Jonathan M. Starkey

The National NeuroAIDS Tissue Consortium Brain Gene Array: Two Types of HIV-Associated Neurocognitive Impairment

Background The National NeuroAIDS Tissue Consortium (NNTC) performed a brain gene expression array to elucidate pathophysiologies of Human Immunodeficiency Virus type 1 (HIV-1)-associated neurocognitive disorders. Methods Twenty-four human subjects in four groups were examined A) Uninfected controls; B) HIV-1 infected subjects with no substantial neurocognitive impairment (NCI); C) Infected with substantial NCI without HIV encephalitis (HIVE); D) Infected with substantial NCI and HIVE. RNA from neocortex, white matter, and neostriatum was processed with the Affymetrix® array platform. Results With HIVE the HIV-1 RNA load in brain tissue was three log10 units higher than other groups and over 1,900 gene probes were regulated. Interferon response genes (IFRGs), antigen presentation, complement components and CD163 antigen were strongly upregulated. In frontal neocortex downregulated neuronal pathways strongly dominated in HIVE, including GABA receptors, glutamate signaling, synaptic potentiation, axon guidance, clathrin-mediated endocytosis and 14-3-3 protein. Expression was completely different in neuropsychologically impaired subjects without HIVE. They had low brain HIV-1 loads, weak brain immune responses, lacked neuronally expressed changes in neocortex and exhibited upregulation of endothelial cell type transcripts. HIV-1-infected subjects with normal neuropsychological test results had upregulation of neuronal transcripts involved in synaptic transmission of neostriatal circuits. Interpretation Two patterns of brain gene expression suggest that more than one pathophysiological process occurs in HIV-1-associated neurocognitive impairment. Expression in HIVE suggests that lowering brain HIV-1 replication might improve NCI, whereas NCI without HIVE may not respond in kind; array results suggest that modulation of transvascular signaling is a potentially promising approach. Striking brain regional differences highlighted the likely importance of circuit level disturbances in HIV/AIDS. In subjects without impairment regulation of genes that drive neostriatal synaptic plasticity reflects adaptation. The array provides an infusion of public resources including brain samples, clinicopathological data and correlative gene expression data for further exploration (http://www.nntc.org/gene-array-project).

Cognitive Enhancement with Rosiglitazone Links the Hippocampal PPARγ and ERK MAPK Signaling Pathways

We previously reported that the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone (RSG) improved hippocampus-dependent cognition in the Alzheimers disease (AD) mouse model, Tg2576. RSG had no effect on wild-type littermate cognitive performance. Since extracellular signal-regulated protein kinase mitogen-activated protein kinase (ERK MAPK) is required for many forms of learning and memory that are affected in AD, and since both PPARγ and ERK MAPK are key mediators of insulin signaling, the current study tested the hypothesis that RSG-mediated cognitive improvement induces a hippocampal PPARγ pattern of gene and protein expression that converges with the ERK MAPK signaling axis in Tg2576 AD mice. In the hippocampal PPARγ transcriptome, we found significant overlap between peroxisome proliferator response element-containing PPARγ target genes and ERK-regulated, cAMP response element-containing target genes. Within the Tg2576 dentate gyrus proteome, RSG induced proteins with structural, energy, biosynthesis and plasticity functions. Several of these proteins are known to be important for cognitive function and are also regulated by ERK MAPK. In addition, we found the RSG-mediated augmentation of PPARγ and ERK2 activity during Tg2576 cognitive enhancement was reversed when hippocampal PPARγ was pharmacologically antagonized, revealing a coordinate relationship between PPARγ transcriptional competency and phosphorylated ERK that is reciprocally affected in response to chronic activation, compared with acute inhibition, of PPARγ. We conclude that the hippocampal transcriptome and proteome induced by cognitive enhancement with RSG harnesses a dysregulated ERK MAPK signal transduction pathway to overcome AD-like cognitive deficits in Tg2576 mice. Thus, PPARγ represents a signaling system that is not crucial for normal cognition yet can intercede to restore neural networks compromised by AD.

Diabetes-Induced Activation of Canonical and Noncanonical Nuclear Factor-κB Pathways in Renal Cortex

Evidence of diabetes-induced nuclear factor-κB (NF-κB) activation has been provided with DNA binding assays or nuclear localization with immunohistochemistry, but few studies have explored mechanisms involved. We examined effects of diabetes on proteins comprising NF-κB canonical and noncanonical activation pathways in the renal cortex of diabetic mice. Plasma concentrations of NF-κB–regulated cytokines were increased after 1 month of hyperglycemia, but most returned to control levels or lower by 3 months, when the same cytokines were increased significantly in renal cortex. Cytosolic content of NF-κB canonical pathway proteins did not differ between experimental groups after 3 months of diabetes, while NF-κB noncanonical pathway proteins were affected, including increased phosphorylation of inhibitor of κB kinase-α and several fold increases in NF-κB–inducing kinase and RelB, which were predominantly located in tubular epithelial cells. Nuclear content of all NF-κB pathway proteins was decreased by diabetes, with the largest change in RelB and p50 (approximately twofold decrease). Despite this decrease, measurable increases in protein binding to DNA in diabetic versus control nuclear extracts were observed with electrophoretic mobility shift assay. These results provide evidence for chronic NF-κB activation in the renal cortex of db/db mice and suggest a novel, diabetes-linked mechanism involving both canonical and noncanonical NF-κB pathway proteins.

Altered Retinoic Acid Metabolism in Diabetic Mouse Kidney Identified by 18O Isotopic Labeling and 2D Mass Spectrometry

Background Numerous metabolic pathways have been implicated in diabetes-induced renal injury, yet few studies have utilized unbiased systems biology approaches for mapping the interconnectivity of diabetes-dysregulated proteins that are involved. We utilized a global, quantitative, differential proteomic approach to identify a novel retinoic acid hub in renal cortical protein networks dysregulated by type 2 diabetes. Methodology/Principal Findings Total proteins were extracted from renal cortex of control and db/db mice at 20 weeks of age (after 12 weeks of hyperglycemia in the diabetic mice). Following trypsinization, 18O- and 16O-labeled control and diabetic peptides, respectively, were pooled and separated by two dimensional liquid chromatography (strong cation exchange creating 60 fractions further separated by nano-HPLC), followed by peptide identification and quantification using mass spectrometry. Proteomic analysis identified 53 proteins with fold change ≥1.5 and p≤0.05 after Benjamini-Hochberg adjustment (out of 1,806 proteins identified), including alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (RALDH1/ALDH1A1). Ingenuity Pathway Analysis identified altered retinoic acid as a key signaling hub that was altered in the diabetic renal cortical proteome. Western blotting and real-time PCR confirmed diabetes-induced upregulation of RALDH1, which was localized by immunofluorescence predominantly to the proximal tubule in the diabetic renal cortex, while PCR confirmed the downregulation of ADH identified with mass spectrometry. Despite increased renal cortical tissue levels of retinol and RALDH1 in db/db versus control mice, all-trans-retinoic acid was significantly decreased in association with a significant decrease in PPARβ/δ mRNA. Conclusions/Significance Our results indicate that retinoic acid metabolism is significantly dysregulated in diabetic kidneys, and suggest that a shift in all-trans-retinoic acid metabolism is a novel feature in type 2 diabetic renal disease. Our observations provide novel insights into potential links between altered lipid metabolism and other gene networks controlled by retinoic acid in the diabetic kidney, and demonstrate the utility of using systems biology to gain new insights into diabetic nephropathy.

Cognitive Enhancing Treatment with a PPARγ Agonist Normalizes Dentate Granule Cell Presynaptic Function in Tg2576 APP Mice

Hippocampal network hyperexcitability is considered an early indicator of Alzheimers disease (AD) memory impairment. Some AD mouse models exhibit similar network phenotypes. In this study we focused on dentate gyrus (DG) granule cell spontaneous and evoked properties in 9-month-old Tg2576 mice that model AD amyloidosis and cognitive deficits. Using whole-cell patch-clamp recordings, we found that Tg2576 DG granule cells exhibited spontaneous EPSCs that were higher in frequency but not amplitude compared with wild-type mice, suggesting hyperactivity of DG granule cells via a presynaptic mechanism. Further support of a presynaptic mechanism was revealed by increased I–O relationships and probability of release in Tg2576 DG granule cells. Since we and others have shown that activation of the peroxisome proliferator-activated receptor gamma (PPARγ) axis improves hippocampal cognition in mouse models for AD as well as benefitting memory performance in some humans with early AD, we investigated how PPARγ agonism affected synaptic activity in Tg2576 DG. We found that PPARγ agonism normalized the I–O relationship of evoked EPSCs, frequency of spontaneous EPSCs, and probability of release that, in turn, correlated with selective expression of DG proteins essential for presynaptic SNARE function that are altered in patients with AD. These findings provide evidence that DG principal cells may contribute to early AD hippocampal network hyperexcitability via a presynaptic mechanism, and that hippocampal cognitive enhancement via PPARγ activation occurs through regulation of presynaptic vesicular proteins critical for proper glutamatergic neurotransmitter release, synaptic transmission, and short-term plasticity.

Detection of Structural and Metabolic Changes in Traumatically Injured Hippocampus by Quantitative Differential Proteomics

Traumatic brain injury (TBI) is a complex and common problem resulting in the loss of cognitive function. In order to build a comprehensive knowledge base of the proteins that underlie these cognitive deficits, we employed unbiased quantitative mass spectrometry, proteomics, and bioinformatics to identify and quantify dysregulated proteins in the CA3 subregion of the hippocampus in the fluid percussion model of TBI in rats. Using stable isotope 18O-water differential labeling and multidimensional tandem liquid chromatography (LC)-MS/MS with high stringency statistical analyses and filtering, we identified and quantified 1002 common proteins, with 124 increased and 76 decreased. The ingenuity pathway analysis (IPA) bioinformatics tool identified that TBI had profound effects on downregulating global energy metabolism, including glycolysis, the Krebs cycle, and oxidative phosphorylation, as well as cellular structure and function. Widespread upregulation of actin-related cytoskeletal dynamics was also found. IPA indicated a common integrative signaling node, calcineurin B1 (CANB1, CaNBα, or PPP3R1), which was downregulated by TBI. Western blotting confirmed that the calcineurin regulatory subunit, CANB1, and its catalytic binding partner PP2BA, were decreased without changes in other calcineurin subunits. CANB1 plays a critical role in downregulated networks of calcium signaling and homeostasis through calmodulin and calmodulin-dependent kinase II to highly interconnected structural networks dominated by tubulins. This large-scale knowledge base lays the foundation for the identification of novel therapeutic targets for cognitive rescue in TBI.

Proteomics and Systems Biology for Understanding Diabetic Nephropathy

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.

Using Power Spectrum Analysis to Evaluate 18O-Water Labeling Data Acquired from Low Resolution Mass Spectrometers

We describe a method for ratio estimations in (18)O-water labeling experiments acquired from low resolution isotopically resolved data. The method is implemented in a software package specifically designed for use in experiments making use of zoom-scan mode data acquisition. Zoom-scan mode data allow commonly used ion trap mass spectrometers to attain isotopic resolution, which makes them amenable to use in labeling schemes such as (18)O-water labeling, but algorithms and software developed for high resolution instruments may not be appropriate for the lower resolution data acquired in zoom-scan mode. The use of power spectrum analysis is proposed as a general approach that may be uniquely suited to these data types. The software implementation uses a power spectrum to remove high-frequency noise and band-filter contributions from coeluting species of differing charge states. From the elemental composition of a peptide sequence, we generate theoretical isotope envelopes of heavy-light peptide pairs in five different ratios; these theoretical envelopes are correlated with the filtered experimental zoom scans. To automate peptide quantification in high-throughput experiments, we have implemented our approach in a computer program, MassXplorer. We demonstrate the application of MassXplorer to two model mixtures of known proteins and to a complex mixture of mouse kidney cortical extract. Comparison with another algorithm for ratio estimations demonstrates the increased precision and automation of MassXplorer.

Ketotifen in the management of chronic urticaria: Resurrection of an old drug

Ketotifen is an oral antiallergic drug developed in 1970 by Sandoz Pharmaceuticals of Switzerland. It is a benzocycloheptathiophene derivative and was initially marketed as an inhibitor of anaphylaxis.1 The pharmacodynamic properties of ketotifen are many, because it is an inhibitor of the release and/or activity of mast cell and basophil mediators, including histamine, neutrophil, and eosinophil chemotactic factors, arachidonic acid metabolites, prostaglandins, and leukotrienes.2 Thus, it inhibits the bronchial response to inhaled histamine, allergen, or aspirin. In addition, the ocular, nasal, and dermal responses to applied allergen in sensitized patients are attenuated with use of ketotifen.3 It also has been found to have some calcium antagonist activity and to inhibit responses to platelet-activating factor from proinflammatory cells, such as eosinophils.2 Additional possible modes of action include its ability to reverse b2-agonisteinduced decreases in b-adrenoreceptor density and to alter the affinity of these receptors and increase intracellular concentrations of cyclic adenosine monophosphate.3 Ketotifen has a chemical structure similar to some first-generation antihistamines, such as cyproheptadine and azatidene.2 With regard to the pharmacokinetic properties of ketotifen, it is readily absorbed from the gastrointestinal tract after oral administration and achieves peak plasma concentrations within 2 to 4 hours of administration. Clearance of the drug from plasma is biphasic, with a half-life of distribution of 3 hours and a half-life of elimination of 22 hours in adults.3 However, the onset of action of ketotifen is slow, and it may take 4 to 6 weeks to achieve full prophylactic value under certain conditions.4 Ketotifen was originally patented by Sandoz in the 1970s. It became very popular for management of asthma in Japan and clinical trials were initiated in the United States in the 1980s for management of asthma and other allergic conditions. One author served on the Food and Drug Administration (FDA) Pulmonarye Allergy Drugs Advisory Committee and chaired that group from 1984 to 1986. Approval for treatment of asthma was not granted, but FDA staff did recommend more extensive evaluations for management of urticaria. Unfortunately, the latter studies have not been performed in a robust fashion. However, oral ketotifen has been used in patients with asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, chronic urticaria, cold-induced urticaria, cholinergic urticaria, exercise-induced urticaria, mastocytosis, and food allergy in Canada, Europe, and Mexico. Approval was granted for ocular administration in the United States for allergic conjunctivitis and later the drug became available over the counter. A review of clintrials.gov shows evaluations of ketotifen by oral administration for fibromyalgia, atopic dermatitis, attenuation of reactions during peanut desensitization, allergic rhinitis, asthma, and post-traumatic joint contractures. The authors became aware that ketotifen could be obtained in the United States from compounding pharmacies and began prescribing it more extensively in their clinics for severe urticaria in 2011. For adults and older children with asthma or allergic disease, the recommended dose of ketotifen is 1 mg twice daily. For young children 6 months to 3 years old, the recommended dose is 0.5 mg twice daily.3 It must be noted that oral ketotifen can be safely compounded in the United States. Pharmacy compounding is a practice in which a licensed pharmacist combines, mixes, or alters ingredients in response to a prescription to create a medication tailored to the medical needs of an individual patient. Pharmacy compounding, if performed properly, can serve an important public health need if a patient cannot be treated with an FDA-approved medication. The issue of safety of compounded drugs in the FDA Compliance Policy for Pharmacy Compounding is primarily for injectable drugs and does not relate to oral compounded drugs. This policy does include a select list of compounding drugs that were withdrawn or Reprints: Kristin C. Sokol, MD, MS, MPH, Division of Allergy and Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555; E-mail: Kcsokol@utmb.edu. Disclosures: Authors have nothing to disclose. Funding Sources: This study was conducted with the support of the Institute for Translational Sciences at the University of Texas Medical Branch, supported in part by a Clinical and Translational Science Award (UL1TR000071) from the National Center for Advancing Translational Sciences, National Institutes of Health.

Concordance of adolescent human papillomavirus vaccination parental report with provider report in the National Immunization Survey-Teen (2008–2013)

OBJECTIVES To examine the accuracy of parental report of HPV vaccination through examination of concordance, with healthcare provider vaccination report as the comparison. METHODS The 2008-2013 National Immunization Survey (NIS)-Teen was used to examine accuracy of parent reports of HPV vaccination for their female daughters aged 13-17years, as compared with provider report of initiation and number of doses. Multivariable logistic regression models were used to examine associations related to concordance of parent and provider report. RESULTS Of 51,746 adolescents, 84% concordance for HPV vaccine initiation and 70% concordance for number of doses was observed. Accuracy varied by race/ethnicity, region, time, and income. The parent report of number of doses was more likely to be accurate among parents of 13 and 14year old females than 17year olds. Accuracy of initiation and number of doses were lower among Hispanic and black adolescents compared to white parents. The odds of over-report was higher among minorities compared to whites, but the odds of underreport was also markedly higher in these groups compared to parents of white teens. Accuracy of parental vaccine report decreased across time. CONCLUSIONS These findings are important for healthcare providers who need to ascertain the vaccination status of young adults. Strengthening existing immunization registries to improve data sharing capabilities and record completeness could improve vaccination rates, while avoiding costs associated with over-vaccination.