspecialties and discuss the implications of those trends for subspecialty viability.
Methods: Data on numbers of residency and fellowship programs and graduates and ABPN certification candidates and diplomates were drawn from several sources, including ABPN records, Web sites of the Accreditation Council for Graduate Medical Education and the American Medical Association, and the annual medical education issues of the Journal of the American Medical Association.
Results: About four-fifths of neurology graduates pursue fellowship training. While most recent neurology and child neurology graduates attempt to become certified by the ABPN, many clinical neurophysiologists elect not to do so. There appears to have been little interest in establishing fellowships in neurodevelopmental disabilities. The pass rate for fellowship graduates is equivalent to that for the “grandfathers” in clinical neurophysiology. Lower percentages of clinical neurophysiologists than specialists participate in maintenance of certification, and maintenance of certification pass rates are high.
Conclusion: The initial enthusiastic interest in training and certification in some of the ABPN neurologic subspecialties appears to have slowed, and the long-term viability of those subspecialties will depend upon the answers to a number of complicated social, economic, and political questions in the new health care era.
ABMS = American Board of Medical Specialties; ABPN = American Board of Psychiatry and Neurology; ACGME = Accreditation Council for Graduate Medical Education; MOC = maintenance of certification; RRC-N = Residency Review Committee in Neurology.