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Biomedical subjects

Ravikumar Aalinkeel

Publications and source records attributed to Ravikumar Aalinkeel.

13 recordsLinked to original sources
PubMed2006

Methamphetamine modulates gene expression patterns in monocyte derived mature dendritic cells: implications for HIV-1 pathogenesis.

BACKGROUND: The US is currently experiencing a grave epidemic of methamphetamine use as a recreational drug, and the risk for HIV-1 infection attributable to methamphetamine use continues to increase. Recent studies show a high prevalence of HIV infection among methamphetamine users. Dendritic cells (DCs) are potent antigen presenting cells that are the initial line of defense against HIV-1 infection. In addition, DCs also serve as reservoirs for HIV-1 and function at the interface between the adaptive and the innate immune systems, which recognize and internalize pathogens and subsequently activate T cells. Exposure to methamphetamine results in modulation of immune functional parameters that are necessary for host defense. Chronic methamphetamine use can cause psychiatric co-morbidity, neurological complications, and can alter normal biological processes and immune functions. Limited information is available on the mechanisms by which methamphetamine may influence immune function. This study explores the effect of methamphetamine on a specific array of genes that may modulate immune function. We hypothesize that methamphetamine treatment results in the immunomodulation of DC functions, leading to dysregulation of the immune system of the infected host. This suggests that methamphetamine has a role as a cofactor in the pathogenesis of HIV-1. METHODS: We used the high-throughput technology of gene microarray analysis to understand the molecular mechanisms underlying the genomic changes that alter normal biological processes when DCs are treated with methamphetamine. Additionally, we validated the results obtained from microarray experiments using a combination of quantitative real-time PCR and Western blot analysis. RESULTS: These data are the first evidence that methamphetamine modulates DC expression of several genes. Methamphetamine treatment alters categories of genes that are associated with chemokine regulation, cytokinesis, signal transduction mechanisms, apoptosis, and cell cycle regulation. This report focuses on a selected group of genes that are significantly modulated by methamphetamine treatment and that have been associated with HIV-1 pathogenesis. DISCUSSION/CONCLUSION: The purpose of this study was to identify genes that are unique and/or specific to the complex immunomodulatory mechanisms that are altered as a result of methamphetamine abuse in HIV-1-infected patients. These studies will help to identify the molecular mechanisms that underlie methamphetamine toxicity, and several functionally important classes of genes have emerged as targets in methamphetamine-mediated immunopathogenesis of HIV-1. Identification of novel DC-specific and methamphetamine-responsive genes that modulate several biological, molecular, and signal transduction functions may serve as methamphetamine- and/or HIV-1-specific drug targets.

Adult↗
PubMed2006

The flavonoid quercetin inhibits proinflammatory cytokine (tumor necrosis factor alpha) gene expression in normal peripheral blood mononuclear cells via modulation of the NF-kappa beta system.

The flavonoids comprise a large class of low-molecular-weight plant metabolites ubiquitously distributed in food plants. These dietary antioxidants exert significant antitumor, antiallergic, and anti-inflammatory effects. The molecular mechanisms of their biological effects remain to be clearly understood. We investigated the anti-inflammatory potentials of a safe, common dietary flavonoid component, quercetin, for its ability to modulate the production and gene expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) by human peripheral blood mononuclear cells (PBMC). Our results showed that quercetin significantly inhibited TNF-alpha production and gene expression in a dose-dependent manner. Our results provide direct evidence of the anti-inflammatory effects of quercetin by PBMC, which are mediated by the inhibition of the proinflammatory cytokine TNF-alpha via modulation of NF-kappabeta1 and Ikappabeta.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2005

RNAi-directed inhibition of DC-SIGN by dendritic cells: prospects for HIV-1 therapy.

Drug-resistant human immunodeficiency virus (HIV) infections are increasing globally, especially in North America. Therefore, it is logical to develop new therapies directed against HIV binding molecules on susceptible host cells in addition to current treatment modalities against virus functions. Inhibition of the viral genome can be achieved by degrading or silencing posttranslational genes using small interfering (si) ribonucleic acids (RNAs) consisting of double-stranded forms of RNA. These siRNAs usually contain 21-23 base pairs (bp) and are highly specific for the nucleotide sequence of the target messenger RNA (mRNA). These siRNAs form a complex with helicase and nuclease enzymes known as "RNA-induced silencing complex" (RISC) that leads to target RNA degradation. Thus, siRNA has become a method of selective destruction of HIV now used by various investigators around the globe. However, given the sequence diversity of the HIV genomes of infected subjects, it is difficult to target a specific HIV sequence. Therefore, targeting nonvariable HIV binding receptors on susceptible cells or other molecules of host cells that are directly or indirectly involved in HIV infections may be an interesting alternative to targeting the virus itself. Thus, the simultaneous use of siRNAs specific for HIV and host cells may be a unique, new approach to the therapy of HIV infections. In this article, we present evidence that siRNA directed at the CD4 independent attachment receptor (DC-SIGN) significantly inhibits HIV infection of dendritic cells (DCs). This effect may be mediated by modulation of p38 mitogen activated protein kinase (MAPK).

Cell Adhesion Molecules↗
PubMed2005

Maternal hyperinsulinemia predisposes rat fetuses for hyperinsulinemia, and adult-onset obesity and maternal mild food restriction reverses this phenotype.

We have previously shown that artificial rearing of newborn female rat pups on a high-carbohydrate (HC) milk formula resulted in chronic hyperinsulinemia and adult-onset obesity (HC phenotype) and that the maternal HC phenotype was transmitted to their progeny (2-HC rats) because of fetal development in the HC female rat. The aims of this study were to investigate 1) the fetal adaptations that predisposed the progeny for the expression of the HC phenotype in adulthood and 2) whether the transfer of the HC phenotype to the progeny could be reversed by maternal food restriction. Fetal parameters such as plasma insulin and glucose levels, mRNA level of preproinsulin gene, pancreatic insulin content, and islet insulin secretory response in vitro were determined. On gestational day 21, 2-HC fetuses were hyperinsulinemic, had increased insulin content and mRNA level of the preproinsulin gene in their pancreata and demonstrated an altered glucose-stimulated insulin secretory response by isolated islets. Modification of the intrauterine environment in HC female rats was achieved by pair feeding them to the amount of diet consumed by age-matched control rats from the time of their weaning. This mild dietary restriction reversed their HC phenotype and also prevented the development of the HC phenotype in their progeny. These findings show that mal-programming of the progeny of the hyperinsulinemic-obese HC female for the expression of the HC phenotype is initiated in utero and that normalization of the maternal environment in HC female rats by mild food restriction resulted in the normal phenotype in their progeny.

Animals↗
PubMed2005

Morphine modulates chemokine gene regulation in normal human astrocytes.

Chemokines and their receptors have been implicated in the pathogenesis of neuroAIDS. Herein we describe the effects of morphine on the gene expression of beta chemokines and their receptors by primary normal human astrocytes (NHA). Our results show that NHA treated with morphine showed significant downregulation of the gene expression of beta chemokines, MCP-1, and MIP-1 beta, while reciprocally upregulating the expression of their specific receptors, CCR2b, CCR3, and CCR5 as detected by real-time quantitative PCR. These morphine-induced effects on NHA cells were reversed by the opioid mu receptor antagonist, naloxone. Further, our results indicate that morphine-induced effects are mediated via the modulation of MAPK and CREB signaling pathways. These results support our hypothesis that opiates act as co-factors in the neuropathogenesis of HIV infection.

Astrocytes↗
PubMed2005

Prostate-specific antigen modulates the expression of genes involved in prostate tumor growth.

Prostate-specific antigen (PSA) is a serine protease that is widely used as a surrogate marker in the early diagnosis and management of prostate cancer. The physiological relevance of tissue PSA levels and their role in prostate tumor growth and metastasis are not known. Free-PSA (f-PSA) was purified to homogeneity from human seminal plasma by column chromatography, eliminating hk2 and all known PSA complexes and retaining its protease activity. Confluent monolayers of prostate cancer cell lines, PC-3M and LNCaP, were treated with f-PSA in a series of in vitro experiments to determine the changes in expression of various genes that are known to regulate tumor growth and metastasis. Gene array, quantitative polymerase chain reaction (QPCR), and enzyme-linked immunosorbent assay (ELISA) results show significant changes in the expression of various cancer-related genes in PC-3M and LNCaP cells treated with f-PSA. In a gene array analysis of PC-3M cells treated with 10 muM f-PSA, 136 genes were upregulated and 137 genes were downregulated. In LNCaP cells treated with an identical concentration of f-PSA, a total of 793 genes was regulated. QPCR analysis reveals that the genes for urokinase-type plasminogen activator (uPA), VEGF, and Pim-1 oncogene, known to promote tumor growth, were significantly downregulated, whereas IFN-gamma, known to be a tumor-suppressor gene, was significantly upregulated in f-PSA-treated PC-3M cells. The effect of f-PSA on VEGF and IFN-gamma gene expression and on protein release in PC-3M cells was distinctly dose-dependent. In vivo studies showed a significant reduction (P = .03) in tumor load when f-PSA was administered in the tumor vicinity of PC-3M tumor-bearing BALB/c nude mice. Our data support the hypothesis that f-PSA plays a significant role in prostate tumor growth by regulating various proangiogenic and antiangiogenic growth factors.

Animals↗
PubMed2005

Morphine exacerbates HIV-1 viral protein gp120 induced modulation of chemokine gene expression in U373 astrocytoma cells.

HIV-1 affects microglia and astroglia, which subsequently contributes to the neurodegenerative changes. Viral proteins cause neurotoxicity by direct action on the CNS cells or by activating glial cells to cause the release of cytokines, chemokines or neurotoxic substances. Opioid abuse has been postulated as a cofactor in the immunopathogenesis of human immunodeficiency virus (HIV) infection and AIDS. HIV-induced pathogenesis is exacerbated by opiate abuse and that the synergistic neurotoxicity is a direct effect of opiates on the CNS. Chemokines and their receptors have been implicated in the pathogenesis of neuroAIDS. Herein we describe the effects of morphine and/or gp120 on the expression of the genes for the beta-chemokine MIP-1beta and its receptors CCR3 and CCR5 by the U373 cells which are a human brain-derived astrocytoma/glioblastoma cell line. Our results indicate that treatment of U373 cells with morphine significantly downregulated the gene expression of the beta chemokine, MIP-1 beta, while reciprocally upregulating the expression of its specific receptors, CCR3 and CCR5 suggesting that the capacity of mu-opioids to increase HIV-1 co-receptor expression may promote viral binding, trafficking of HIV-1-infected cells, and enhanced disease progression. Additionally, opiates can enhance the cytotoxicity of HIV-1 viral protein gp120 via mechanisms that involve intracellular calcium modulation resulting in direct actions on astroglia, making them an important cellular target for HIV-opiate interactions.

AIDS Dementia Complex↗
PubMed2004

Gene expression of angiogenic factors correlates with metastatic potential of prostate cancer cells.

We hypothesize that expression of proangiogenic genes correlates with the metastatic potential of prostate cancer cells. LNCaP, DU-145, and PC-3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as we demonstrated by their capacity to invade an extracellular matrix, an established tumor invasion assay. The constitutive gene expression of the proangiogenic factors, vascular endothelial growth factor, intercellular adhesion molecule-1, interleukin-8, and transforming growth factor-beta2, was significantly greater in the more metastatic DU-145 and PC-3 cells as compared with LNCaP cells. Matrix metalloproteinase (MMP)-9 is thought to contribute to the invasive phenotype of tumor cells. PC-3 cells showed increased expression of MMP-9 and membrane type 4-MMP as compared with LNCaP and DU-145. Tissue inhibitors of metalloproteinase 1 and 4 gene expression were elevated in DU-145 and PC-3 cells, but paradoxically, LNCaP cells had undetectable levels of these genes. We transfected and overexpressed MMP-9 in poorly metastatic LNCaP cells and measured their invasive activity. Transient expression of human MMP-9 in LNCaP cells produced a 3-5-fold increase in MMP-9 activity with a comparable increase in invasiveness. Antisense ablation of the expression of MMP-9 in DU-145 and PC-3 cells produced concomitant inhibition of the gene expression of the proangiogenic factors, vascular endothelial growth factor, and intercellular adhesion molecule-1 (ICAM-1). Treatment of DU-145 and PC-3 cells with a selective chemical inhibitor of MMP-9 proteinase activity also inhibited their invasive activity. These results support our hypothesis that metastatic potential of prostate cancer cells correlates with expression of proangiogenic factors.

Angiogenesis Inducing Agents↗
PubMed2004

Inhibition of prostate cancer cell colony formation by the flavonoid quercetin correlates with modulation of specific regulatory genes.

The natural product quercetin is a flavonoid found in many fruits and vegetables. Previous research has shown that quercetin has antitumor, anti-inflammatory, antiallergic, and antiviral activities. In the present investigation we studied the effect of quercetin on the ability of prostate cancer cell lines with various degrees of aggressive potential to form colonies in vitro. Specifically, we examined the molecular mechanisms underlying this effect, including the expression of cell cycle and tumor suppressor genes as well as oncogenes. We observed that quercetin at concentrations of 25 and 50 micro M significantly inhibited the growth of the highly aggressive PC-3 prostate cancer cell line and the moderately aggressive DU-145 prostate cancer cell line, whereas it did not affect colony formation by the poorly aggressive LNCaP prostate cancer cell line or the normal fibroblast cell line BG-9. Using the gene array methodology, we found that quercetin significantly inhibited the expression of specific oncogenes and genes controlling G(1), S, G(2), and M phases of the cell cycle. Moreover, quercetin reciprocally up-regulated the expression of several tumor suppressor genes. In conclusion, our results demonstrate that the antitumor effects of quercetin directly correlate with the aggressive potential of prostate cancer cells and that the mechanism(s) of quercetin-mediated antitumor effects may involve up-regulation of tumor suppressor genes and reciprocal down-regulation of oncogenes and cell cycle genes. The results of these studies provide a scientific basis for the potential use of flavonoids as nutraceuticals in the chemoprevention of cancer.

Antineoplastic Agents, Phytogenic↗
PubMed2003

Effector cell mediated cytotoxicity measured by intracellular Granzyme B release in HIV infected subjects.

CD8+ cytotoxic T lymphocyte (CTL) activity is currently believed to be one of the key immunologic mechanisms responsible for the prevention or attenuation of HIV-1 infection. The induction of CD8+ T cell activation may also result in the production of soluble or non-classical lytic factors that are associated with protection from infection or slower disease progression. Traditionally, CD8+ CTL responses have been measured by the classic chromium release assay, monitoring the ability of T cells (Effector cells) to lyse radiolabelled HLA - matched "target cells" that express the appropriate antigen-MHC complex. This method is not only labor intensive, semi quantitative assay at best, but also needs fresh, non-cryopreserved cells. Recently, cytokine specific ELISPOT assays or tetrameric MHC-I/ peptide complexes have utilized to directly quantitate circulating CD8+ effector cells, and these assays are more sensitive, quantitative and reproducible than the traditional CTL lysis assay and can also be performed on cryopreserved cells. Although these are reproducible assays for the assessment of soluble antiviral activity secreted by activated T cell populations they can be extremely expensive to perform. We have used FACS Analysis to measure Granzyme B release as a function of cell mediated cytotoxicity. This method helps quantitate the CTL activity and also identifies the phenotype of the cells elucidating this immune response. The method described not only monitors immunological response but also is also simple to perform, precise and extremely time efficient and is ideal for screening a large number of samples.

Journal Article↗
PubMed2003

Programming of islet functions in the progeny of hyperinsulinemic/obese rats.

Neonatal female rat pups that were raised artificially on a high-carbohydrate (HC) milk formula during their suckling period developed hyperinsulinemia immediately, maintained chronic hyperinsulinemia in the postweaning period on laboratory diet, and developed obesity in adulthood. Pups (second-generation HC [2-HC]) born to such female rats (first-generation HC [1-HC]) spontaneously developed chronic hyperinsulinemia and adult-onset obesity (HC phenotype) without the requirement for any dietary intervention in their suckling period. Leftward shift in the insulin secretory response to a glucose stimulus, increase in hexokinase activity, and increased preproinsulin gene transcription were observed in islets from 28-day-old 2-HC rats, and these adaptations are similar to those reported for islets from 12-day-old and 100-day-old 1-HC rats. Unlike 1-HC islets, the ability to secrete moderate amounts of insulin in the absence of glucose and calcium and the incretin input for augmentation of insulin secretion were not observed in 2-HC islets. These results show that a dietary modification in the early postnatal life of the 1-HC female rat sets up a vicious cycle of spontaneous transfer of the HC phenotype to its progeny, implicating a new component to the growing list of factors that contribute to the fetal origins of adult-onset diseases.

Animals↗
PubMed2002

Glucose lowering effect of aqueous extract of Enicostemma littorale Blume in diabetes: a possible mechanism of action.

Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia. Enicostemma littorale Blume is a small herb and recently we have reported its blood glucose lowering potential in alloxan induced diabetic rats. A single dose of aqueous extract of E. littorale (15 g dry plant equivalent extract per kg) had shown significant increase in the serum insulin levels in alloxan-induced diabetic rats at 8 h. The insulinotropic action of aqueous extract of E. littorale was further investigated using rat pancreatic islets. Extract has the potential to enhance glucose-induced insulin release at 11.1 mM glucose from isolated rat pancreatic islets and was partially able to reverse the effect of diazoxide (0.25 mM). Incubation with Ca(2+) chelator (EGTA) and Ca(2+) channel blocker (nimodipine) did not affect the glucose-induced insulin release augmented by the extract. Above results suggest the glucose lowering effect of aqueous extract of E. littorale to be associated with potentiation of glucose-induced insulin release through K(+)-ATP channel dependent pathway but did not require Ca(2+) influx.

Animals↗