Kimberly A. Carlson

Macrophage proteomic fingerprinting predicts HIV-1-associated cognitive impairment

Background: Specific proteins produced from monocytes may be linked to the pathogenesis and aid in the diagnosis of HIV-1–associated dementia (HAD). Objective: The authors assessed whether a diagnostic phenomic protein profile could be obtained from monocyte-derived macrophages (MDM) from HIV-1–infected patients with cognitive impairment. Methods: Twenty-one HIV-1–infected Hispanic women and 10 seronegative controls matched by age and sex were followed at the University of Puerto Rico Medical Sciences Campus, where neuropsychological, immune, and viral parameters were tested. Monocytes were recovered by Percoll gradient centrifugation from peripheral blood mononuclear cells. MDM lysates were prepared after 7 days of cultivation and protein profiles analyzed by surface enhanced laser desorption/ionization (SELDI)–time of flight (TOF) ProteinChip tests. Classification trees were prepared for statistical analyses. Results: A total of 177 protein peaks from 2 to 80 kDa were evaluated in 31 patient MDM lysates by SELDI-TOF ProteinChip assays. Select protein peaks, at 5028 and 4320 Da, separated HIV-1–infected from HIV-1–seronegative subjects with a sensitivity of 100% and a specificity of 80%. Thirty-eight peaks were used to differentiate HIV-1–infected subjects with and without cognitive impairment. A 4348 Da protein separated the two groups with a sensitivity of 100% and a specificity of 75%. Conclusions: The identification of unique phenomic MDM profiles from cognitively impaired HIV-1–infected patients supports the hypothesis that changes in monocyte function parallel the development of HAD.

Inhibition of long-term potentiation by interleukin-8: Implications for human immunodeficiency virus-1-associated dementia

Human immunodeficiency virus type 1 (HIV‐1)‐infected mononuclear phagocytes (MP; brain macrophages and microglia) secrete a number of toxic factors that affect the pathogenesis of HIV‐1‐associated dementia (HAD). The identification and relative role of each MP toxin for neuronal dysfunction during HAD are not well understood. Interleukin‐8 (IL‐8), a CXC chemokine involved in leukocyte activation and chemotaxis, is constitutively produced by MP, and elevated levels of IL‐8 mRNA were detected in the brains of patients with HIV‐1 encephalitis (HIVE) by both ribonuclease protection assays and real‐time PCR. To determine the role that IL‐8 might play in the neuronal dysfunction in HAD, we studied its effect on synaptic transmission and plasticity in the CA1 region of hippocampus, the seat of learning and memory. Bath application of IL‐8 (50 ng/ml) to rat hippocampal slices had no effect on basal synaptic transmission. However, IL‐8 was shown to inhibit long‐term potentiation (LTP) in a concentration‐dependent manner. In control and IL‐8‐treated slices, the LTP magnitudes were 167.8% ± 11.9% (mean ± SE; n = 17) and 122.2% ± 16.2% of basal levels (n = 13), respectively. These differences were statistically significant (P < 0.05). Preincubation of hippocampal slices with a monoclonal CXCR2 antibody (2 μg/ml) but not control IgG (2 μg/ml) blocked IL‐8‐induced inhibition of LTP. The expression of CXCR2 receptors in the CA1 region was shown by Western blot assays. The induction of IL‐8 in HAD, its inhibition of LTP, and the expression of its receptor, CXCR2, in the hippocampus all suggest that it plays a role in the cognitive dysfunction associated with HAD.

Molecular Characterization of a Putative Antiretroviral Transcriptional Factor, OTK18

Elucidation of the factors involved in host defense against human immunodeficiency viral infection remains pivotal if viral control may be achieved. Toward these ends, we investigated the function of a putative antiretroviral factor, OTK18, isolated by differential display of mRNA from HIV type 1-infected primary human monocyte-derived macrophages. Molecular and immunohistochemical analyses showed that the OTK18 nucleotide sequence contains 13 adjacent C2H2-type zinc finger motifs, a Krüppel-associated box, and is localized to both cytosol and nucleus. Mutational analyses revealed that both the Krüppel-associated box and zinc finger regions of OTK18 are responsible for the transcriptional suppressive activities of this gene. OTK18 was copiously expressed in macrophages following HIV type I infection and diminished progeny virion production. A mechanism for this antiretroviral activity was by suppression of HIV type 1 Tat-induced viral long terminal repeat promoter activity. Our findings suggest that one possible function of OTK18 is as a HIV type 1-inducible transcriptional suppresser.

Peripheral nerve induces macrophage neurotrophic activities: regulation of neuronal process outgrowth, intracellular signaling and synaptic function

Rat cortical neurons cultured in conditioned media from human monocyte-derived macrophages (MDM) show increased neuronal protein synthesis, neurite outgrowth, mitogen-activating protein kinase activity, and synaptic function. Neurotrophic properties of human MDM-conditioned media are significantly enhanced by human peripheral nerve and to a more limited extent by CD40 ligand pre-stimulation. Such positive effects of MDM secretions on neuronal function parallel the secretion of brain-derived neurotrophic factor (BDNF). MDM activation cues may serve to balance toxic activities produced during neurodegenerative diseases and thus, under certain circumstances, mitigate neuronal degeneration.

OTK18 expression in brain mononuclear phagocytes parallels the severity of HIV-1 encephalitis

OTK18 was isolated by mRNA differential display of human monocyte-derived macrophages (MDM) infected with human immunodeficiency virus type one (HIV-1). Northern blot and real-time reverse transcription polymerase chain reaction showed low levels of OTK18 expression in human tissue, which markedly increased during advanced HIV-1 encephalitis (HIVE). Immunocytochemistry, using rabbit polyclonal antisera, showed OTK18 localized to brain mononuclear phagocytes (MP) in moderate to severe HIV-1 encephalitis. OTK18 expression was selective and not found in HIV-1-infected brain tissue with limited neuropathological abnormalities, nor in cytomegalovirus encephalitis, multiple sclerosis, Alzheimers disease, or uninfected control brains. Thus, OTK18 expression in brain mononuclear phagocytes is a signature for advanced HIV-1 encephalitis.

Proteomic fingerprinting of HIV-1-infected human monocyte-derived macrophages: a preliminary report

Mononuclear phagocytes (MP; blood monocytes, alveolar, lymph node, and brain macrophages and microglia) are vehicles for dissemination and principle target cells for human immunodeficiency virus type 1 (HIV-1) infection. Notably, viral persistence in macrophages occurs despite ongoing phagocytic, intracellular killing, innate and adaptive immune responses. To assess potential pathways for how HIV-1 may bypass antiviral MP responses, we used proteomic tests to evaluate protein fingerprints of HIV-1-infected human monocyte-derived macrophages 7 days after viral infection. By using weak cation exchange chips, 58 proteins were found up- or down-regulated after HIV-1(ADA) infection. Several of these proteins were identified by microsequencing. It is probable that cellular proteins identified by proteomic fingerprinting could assist in unraveling how persistent viral infection occurs in MP lineage cells. Moreover, this evolving technology can be utilized to unravel changes in immune activities initiated by interactions between virus, environmental cues and drugs of abuse.

Differential gene expression related to Nora virus infection of Drosophila melanogaster.

Nora virus is a recently discovered RNA picorna-like virus that produces a persistent infection in Drosophila melanogaster, but the antiviral pathway or change in gene expression is unknown. We performed cDNA microarray analysis comparing the gene expression profiles of Nora virus infected and uninfected wild-type D. melanogaster. This analysis yielded 58 genes exhibiting a 1.5-fold change or greater and p-value less than 0.01. Of these genes, 46 were up-regulated and 12 down-regulated in response to infection. To validate the microarray results, qRT-PCR was performed with probes for Chorion protein 16 and Troponin C isoform 4, which show good correspondence with cDNA microarray results. Differential regulation of genes associated with Toll and immune-deficient pathways, cytoskeletal development, Janus Kinase-Signal Transducer and Activator of Transcription interactions, and a potential gut-specific innate immune response were found. This genome-wide expression profile of Nora virus infection of D. melanogaster can pinpoint genes of interest for further investigation of antiviral pathways employed, genetic mechanisms, sites of replication, viral persistence, and developmental effects.

Genome-Wide Gene Expression in relation to Age in Large Laboratory Cohorts of Drosophila melanogaster.

Aging is a complex process characterized by a steady decline in an organisms ability to perform life-sustaining tasks. In the present study, two cages of approximately 12,000 mated Drosophila melanogaster females were used as a source of RNA from individuals sampled frequently as a function of age. A linear model for microarray data method was used for the microarray analysis to adjust for the box effect; it identified 1,581 candidate aging genes. Cluster analyses using a self-organizing map algorithm on the 1,581 significant genes identified gene expression patterns across different ages. Genes involved in immune system function and regulation, chorion assembly and function, and metabolism were all significantly differentially expressed as a function of age. The temporal pattern of data indicated that gene expression related to aging is affected relatively early in life span. In addition, the temporal variance in gene expression in immune function genes was compared to a random set of genes. There was an increase in the variance of gene expression within each cohort, which was not observed in the set of random genes. This observation is compatible with the hypothesis that D. melanogaster immune function genes lose control of gene expression as flies age.

Extended longevity lines of Drosophila melanogaster: abundance of yolk protein gene mRNA in fat body and ovary

Lines of Drosophila melanogaster selected for late-life female reproduction typically exhibit correlated responses of reduced early fecundity and increased longevity. This relationship suggests a tradeoff between reproductive effort and somatic maintenance, which in turn, underlies some evolutionary theories of senescence. The mechanistic basis of the apparent tradeoff between increased longevity and reduced early-age fecundity has remained obscure. The present manuscript addresses the issues of whether the reduced early-age fecundity in selected lines corresponds to reduced yolk-protein mRNA production, and whether long-lived flies exhibit somatic maintenance in terms of relatively reduced yolk-protein mRNA production in the fat body. Yolk protein is one of the most abundant proteins used for female reproduction. By comparing a set of lines selected for late life reproduction with the corresponding control lines, we show that that yolk-protein gene mRNA relative abundance during the first four days posteclosion did not correspond to reduced early-life fecundity in the selected lines. In D. melanogaster, yolk protein is produced in the fat body and ovarian follicle cells. On the fourth day posteclosion, relatively more yolk-protein gene mRNA was present in the fat body. On day 1 posteclosion, supplemental yeast did not alter relative yolk-protein gene mRNA abundance. However, on day 4 posteclosion, supplemental yeast stimulated yolk-protein gene mRNA production in the fat body, which suggests an underlying mechanism for the nutrition-based phenotypic plasticity of fecundity previously documented in these lines. On medium without supplemental yeast, the relatively low abundance of fat body yolk-protein gene mRNA in the selected lines on day 4 posteclosion corresponds to a prediction derived from the disposable soma theory.

Drosophila melanogaster as a complementary system for studying HIV-1-related genes and proteins.

Human immunodeficiency virus type 1 (HIV‐1) persists as a pandemic even though new information about the virus is being discovered on a daily basis. If the brain becomes infected, HIV‐1 encephalitis or HIV‐1‐associated dementia may develop. There is much to be learned about the modes of action and mechanisms of genes and proteins, and their interactions that underlie HIV‐1 infection. Drosophila melanogaster has been used successfully to study genes and proteins related to HIV‐1 infection, including but not limited to the disturbance of antimicrobial responses by viral protein U and the identification of D. melanogaster analogs to the serine palmitoyltransferase 5 and 6 proteins that play a role in activation of transcription by the HIV‐1 Tat protein in human cells. We believe that utilizing D. melanogaster as a complementary system for the study of genes and proteins related to HIV‐1 infection will provide useful information that will lead to new studies designed to enhance our understanding of the mechanistic roles of these molecules. In the present study, we focus on the utilization of D. melanogaster as a complementary system for studying HIV‐1 related genes and proteins, why this research should be extended, and why this complementary system is an important method for enhancing our understanding of the genetics involved in HIV‐1 infection.