J. Russ Carmical

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.

Mutagenic potential of adenine N6 adducts of monoepoxide and diolepoxide derivatives of butadiene

To determine the biological effects of specific DNA adducts resulting from the interaction of 1,3‐butadiene metabolites with DNA, deoxyoligonucleotides have been synthesized with four different adducts at the N6 position of adenine, centrally located within the human N‐ras codon 61. The adducts are those arising from adduction by either the R or S stereoisomer of the monoepoxide (BDO) or the (R,R) or (S,S) isomer of the diolepoxide (BDE). The diolepoxide can arise from partial hydrolysis of the diepoxide (BDO2) or from epoxidation of hydrolyzed monoepoxide. These adducted oligonucleotides were used in in vivo and in vitro assays designed both to determine their mutagenic potency and to examine specific interactions with Escherichia coli polymerases. Each adducted oligonucleotide was ligated into a single‐stranded vector M13mp7L2 that was subsequently used to transfect E. coli. The resulting mutagenic spectrum for these modified DNAs was stereoisomer specific. Both monoepoxide lesions were nonmutagenic, but the mutagenic spectra for the modified DNAs containing BDE adducts were stereoisomer specific. The mutations generated by adducts of the R,R enantiomer of the diolepoxide were exclusively A → G, whereas adducts of the S,S enantiomer of the diolepoxide yielded exclusively A → C mutations. None of the four modifications resulted in significant blocks to in vivo phage replication, as evidenced by no decrease in plaque‐forming ability. Consistent with these data, when each of three purified E. coli polymerases was used to replicate DNAs containing these adducted deoxyoligonucleotides, the individual polymerases appeared to be virtually unaffected, such that all lesions were readily bypassed. Whereas previous animal model studies identified the mutagenic spectrum related to butadiene exposure, these studies begin to establish the specific lesions responsible for mutagenesis. This is the first report of stereoselectivity related to butadiene‐induced mutagenesis. Environ. Mol. Mutagen. 35:48–56, 2000

Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

ABSTRACT Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection.

A novel DNA N-glycosylase activity of E. coli T4 endonuclease V that excises 4,6-diamino-5-formamidopyrimidine from DNA, a UV-radiation- and hydroxyl radical-induced product of adenine

We report on a novel activity of T4 endonuclease V. This enzyme is well known to be specific for the excision of pyrimidine dimers from UV-irradiated DNA. In this work, we show that T4 endonuclease V excises 4,6-diamino-5-formamidopyrimidine from DNA. 4,6-Diamino-5-formamidopyrimidine is formed as a product of adenine in DNA upon action of hydroxyl radicals and upon UV-irradiation. DNA substrates were prepared by UV-or gamma-irradiation of DNA in aqueous solution. DNA substrates were incubated either with active T4 endonuclease V or with heat-inactivated T4 endonuclease V or without the enzyme. After incubation, DNA was precipitated and supernatant fractions were separated. Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results provide evidence for the excision of 4,6-diamino-5-formamidopyrimidine by T4 endonuclease V from both gamma-and UV-irradiated DNA. Kinetics of excision were also determined. Fifteen other pyrimidine- and purine-derived base lesions that were identified in DNA samples were not substrates for this enzyme. It was concluded that, in addition to its well known activity for pyrimidine photodimers, T4 endonuclease V possesses an N-glycosylase activity for a major UV-radiation- and hydroxyl radical-induced monomeric product in DNA.

Early Immunologic Events at the Tick-Host Interface

Ixodes species ticks are competent vectors of tick-borne viruses including tick-borne encephalitis and Powassan encephalitis. Tick saliva has been shown to facilitate and enhance viral infection. This likely occurs by saliva-mediated modulation of host responses into patterns favorable for viral infection and dissemination. Because of the rapid kinetics of tick-borne viral transmission, this modulation must occur as early as tick attachment and initiation of feeding. In this study, cutaneous bite-site lesions were analyzed using Affymetrix mouse genome 430A 2.0 arrays and histopathology at 1, 3, 6, and 12 hours after uninfected Ixodes scapularis nymphal tick attachment. At 1 and 3 hrs after attachment, the gene expression profile is markedly different than at later time points. Upregulated gene ontology term clusters enriched at 1 and 3 hrs were related to post-translational modification. At 6 and 12 hrs, cytoskeletal rearrangements, DNA replication/cell division, inflammation, and chemotaxis were prominent clusters. At 6 and 12 hrs, extracellular matrix, signaling, and DNA binding clusters were downregulated. Histopathological analysis shows minimal inflammation at 1 and 3 hrs but an appreciable neutrophil infiltrate at 6 and 12 hrs. In addition, putative hyperemia, localized necrosis, and increased ECM deposition were identified. Putting the gene expression and histopathology analysis together suggests early tick feeding is characterized by modulation of host responses in resident cells that merges into a nascent, neutrophil-driven immune response by 12 hrs post-attachment.

Variable penetrance of metabolic phenotypes and development of high-fat diet-induced adiposity in NEIL1-deficient mice

Exposure to chronic and acute oxidative stress is correlated with many human diseases, including, but not limited to, cancer, heart disease, diabetes, and obesity. In addition to cellular lipids and proteins, cellular oxidative stress can result in damage to DNA bases, especially in mitochondrial DNA. We previously described the development of spontaneous late-onset obesity, hepatic steatosis, hyperinsulinemia, and hyperleptinemia in mice that are deficient in the DNA glycosylase nei-like 1 (NEIL1), which initiates base excision repair of several oxidatively damaged bases. In the current study, we report that exposure to a chronic oxidative stress in the form of a high-fat diet greatly accelerates the development of obesity in neil1(-/-) mice. Following a 5-wk high-fat diet challenge, neil1(-/-) mice gained significantly more body weight than neil1(+/+) littermates and had increased body fat accumulation and moderate to severe hepatic steatosis. Analysis of oxygen consumption by indirect calorimetry indicated a modest reduction in total oxygen consumption in neil1(-/-) mice that was abolished upon correction for lean body mass. Additionally, hepatic expression of several inflammatory genes was significantly upregulated in neil1(-/-) mice following high-fat diet challenge compared with chow-fed or neil1(+/+) counterparts. A long-term high-fat diet also induced glucose intolerance as well as a significant reduction in mitochondrial DNA and protein content in neil1(-/-) mice. Collectively, these data indicate that NEIL1 deficiency results in an increased susceptibility to obesity and related complications potentially by lowering the threshold for tolerance of cellular oxidative stress in neil1(-/-) mice.

THE CD133-POSITIVE COLON CANCER CELL PHENOTYPE IS MORE INTERACTIVE WITH THE TUMOR MICROENVIRONMENT COMPARED TO CD133-NEGATIVE CELL

Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133-negative (CD133−) cells. We hypothesized that CD133-positive (CD133+) cells, compared with CD133−, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma-associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and CD133− cells using fluorescence-activated cell sorter. The CD133+ cells formed large tumors in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133− cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs CD133− cells. RT-PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs – cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin β8 and fibroblast growth factor receptor 2. The CAF highly express the respective ligands: stromal-derived factor-1 (SDF-1), vitronectin and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in intracellular calcium in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension compared with only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew more colonies compared with vehicle, as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared with CD133−, cells is due to their increased ability to interact with their neighboring CAF.Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133− cells. We hypothesized that CD133+ cells, compared to CD133−, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma –associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and – cells using FACS. The CD133+ cells formed large tumors in NOD-SCID mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133− cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change, and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs. − cells. RT PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs − cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin β8 and fibroblast growth factor receptor 2 (FGFR2). The CAF highly express the respective ligands: SDF-1, vitronectin, and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in [Ca2+]I in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension, compared to only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew both more colonies compared to vehicle as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared to CD133−, cells is due to their increased ability to interact with their neighboring CAF.

T4 ENDONUCLEASE V EXISTS IN SOLUTION AS A MONOMER AND BINDS TO TARGET SITES AS A MONOMER

Endonuclease V, a N-glycosylase/lyase from T4 bacteriophage that initiates the repair of cyclobutane pyrimidine dimers in DNA, has been reported to form a monomer-dimer equilibrium in solution [Nickell and Lloyd (1991) Biochemistry 30, 8638], although the enzyme has only been crystallized in the absence of substrate as a monomer [Morikawa et al. (1992) Science 256, 523]. In this study, analytical gel filtration and sedimentation equilibrium techniques were used to rigorously characterize the association state of the enzyme in solution. In contrast to the previous report, at 100 mM KCl endonuclease V was found to exist predominantly as a monomer in solution by both of these techniques; no evidence for dimerization was seen. To characterize the oligomeric state of the enzyme at its target sites on DNA, the enzyme was bound to oligonucleotides containing a single site specific pyrimidine dimer or tetrahydrofuran residue. These complexes were analyzed by nondenaturing gel electrophoresis at various acrylamide concentrations in order to determine the molecular weights of the enzyme-DNA complexes. The results from these experiments demonstrate that endonuclease V binds to cyclobutane pyrimidine dimer and tetrahydrofuran site containing DNA as a monomer.

CD133|[plus]| colon cancer cells are more interactive with the tumor microenvironment than CD133|[minus]| cells

Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133-negative (CD133−) cells. We hypothesized that CD133-positive (CD133+) cells, compared with CD133−, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma-associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and CD133− cells using fluorescence-activated cell sorter. The CD133+ cells formed large tumors in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133− cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs CD133− cells. RT-PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs – cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin β8 and fibroblast growth factor receptor 2. The CAF highly express the respective ligands: stromal-derived factor-1 (SDF-1), vitronectin and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in intracellular calcium in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension compared with only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew more colonies compared with vehicle, as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared with CD133−, cells is due to their increased ability to interact with their neighboring CAF.Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133− cells. We hypothesized that CD133+ cells, compared to CD133−, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma –associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and – cells using FACS. The CD133+ cells formed large tumors in NOD-SCID mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133− cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change, and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs. − cells. RT PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs − cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin β8 and fibroblast growth factor receptor 2 (FGFR2). The CAF highly express the respective ligands: SDF-1, vitronectin, and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in [Ca2+]I in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension, compared to only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew both more colonies compared to vehicle as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared to CD133−, cells is due to their increased ability to interact with their neighboring CAF.