Galectin-1 suppresses methamphetamine induced neuroinflammation in human brain microvascular endothelial cells: Neuroprotective role in maintaining blood brain barrier integrity.

Methamphetamine (Meth) abuse can lead to the breakdown of the blood-brain barrier (BBB) integrity leading to compromised CNS function. The role of Galectins in the angiogenesis process in tumor-associated endothelial cells (EC) is well established; however no data are available on the expression of Galectins in normal human brain microvascular endothelial cells and their potential role in maintaining BBB integrity. We evaluated the basal gene/protein expression levels of Galectin-1, -3 and -9 in normal primary human brain microvascular endothelial cells (BMVEC) that constitute the BBB and examined whether Meth altered Galectin expression in these cells, and if Galectin-1 treatment impacted the integrity of an in-vitro BBB. Our results showed that BMVEC expressed significantly higher levels of Galectin-1 as compared to Galectin-3 and -9. Meth treatment increased Galectin-1 expression in BMVEC. Meth induced decrease in TJ proteins ZO-1, Claudin-3 and adhesion molecule ICAM-1 was reversed by Galectin-1. Our data suggests that Galectin-1 is involved in BBB remodeling and can increase levels of TJ proteins ZO-1 and Claudin-3 and adhesion molecule ICAM-1 which helps maintain BBB tightness thus playing a neuroprotective role. Galectin-1 is thus an important regulator of immune balance from neurodegeneration to neuroprotection, which makes it an important therapeutic agent/target in the treatment of drug addiction and other neurological conditions.

Nanotherapeutic approach for opiate addiction using DARPP-32 gene silencing in an animal model of opiate addiction.

Opiates act on the dopaminergic system of the brain and perturb 32 kDa dopamine and adenosine 3', 5'-monophosphate-regulated phosphoprotein (DARPP-32) function. The DARPP-32 mediated inhibition of protein phosphatase-1 (PP-1) and modulation of transcriptional factor CREB is critical to the changes in neuronal plasticity that result in behavioral responses during drug abuse. To investigate the role of DARPP-32 mediated signaling on withdrawal behavior in a rat model of opiate addiction, we used intracerebral administration of gold nanorods (GNR) complexed to DARPP-32 siRNA to silence DARPP-32 gene expression and measure its effects on the opiate withdrawal syndrome. We hypothesized that DARPP-32 siRNA will suppress the neurochemical changes underlying the withdrawal syndrome and therefore prevent conditioned place aversion by suppressing or removing the constellation of negative effects associated with withdrawal, during the conditioning procedure. Our results showed that opiate addicted animals treated with GNR-DARPP-32 siRNA nanoplex showed lack of condition place aversive behavior consequent to the downregulation of secondary effectors such as PP-1 and CREB which modify transcriptional gene regulation and consequently neuronal plasticity. Thus, nanotechnology based delivery systems could allow sustained knockdown of DARPP-32 gene expression which could be developed into a therapeutic intervention for treating drug addiction by altering reward and motivational systems and interfere with conditioned responses.

Isolation and characterization of nine microsatellite loci from Bemisia tabaci (Hemiptera: Aleyrodidae) biotype B.

Nine microsatellites were isolated from Bemisia tabaci (Gennadius) biotype B and screened across 60 individuals from two populations (biotype B) to examine polymorphism. Two to 12 alleles were observed per locus. Observed and expected heterozygosities ranged from 0.033 to 0.967 and 0.033 to 0.854, respectively. There was no significant deviation from Hardy-Weinberg equilibrium and no significant linkage disequilibrium between loci. One locus showed evidence for null alleles. These loci will be useful in future studies of the genetic structure of worldwide biotypes and gene flow analyses between and within biotypes of B. tabaci.

Genomic and proteomic analysis of the effects of cannabinoids on normal human astrocytes.

Delta-9-tetrahydrocannabinol (Delta(9)-THC), the main psychoactive component of marijuana, is known to dysregulate various immune responses. Cannabinoid (CB)-1 and -2 receptors are expressed mainly on cells of the central nervous system (CNS) and the immune system. The CNS is the primary target of cannabinoids and astrocytes are known to play a role in various immune responses. Thus we undertook this investigation to determine the global molecular effects of cannabinoids on normal human astrocytes (NHA) using genomic and proteomic analyses. NHA were treated with Delta(9)-THC and assayed using gene microarrays and two-dimensional (2D) difference gel electrophoresis (DIGE) coupled with mass spectrometry (MS) to elucidate their genomic and proteomic profiles respectively. Our results show that the expression of more than 20 translated protein gene products from NHA was differentially dysregulated by treatment with Delta(9)-THC compared to untreated, control NHA.

Association of drug abuse with inhibition of HIV-1 immune responses: studies with long-term of HIV-1 non-progressors.

Recreational drug use has been proposed to affect the course of human immunodeficiency virus (HIV) infections. To investigate the effects of substance abuse on HIV infections, we compared virus-specific cytotoxic T lymphocyte (CTL) responses and the expression of IL-16, TGF-beta1, and CXCR4 in three different cohorts of HIV-infected patients: (1) long-term nonprogressors (LT-NPs) of HIV infection who do not use recreational drugs; (2) nondrugs using normal progressors (NPs), and (3) drugs using NPs. Our results show that LT-NPs manifest increased CTL activity and IL-16 expression and decreased expression of TGF-beta1 and CXCR4 compared to NPs, regardless of recreational drug usage. Furthermore, drugs using NPs showed significantly lower levels of CTL and IL-16 expression and increased TGF-beta1 and CXCR4 expression compared to nondrugs using NPs. Our results suggest that recreational drug use may reduce CTL and IL-16 expression and increase the expression of TGF-beta1 and CXCR4, all of which may facilitate progression of HIV infections.

Sensitization of human colon cancer cells to TRAIL-mediated apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL), a novel member of the tumor necrosis factor (TNF) family, is thought to induce apoptosis preferentially in cancer cells; however, increasing evidence suggests that a number of cancers are resistant to TRAIL treatment. FLICE-like inhibitory protein (FLIP), which structurally resembles caspase-8, can act as an inhibitor of apoptosis when expressed at high levels in certain cancer cells. The purpose of our present study was to determine whether human colon cancer cells are sensitive to TRAIL treatment and, if not, to identify potential mechanisms of resistance. Colon cancer cells of different metastatic potential (KM12C, KML4A, and KM20) were found to be resistant to the effects of TRAIL when used as a single agent. FLIP expression levels were increased in all three KM cell lines. Treatment with either actinomycin D (Act D;10 :g/ml) or cycloheximide (CHX; 10 :g/ml) decreased FLIP expression levels in all three cell lines. The decrease in cellular levels of FLIP was associated with sensitization to TRAIL-mediated apoptosis, as demonstrated by enhanced cell death and caspase-3 activity compared with either Act D or CHX alone. Our findings suggest that reduction of FLIP levels by Act D or CHX renders TRAIL-resistant human colon cancer cells sensitive to TRAIL-mediated apoptosis. The combination of TRAIL along with agents such as Act D or CHX, which target proteins that prevent cell death, may provide a more effective and less toxic regimen for treatment of resistant colon cancers.

Intravenous immunoglobulin treatment of immunodeficiency disorders.

ADDs can occur as primary genetic disorders or may develop secondary to various other conditions, including infections, trauma, malnutrition, and protein-losing states. Although antibiotics are the first-line therapy for acute infection, using them prophylactically can select for resistant organisms. IM ISG and fresh frozen plasma were the principal agents for antibody-replacement therapy until the advent of IVIG 2 decades ago. IVIG is now the definitive product for antibody-replacement therapy. Although IVIG has a long history of safety regarding the infectious pathogens, the identification of more than 100 patients with non-A, non-B hepatitis apparently acquired from a single product prompted additional modifications, improving the safety profile of IVIG. Despite the excellent safety record of IVIG, the unexpected occurrence of hepatitis in some recipients served as a reminder that IVIG is a biologic product derived from human plasma. Newer products are being developed that may supplement polyvalent IVIG including humanized MAbs and hyperimmune IVIG preparations to address specific clinical requirements.

The stress hormone, cortisol, synergizes with HIV-1 gp-120 to induce apoptosis of normal human peripheral blood mononuclear cells.

Both quantitative and qualitative defects in immune functions in patients with AIDS may result from induction of programmed cell death or apoptosis of CD4 T lymphocytes. We postulate that neurohormones may interact with gp-120 that is shed during active HIV infection and cause apoptosis of immunocompetent cells leading to immunopathogenesis of HIV infections. In this study, we investigated the synergistic effect of cortisol plus HIV gp-120 in inducing apoptosis of lymphocytes from normal subjects. Total peripheral blood mononuclear cells and isolated CD4+ T-cells were treated with cortisol or gp-120 separately and in combination and RNA and DNA were extracted. RNA was reverse transcribed and amplified with specific primers for Fas and Fas ligand and analyzed on agarose gels. DNA was analyzed by gel electrophoresis for ladder formation, the hallmark for apoptosis, and Fas antigen expression by confocal microscopy. Results demonstrate that cortisol and gp-120 induce apoptosis of lymphocytes from normal donors as demonstrated by DNA ladder formation, TUNEL staining and Fas gene expression. Concentrations of cortisol and gp-120 that did not produce apoptosis when used separately, induced significant apoptosis when used in combination. Further, gp-120 induced DNA fragmentation was significant in the CD4+ T-cell subpopulation compared to the CD47 subpopulation. This study suggests that the stress-associated neurohormone, cortisol, synergizes with HIV peptides in causing apoptosis of normal lymphocytes. The synergistic effect of cortisol and gp- 120 in inducing apoptosis of lymphocytes is consistent with a model proposing that stress-associated and circulating HIV-1 derived soluble products may cause progression of HIV infections.

Reversal of human immunodeficiency virus type 1 protein-induced inhibition of natural killer cell activity by alpha interferon and interleukin-2.

A recombinant fusion peptide, Env-Gag, derived from the human immunodeficiency virus type 1 (HIV-1) genome corresponding to a defined portion of the envelope (Env) and internal core (Gag) proteins was examined for immunoregulatory effects on the cytotoxic activity of natural killer (NK) cell-enriched, large granular lymphocytes (LGL) from healthy donors. Percoll-separated, NK cell-enriched LGL precultured for 24 h with Env-Gag at 10- and 50-ng/ml concentrations, which significantly stimulated lymphocyte proliferation, caused significant suppression of NK cell activity. Denatured Env-Gag did not cause any effect on the NK cell activity of LGL. Two other control peptides, one derived from the Escherichia coli vector used to clone the HIV Env-Gag fusion peptide and the other derived from a non-HIV-1 viral antigen (rubeola virus), did not produce any observable effect on the NK cell activity of LGL, demonstrating the specificity of the effect produced by Env-Gag. Subsequent treatment of LGL with alpha interferon (IFN-alpha) or interleukin 2 (IL-2) alone partially reversed the Env-Gag-induced suppression of NK cell activity. However, LGL treated with both IFN-alpha and IL-2 completely reversed the suppression of NK cell cytotoxicity by Env-Gag. The combined effect of IFN-alpha and IL-2 in enhancing NK cell activity may provide a novel therapeutic approach to the restoration of depressed NK cell activity observed in HIV-infected patients.

Cocaine differentially modulates chemokine production by mononuclear cells from normal donors and human immunodeficiency virus type 1-infected patients.

Earlier studies have supported a significant role for cocaine in the susceptibility to and the progression of human immunodeficiency virus type 1 (HIV-1) infection. Recently, several unique HIV-1 entry coreceptors (e.g., CCR5 and CCR3) and a trio of HIV-1-specific suppressor chemokines, namely, RANTES (regulated-upon-activation T expressed and secreted), macrophage inflammatory protein 1alpha (MIP-1alpha) and MIP-1beta, were identified. Although cocaine has been linked to the immunopathogenesis of HIV-1 infection, the corresponding cellular and molecular mechanism(s) have not been well defined. We hypothesize that cocaine mediates these pathologic effects through the downregulation of HIV-1-suppressing chemokines and/or upregulating HIV-1 entry coreceptors in HIV-1-infected subjects, resulting in disease progression to AIDS. Our results show that cocaine selectively downregulates endogenous MIP-1beta secretion by normal peripheral blood mononuclear cells (PBMC), while cocaine did not affect the MIP-1beta production by PBMC from AIDS patients. Cocaine also selectively suppresses lipopolysaccharide-induced MIP-1beta production by PBMC from HIV-infected patients. Further, cocaine significantly downregulates endogenous MIP-1beta gene expression, while it upregulates HIV-1 entry coreceptor CCR5 by normal PBMC. These studies suggests a role for cocaine as a cofactor in the pathogenesis of HIV infection and support the premise that cocaine increases susceptibility to and progression of HIV-1 infection by inhibiting the synthesis of HIV-1 protective chemokines and/or upregulating the HIV-1 entry coreceptor, CCR5.

The role of NF-kappaB/IkappaB proteins in cancer: implications for novel treatment strategies.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors are involved in multiple cellular processes, including cytokine gene expression, cellular adhesion, cell cycle activation, apoptosis and oncogenesis. Constitutive activation of NF-kappaB has been described in a number of solid tumors and this activation appears to affect cancer cell survival. Inhibition of NF-kappaB has been shown to enhance the sensitivity of some cancer cell lines to antineoplastic- or radiation-induced apoptosis. Furthermore, suppression of NF-kappaB results in attenuation of cancer cachexia in a mouse tumor model. Studies are underway to further delineate the role of NF-kappaB in cancer cell survival, growth and resistance to standard chemotherapy and radiation regimens. Moreover, the effects of novel therapeutic agents which specifically target NF-kappaB proteins are currently being assessed in experimental models of cancer cell growth both in vitro and in vivo. In this review, we discuss the possible involvement of NF-kappaB in the growth of various solid tumors and potential future treatment strategies based on NF-kappaB inhibition.

An RGD containing peptide from HIV-1 Tat-(65-80) modulates protooncogene expression in human bronchoalveolar carcinoma cell line, A549.

Tat (transactivator of transcription) is essential for HIV-1 replication in vivo and in vitro. Tat-(65-80), an RGD containing domain, has been shown to regulate proliferative function of a variety of cell lines, including a human adenocarcinoma cell line, A549. The exact cellular and molecular mechanisms by which these effects are mediated, remain unknown. To evaluate the hypothesis that Tat-(65-80) modulates the expression of immediate early genes (IEG) c-jun, c-myc, c-fos and the tumor suppressor gene p53, serum starved A549 cells were incubated with Tat-(65-80) or heat-inactivated Tat-(65-80) at 10 ng/ml. Total cellular RNA was isolated from the cells at various time points (0-24 hours). In each case, 5 micrograms of RNA was reverse transcribed in 20 microliters of reaction volume. Equal amounts of cDNA were subjected to polymerase chain reaction (PCR) and analyzed by electrophoresis. The photographic negatives of the ethidium bromide stained gels were quantitated by densitometric scanning and normalized to corresponding beta-actin PCR products. Treatment with Tat-(65-80) showed a twofold induction of c-jun at 0.5 h. Peak expression occurred at 60 minutes and remained above baseline at 24 hours (h). c-myc was increased at 0.5 h, reached a twofold increase at 2 h and remained above baseline at 24 h. c-fos increased seven fold at 0.5 h and declined subsequently to baseline at 8 h. p-53 gene was reduced fivefold at 0.5 h and remained downregulated thereafter. These results show that Tat-(65-80) can modulate growth related genes in human lung epithelial cells.

Immunoregulatory effects of morphine on human lymphocytes.

It is now well established that parenteral drug abuse is a significant risk factor for contracting human immunodeficiency virus type 1 (HIV-1) infection and subsequently developing AIDS. Earlier studies have shown that morphine can modulate various immune responses and therefore support the premise that morphine is a cofactor in susceptibility to and progression of HIV infection. Dysregulation of interferon (IFN) production, nonspecific apoptosis of T cells, and the immune response to soluble HIV gene products have been associated with potential mechanisms of pathogenesis in HIV disease. The present study was undertaken to examine the immunomodulatory role of morphine on HIV protein-induced lymphocyte proliferative responses, Sendai and Newcastle disease virus-induced alpha IFN (IFN-alpha) and IFN-beta production by lymphocytes and fibroblast cells, respectively, and induction of apoptosis of normal lymphocytes in vitro. Our results demonstrate that HIV protein-induced human lymphocyte proliferative responses were significantly inhibited by morphine in a dose-dependent manner. Furthermore, morphine significantly inhibited both IFN-alpha and IFN-beta production by normal lymphocytes and fibroblasts but induced apoptosis of normal lymphocytes. Inhibition of IFN-alpha production by morphine could be reversed by the opiate receptor antagonist naloxone. This suggests that the immunomodulatory effects of morphine are mediated through the opioid receptor. These studies support a role of morphine as a cofactor in the pathogenesis of HIV infection and describe some of the possible pathologic mechanisms which underlie the immunoregulatory effects of morphine.

Molecular mechanisms in the pathogenesis of AIDS encephalopathy.

While progress has been made in our knowledge of the natural history of HIV infections, an understanding of the molecular pathogenesis of AIDS encephalopathy remains to be determined. Previously we demonstrated that apoptosis and a deficiency of natural killer (NK) cell activity may play significant roles in the progression of HIV infections. We also reported that intracerebral co-injection of a recombinant HIV-1 fusion protein plus an excitatory amino acid agonist into neonatal rats synergistically produced brain pathology. Here we examine: 1) the effects of the HIV-1 envelope protein, gp-120, on neonatal rat astrocytes in vitro; 2) the spontaneous apoptosis of human neonatal and adult mononuclear leukocytes and, 3) the selective inhibition of NK activity cell from adult AIDS patients by the HIV-1 protein that caused brain pathology in neonatal rats. We demonstrate that gp-120 suppresses fas gene expression, a marker for apoptosis, by neonatal rat astrocytes. Neonatal human mononuclear leukocytes manifest spontaneous apoptosis as measured by DNA ladder formation while cells from adult donors do not. Direct addition of the HIV-1 protein to mononuclear cells in vitro selectively suppressed the NK cell activity from AIDS patients. These results support our premise that HIV-1 proteins are involved in the pathogenesis of AIDS encephalopathy.

Inhibition of natural killer cell activities from normal donors and AIDS patients by envelope peptides from human immunodeficiency virus type I.

Recombinant and synthetic peptides derived from the human immunodeficiency virus type I (HIV-1) genome corresponding to portions of the envelope (env) and internal core protein (gag) were examined for their immunoregulatory effects on the natural killer (NK) cell activity of lymphocytes from healthy donors and from patients with the acquired immunodeficiency syndrome (AIDS). Two recombinant peptides (env-gag and Env 80-DHFR) and three chemically synthesized peptides (env 487-511, env 578-608 and env 647-659) were used. Normal lymphocytes precultured for 24 to 72 hrs. with either env-gag, env 487-511, or env 647-659 at 5 and 50 ng/ml concentrations which significantly stimulated lymphocyte proliferation, produced significant suppression of NK activities. Two control peptides, one derived from the E. coli vector used to clone the HIV env-gag fusion peptide and another, a non-HIV-1 viral antigen (rubeola virus) did not produce any observable effect on NK activity of normal lymphocytes demonstrating the specificity of the reaction. Env-gag peptide also inhibited the NK activities of Percoll-separated, NK-enriched large granular lymphocytes. In target binding assays, lymphocytes precultured with env-gag significantly suppressed the target binding capacity of effector cells and produced significantly lower levels of natural killer cytotoxic factor (NKCF). In kinetic studies, lymphocytes from normal donors preincubated with env-gag for 24 to 72 hrs. produced significant inhibition of their NK activity and an even greater inhibitory effect on NK activities was observed when lymphocytes from AIDS patients were preincubated with HIV peptides. Thus HIV-1 peptides, which we previously demonstrated could regulate B- and T-lymphocyte activities, are also capable of regulating the NK activities of lymphocytes from HIV-1-infected and normal individuals.

Management of a maxillary canine with dens invaginatus and a vital pulp.

A case is presented that demonstrates successful management of a maxillary canine with dens invaginatus (Oehlers' type 3 invagination) with associated chronic periradicular periodontitis and a vital pulp. Debridement and obturation of the invaginated space resulted in resolution of the associated periradicular radiolucency. Pulp vitality was retained after endodontic treatment of the invagination.

Alcohol inhibits lipopolysaccharide-induced tumor necrosis factor alpha gene expression by peripheral blood mononuclear cells as measured by reverse transcriptase PCR in situ hybridization.

We recently showed that alcohol significantly suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) production by whole blood and total mononuclear cells from healthy subjects as measured by bioassay. In the current study, we further examined the effect of alcohol on LPS-induced TNF-alpha gene expression by semiquantitative solution PCR and in situ reverse transcriptase PCR (RT-PCR) hybridization methods. Peripheral blood mononuclear cells were cultured with LPS (10 micrograms/ml) for 4 to 8 h with or without different concentrations of ethanol (0.1, 0.2, and 0.3% [vol/vol]). Total RNA from treated and untreated cultures was extracted and used for solution PCR analysis. Treated and untreated cells were subjected to both conventional in situ hybridization and RT-PCR in situ hybridization. In solution RT-PCR in vitro analysis, alcohol significantly suppressed TNF-specific message. In conventional in situ hybridization, the effect of alcohol on TNF-alpha gene expression was poorly detected. However, when cells were subjected to RT-PCR prior to in situ hybridization, cells treated with alcohol significantly suppressed expression of the message for TNF-alpha. These studies confirm our earlier finding that alcohol suppressed the production of TNF-alpha by LPS-induced whole blood cells and peripheral blood mononuclear cells. Furthermore, these studies also demonstrate that the RT-PCR in situ technique is a powerful tool for detecting and amplifying specific genes in whole cells when limited numbers of cells are available for RNA extraction.