Alteration of superoxide- and nitric oxide-mediated antimicrobial function of macrophages by in vivo cocaine exposure

Cocaine is a popular drug of abuse and despite impressive advances in the understanding of its physiological, pharmacological, and toxic effects, its mechanism of immunosuppression at the cellular level is not well understood. In this paper we report the role of effector molecules like superoxide and nitric oxide in the antibacterial function of macrophages exposed to acute and chronic doses of cocaine in vivo. Bacterial killing by acute cocaine-exposed macrophages (ACE-Mphis) increased significantly, with a concomitant rise in respiratory burst and generation of superoxide and nitric oxide, compared with control macrophages. In contrast, chronic cocaine-exposed macrophages (CCE-Mphis) exhibited limited antimicrobial activity, which correlated closely with diminished respiratory burst and reduced production of superoxide and nitric oxide. Further, a killing assay was carried out in the presence of N(G)-methyl-L-arginine acetate, an inhibitor of iNOS, to evaluate the role of nitric oxide in the killing process. The results obtained indicate that while about 30% killing of input bacteria by control and ACE-Mphis was attributable to NO-mediated killing, only about 6% killing from NO was found with CCE-Mphis. The findings indicate that acute exposure to cocaine possibly caused upregulation of enzymes responsible for the generation of ROI (reactive oxygen intermediates) and RNI (reactive nitrogen intermediates), leading to enhanced antimicrobial function. On the other hand, chronic exposure to cocaine impaired the oxygen-dependent microbicidal capacity of macrophages, possibly through impaired expression of enzymes responsible for ROI and RNI formation. Proinflammatory cytokines may play a key role in cocaine-mediated immunosuppression, since exposure of macrophages to cocaine impairs the ability of the cells to produce these cytokines.     

Ack1-mediated androgen receptor phosphorylation modulates radiation resistance in castration-resistant prostate cancer

Androgen deprivation therapy has been the standard of care in prostate cancer due to its effectiveness in initial stages. However, the disease recurs, and this recurrent cancer is referred to as castration-resistant prostate cancer (CRPC). Radiotherapy is the treatment of choice; however, in addition to androgen independence, CRPC is often resistant to radiotherapy, making radioresistant CRPC an incurable disease. The molecular mechanisms by which CRPC cells acquire radioresistance are unclear. Androgen receptor (AR)-tyrosine 267 phosphorylation by Ack1 tyrosine kinase (also known as TNK2) has emerged as an important mechanism of CRPC growth. Here, we demonstrate that pTyr(267)-AR is recruited to the ATM (ataxia telangiectasia mutated) enhancer in an Ack1-dependent manner to up-regulate ATM expression. Mice engineered to express activated Ack1 exhibited a significant increase in pTyr(267)-AR and ATM levels. Furthermore, primary human CRPCs with up-regulated activated Ack1 and pTyr(267)-AR also exhibited significant increase in ATM expression. The Ack1 inhibitor AIM-100 not only inhibited Ack1 activity but also was able to suppress AR Tyr(267) phosphorylation and its recruitment to the ATM enhancer. Notably, AIM-100 suppressed Ack1 mediated ATM expression and mitigated the growth of radioresistant CRPC tumors. Thus, our study uncovers a previously unknown mechanism of radioresistance in CRPC, which can be therapeutically reversed by a new synergistic approach that includes radiotherapy along with the suppression of Ack1/AR/ATM signaling by the Ack1 inhibitor, AIM-100.     

Morphine and galectin-1 modulate HIV-1 infection of human monocyte-derived macrophages

Morphine is a widely abused, addictive drug that modulates immune function. Macrophages are a primary reservoir of HIV-1; therefore, they play a role in the development of this disease, as well as impact the overall course of disease progression. Galectin-1 is a member of a family of β-galactoside-binding lectins that are soluble adhesion molecules and that mediate direct cell-pathogen interactions during HIV-1 viral adhesion. Because the drug abuse epidemic and the HIV-1 epidemic are closely interrelated, we propose that increased expression of galectin-1 induced by morphine may modulate HIV-1 infection of human monocyte-derived macrophages (MDMs). In this article, we show that galectin-1 gene and protein expression are potentiated by incubation with morphine. Confirming previous studies, morphine alone or galectin-1 alone enhance HIV-1 infection of MDMs. Concomitant incubation with exogenous galectin-1 and morphine potentiated HIV-1 infection of MDMs. We used a nanotechnology approach that uses gold nanorod-galectin-1 small interfering RNA complexes (nanoplexes) to inhibit gene expression for galectin-1. We found that nanoplexes silenced gene expression for galectin-1, and they reversed the effects of morphine on galectin-1 expression. Furthermore, the effects of morphine on HIV-1 infection were reduced in the presence of the nanoplex.     

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.¹ Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.² These include RPE65 gene replacement therapy in patients with Leber''s congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt''s disease.^3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.^5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.     

Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association

The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.     

Combination of dipeptidylpeptidase IV inhibitor and low dose thiazolidinedione: preclinical efficacy and safety in db/db mice

Thiazolidinediones (TZDs) are currently the most efficacious class of oral antidiabetics. However, they carry the burden of weight gain and haemodilution, which may lead to cardiovascular complications. The present study was designed to ascertain whether a combination of dipeptidyl peptidase IV (DPP IV) inhibitor with low dose of a thiazolidinedione absolves TZD associated weight gain and oedema without compromising its efficacy. In this study, we examined the efficacy and safety of lower dose (1 mg/kg/day) of rosiglitazone, a thiazolidinedione, in combination with 5 mg/kg/day dose of LAF-237 (vildagliptin), a known DPP IV inhibitor, in aged db/db mice after 14 days of treatment and compared the combination with therapeutic dose (10 mg/kg) of rosiglitazone. The combination therapy showed similar efficacy as that of 10 mg/kg/day rosiglitazone in lowering random blood glucose (53.8%, p<0.001 and 54.3%, p<0.001 respectively), AUC ((0-120) min) during oral glucose tolerance test (OGTT) (38.6 %, p<0.01; 38.3%, p<0.01 respectively) and triglyceride levels (63.9% and 61% respectively; p<0.01). Plasma active glucagon like peptide-1 (GLP-1) and insulin levels were found to be elevated significantly (p<0.01 and p<0.05 respectively) in both LAF-237 and combination treated groups following oral glucose load. LAF-237 alone had no effect on random glucose and glucose excursion during OGTT in severely diabetic db/db mice. Interestingly, the combination treatment showed no significant increase in body weight as compared to the robust weight gain by therapeutic dose of rosiglitazone. Rosiglitazone at 10 mg/kg/day showed significant reduction (p<0.05) in haematocrit, RBC count, haemoglobin pointing towards haemodilution associated with increased mRNA expression of Na(+), K(+)-ATPase-alpha and epithelial sodium channel gamma (ENaCgamma) in kidney. The combination therapy escaped these adverse effects. The results suggest that combination of DPP IV inhibitor with low dose of thiazolidinedione can interact synergistically to represent a therapeutic advantage for the clinical treatment of type 2 diabetes without the adverse effects of haemodilution and weight gain associated with thiazolidinediones.