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Ischemic stroke-induced neuronal cell death results in the permanent disabling of brain function. Apoptotic mechanisms are thought to play a prominent role in neuronal injury and ample evidence implicates Fas signaling in mediating cell death. In this study, we describe the neuroprotective effects of a Fas-blocking peptide (FBP) that by obstructing Fas signaling in cerebral ischemia inhibits apoptosis. Using an intranasal administration route in a rat model of focal cerebral ischemia, we demonstrate that nose-to-brain delivery of FBP after middle cerebral artery occlusion (MCAO) surgery results in the delivery and retention of FBP in Fas-expressing ischemic areas of the brain. A single intranasal administration of 2 mg/kg FBP resulted in significantly reduced neuronal cell death by inhibiting Fas-mediated apoptosis leading to decreased infarct volumes, reduced neurologic deficit scores and recovery from cerebral ischemia. Intranasally delivered FBP might be a promising strategy for the treatment of cerebral ischemic stroke.

The HIV-1 pandemic affecting over 37 million people worldwide continues, with nearly one-half of the infected population on highly active antiretroviral therapy (HAART). Major therapeutic challenges remain because of the emergence of drug-resistant HIV-1 strains, limitations because of safety and toxicity with current HIV-1 drugs, and patient compliance for lifelong, daily treatment regimens. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral polymerase have been a key component of the current HIV-1 combination drug regimens; however, these issues hamper them. Thus, the development of novel more effective NNRTIs as anti-HIV-1 agents with fewer long-term liabilities, efficacy on new drug-resistant HIV-1 strains, and less frequent dosing is crucial. Using a computational and structure-based design strategy to guide lead optimization, a 5 µM virtual screening hit was transformed to a series of very potent nanomolar to picomolar catechol diethers. One representative, compound I, was shown to have nanomolar activity in HIV-1-infected T cells, potency on clinically relevant HIV-1 drug-resistant strains, lack of cytotoxicity and off-target effects, and excellent in vivo pharmacokinetic behavior. In this report, we show the feasibility of compound I as a late-stage preclinical candidate by establishing synergistic antiviral activity with existing HIV-1 drugs and clinical candidates and efficacy in HIV-1-infected humanized [human peripheral blood lymphocyte (Hu-PBL)] mice by completely suppressing viral loads and preventing human CD4 + T-cell loss. Moreover, a long-acting nanoformulation of compound I [compound I nanoparticle (compound I-NP)] in poly(lactide-coglycolide) (PLGA) was developed that shows sustained maintenance of plasma drug concentrations and drug efficacy for almost 3 weeks after a single dose., Competing Interests: The authors declare no conflict of interest.

The number of humanized mouse models for the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and other infectious diseases has expanded rapidly over the past 8 years. Highly immunodeficient mouse strains, such as NOD/SCID/gamma chain(null) (NSG, NOG), support better human hematopoietic cell engraftment. Another improvement is the derivation of highly immunodeficient mice, transgenic with human leukocyte antigens (HLAs) and cytokines that supported development of HLA-restricted human T cells and heightened human myeloid cell engraftment. Humanized mice are also used to study the HIV reservoir using new imaging techniques. Despite these advances, there are still limitations in HIV immune responses and deficits in lymphoid structures in these models in addition to xenogeneic graft-versus-host responses. To understand and disseminate the improvements and limitations of humanized mouse models to the scientific community, the NIH sponsored and convened a meeting on April 15, 2015 to discuss the state of knowledge concerning these questions and best practices for selecting a humanized mouse model for a particular scientific investigation. This report summarizes the findings of the NIH meeting.

Adipose tissue macrophage (ATM)-mediated inflammation is a key feature contributing to the adverse metabolic outcomes of dietary obesity. Recruitment of macrophages to obese adipose tissues (AT) can occur through the engagement of CCR2, the receptor for MCP-1 (monocyte chemoattractant protein-1), which is expressed on peripheral monocytes/macrophages. Here, we show that i.p. administration of a rabies virus glycoprotein-derived acetylcholine receptor-binding peptide effectively delivers complexed siRNA into peritoneal macrophages and ATMs in a mouse model of high-fat diet-induced obesity. Treatment with siRNA against CCR2 inhibited macrophage infiltration and accumulation in AT and, therefore, proinflammatory cytokines produced by macrophages. Consequently, the treatment significantly improved glucose tolerance and insulin sensitivity profiles, and also alleviated the associated symptoms of hepatic steatosis and reduced hepatic triglyceride production. These results demonstrate that disruption of macrophage chemotaxis to the AT through cell-targeted gene knockdown strategies can provide a therapeutic intervention for obesity-related metabolic diseases. The study also highlights a siRNA delivery approach for targeting specific monocyte subsets that contribute to obesity-associated inflammation without affecting the function of other tissue-resident macrophages that are essential for host homeostasis and survival.

Dendritic cells can capture and transfer retroviruses in vitro across synaptic cell-cell contacts to uninfected cells, a process called trans-infection. Whether trans-infection contributes to retroviral spread in vivo remains unknown. Here, we visualize how retroviruses disseminate in secondary lymphoid tissues of living mice. We demonstrate that murine leukemia virus (MLV) and human immunodeficiency virus (HIV) are first captured by sinus-lining macrophages. CD169/Siglec-1, an I-type lectin that recognizes gangliosides, captures the virus. MLV-laden macrophages then form long-lived synaptic contacts to trans-infect B-1 cells. Infected B-1 cells subsequently migrate into the lymph node to spread the infection through virological synapses. Robust infection in lymph nodes and spleen requires CD169, suggesting that a combination of fluid-based movement followed by CD169-dependent trans-infection can contribute to viral spread., (Copyright © 2015, American Association for the Advancement of Science.)

Cell-penetrating peptides (CPPs), such as nona-arginine (9R), poorly translocate siRNA into cells. Our studies demonstrate that attaching 9R to ligands that bind cell surface receptors quantitatively increases siRNA uptake and importantly, allows functional delivery of complexed siRNA. The mechanism involved accumulation of ligand-9R:siRNA microparticles on the cell membrane, which induced transient membrane inversion at the site of ligand-9R binding and rapid siRNA translocation into the cytoplasm. siRNA release also occurred late after endocytosis when the ligand was attached to the L isoform of 9R, but not the protease-resistant 9DR, prolonging mRNA knockdown. This critically depended on endosomal proteolytic activity, implying that partial CPP degradation is required for endosome-to-cytosol translocation. The data demonstrate that ligand attachment renders simple polycationic CPPs effective for siRNA delivery by restoring their intrinsic property of translocation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Despite antiretroviral therapy (ART), human immunodeficiency virus (HIV)-1 persists in a stable latent reservoir, primarily in resting memory CD4(+) T cells. This reservoir presents a major barrier to the cure of HIV-1 infection. To purge the reservoir, pharmacological reactivation of latent HIV-1 has been proposed and tested both in vitro and in vivo. A key remaining question is whether virus-specific immune mechanisms, including cytotoxic T lymphocytes (CTLs), can clear infected cells in ART-treated patients after latency is reversed. Here we show that there is a striking all or none pattern for CTL escape mutations in HIV-1 Gag epitopes. Unless ART is started early, the vast majority (>98%) of latent viruses carry CTL escape mutations that render infected cells insensitive to CTLs directed at common epitopes. To solve this problem, we identified CTLs that could recognize epitopes from latent HIV-1 that were unmutated in every chronically infected patient tested. Upon stimulation, these CTLs eliminated target cells infected with autologous virus derived from the latent reservoir, both in vitro and in patient-derived humanized mice. The predominance of CTL-resistant viruses in the latent reservoir poses a major challenge to viral eradication. Our results demonstrate that chronically infected patients retain a broad-spectrum viral-specific CTL response and that appropriate boosting of this response may be required for the elimination of the latent reservoir.

Effective delivery of DNA or siRNA into primary cells demands an efficient delivery system. However, the significant differences in physical and molecular characteristics of the two molecules generally necessitate distinct delivery systems or considerable differences in carrier formulation protocols for effective transfection. Arginine-grafted bioreducible poly (disulfide amine) (ABP) is a redox-sensitive, bioreducible, positively charged polymer which complexes with siRNA and DNA via charge interactions to form nanoplexes. ABP effectively mediates cytoplasmic delivery of both DNA and siRNA into multiple cell types, including primary cells like myoblast, human umbilical vein endothelial cells (HUVECs), and primary rat aorta vascular smooth muscle cells (SMCs) eliciting functional activity. In this chapter, we provide the detailed protocols for the synthesis of ABP as well as transfection of both siRNA and DNA using ABP.

Biodegradable poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) encapsulating triplex-forming peptide nucleic acids (PNAs) and donor DNAs for recombination-mediated editing of the CCR5 gene were synthesized for delivery into human peripheral blood mononuclear cells (PBMCs). NPs containing the CCR5-targeting molecules efficiently entered PBMCs with low cytotoxicity. Deep sequencing revealed that a single treatment with the formulation resulted in a targeting frequency of 0.97% in the CCR5 gene and a low off-target frequency of 0.004% in the CCR2 gene, a 216-fold difference. NP-treated PBMCs efficiently engrafted immunodeficient NOD-scid IL-2rγ(-/-) mice, and the targeted CCR5 modification was detected in splenic lymphocytes 4 weeks posttransplantation. After infection with an R5-tropic strain of HIV-1, humanized mice with CCR5-NP-treated PBMCs displayed significantly higher levels of CD4(+) T cells and significantly reduced plasma viral RNA loads compared with control mice engrafted with mock-treated PBMCs. This work demonstrates the feasibility of PLGA-NP-encapsulated PNA-based gene-editing molecules for the targeted modification of CCR5 in human PBMCs as a platform for conferring HIV-1 resistance.Molecular Therapy-Nucleic Acids (2013) 2, e135; doi:10.1038/mtna.2013.59; published online 19 November 2013.

The human malaria parasite Plasmodium falciparum is absolutely dependent on the acquisition of host pantothenate for its development within human erythrocytes. Although the biochemical properties of this transport have been characterized, the molecular identity of the parasite-encoded pantothenate transporter remains unknown. Here we report the identification and functional characterization of the first protozoan pantothenate transporter, PfPAT, from P. falciparum. We show using cell biological, biochemical, and genetic analyses that this transporter is localized to the parasite plasma membrane and plays an essential role in parasite intraerythrocytic development. We have targeted PfPAT to the yeast plasma membrane and showed that the transporter complements the growth defect of the yeast fen2Δ pantothenate transporter-deficient mutant and mediates the entry of the fungicide drug, fenpropimorph. Our studies in P. falciparum revealed that fenpropimorph inhibits the intraerythrocytic development of both chloroquine- and pyrimethamine-resistant P. falciparum strains with potency equal or better than that of currently available pantothenate analogs. The essential function of PfPAT and its ability to deliver both pantothenate and fenpropimorph makes it an attractive target for the development and delivery of new classes of antimalarial drugs.

Small interfering RNA (siRNA) represent an interesting class of developmental nucleic acid-based therapeutics. Cationic carriers for deoxyribonucleic acids (DNA) are potential vehicles for siRNA delivery. However, in contrast to supercoiled plasmid DNA, the physical properties of siRNA molecules induces the formation of larger, loosely-packed complexes with most polycationic carriers, and consequently, poor target silencing. Here, we investigate a gene delivery agent, arginine-grafted bioreducible poly (disulfide amine) polymer (ABP) for siRNA delivery as it contains arginine residues with siRNA binding properties. ABP combines the attributes of polycations and poly disulfide-amines namely- excellent cell-penetrability and rapid release after disulphide bond reduction in the intracellular compartment. ABP bound siRNA, assembled into stable 150 nm sized nanoparticles and efficiently released complexed siRNA upon cellular entry. We investigated the utility of ABP in a combinatorial RNAi strategy for solid cancer therapy. Systemic administration of ABP-siRNA resulted in a preferential and enhanced accumulation of carrier-siRNA complexes in the tumor tissue. Two administrations of the formulation with a siRNA cocktail targeting Bcl-2, VEGF and Myc at 0.3 mg total siRNA/kg body weight could effectively regress advanced stage tumors. Our results establish the promise of ABP as a common systemic delivery platform for both siRNA and DNA therapeutics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Humanized mice have recently emerged as powerful translational animal models for studying human hematopoiesis, immune interactions, and diseases of the human immune system. Several important advances in the humanized mouse technology have been reported over the last few years, thereby resulting in improved engraftment, high levels of human chimerism, and sustained human hematopoiesis. This chapter describes the detailed procedures for generating various humanized mouse models including hu-PBL, hu-HSC, and BLT models and discusses considerations for choosing the appropriate model system.

Cellular targeting and intracellular delivery of small interfering RNA (siRNA) remain a critical barrier to the clinical application of RNA interference. This chapter provides an overview of various delivery agents employing protein ligands mediating cell-specific delivery of siRNA. Specifically, the chapter details methodologies for the conjugation of antibody or peptide ligands to i) the cationic peptide-oligo-9-arginine (ii) the polymer poly(lactic-co-glycolic acid) (PLGA) and (iii) a lipid-vesicle (liposome)., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The ability to block the expression of any disease-causing gene or disease-related protein highlights the potential use of RNAi technology in the therapy of 'undruggable' human diseases. However, considering the risks associated with RNAi therapy, targeting and restricting the action of siRNA to specific cells could greatly minimize toxic side effects. However, this is a major challenge, as many primary cell types are highly recalcitrant to siRNA uptake. This review discusses advances in siRNA targeting methods for human T-cells, with an emphasis on the potential use of an RNAi-based therapy for the treatment of HIV/AIDS.

RNA interference (RNAi) has the potential to permit the downregulation of virtually any gene. While transgenic RNAi enables stable propagation of the resulting phenotype to progeny, the dominant nature of RNAi limits its use to applications where the continued suppression of gene expression does not disturb normal cell functioning. This is of particular importance when the target gene product is essential for cell survival, development or differentiation. It is therefore desirable that knockdown be externally regulatable. This review is aimed at providing an overview of the approaches for conditional RNAi in mammalian systems, with a special mention of studies employing these approaches to target therapeutically/biologically relevant molecules, their advantages and disadvantages, and a pointer towards approaches best suited for RNAi-based gene therapy.

RNA interference (RNAi), initially recognized as a natural antiviral mechanism in plants, has rapidly emerged as an invaluable tool to suppress gene expression in a sequence-specific manner in all organisms, including mammals. Its potential to inhibit the replication of a variety of viruses has been demonstrated in vitro and in vivo in mouse and monkey models. These results have generated profound interest in the use of this technology as a potential treatment strategy for viral infections for which vaccines and drugs are unavailable or inadequate. In this review, we discuss the progress made within the past 2-3 years towards harnessing the potential of RNAi for clinical application in viral infections and the hurdles that have yet to be overcome.

The organization of flaviviral replicase proteins within the membrane-bound replication complexes of West Nile (WNV), dengue (DENV) and Japanese encephalitis viruses (JEV) was probed by investigating the combined effect of detergents and trypsin on both viral replicase activity and profile of metabolically labelled viral proteins. While trypsin treatment of virus-induced membrane fractions degraded the vast majority of replicase proteins, viral RNA-dependent RNA polymerase (RdRp) activity remained completely unaffected. Solubilization of the membranes with deoxycholate (DOC) however rendered the replicase accessible to trypsin. Triton X-100 (TX100) treatment reduced RdRp activity by half in WNV but totally destroyed RdRp activity in JEV. TX100 also dissociated NS1' in addition to NS1 from NS5 and NS3 inJEV. Antibodies to NS3 coprecipitated NS1' along with NS5 only from DOC-solubilized but not from TX100-treated extracts, the former of which alone retained RdRp activity. Exogenous addition of recombinant NS1' to TX100 treated JEV-induced membranes restored the defect in the release step of RNA synthesis. Our results suggest for the first time a direct role for JEV NS1' in viral RNA synthesis in vitro.

Japanese encephalitis virus (JEV) is the principal cause of viral encephalitis. The predominance of children among patients with encephalitis in areas where JEV is endemic that do not have immunization programs suggests that acquired immunity is critical in protecting adults against symptomatic JEV encephalitis. We characterized and compared the T cell response to nonstructural (NS) protein 3 between healthy individuals naturally exposed to JEV and patients in the convalescent phase of JEV encephalitis. The NS3 protein, used as a fusion to the 11-amino acid protein transduction domain of human immunodeficiency virus Tat, elicited CD4(+) and T helper-dependent CD8(+) T cell responses. The production of interferon (IFN)- gamma by responding T cells was significantly higher in healthy donors than in patients, correlating strongly with acquired protective immunity to JEV. Furthermore, a striking inverse association between IFN- gamma levels and the severity of postencephalitic sequelae in patients implicated a role of IFN- gamma in recovery.

The search to identify Mycobacterium tuberculosis antigens capable of conferring protective immunity against tuberculosis has received a boost owing to the resurgence of tuberculosis over the past two decades. It has long been recognized that lymphoid cells are required for protection against M. tuberculosis. While traditionally the CD4(+) populations of T cells were believed to predominantly serve this protective function, a pivotal role for CD8(+) T cells in this task has been increasingly appreciated. We show that the 50- to 55-kDa Apa protein, specified by the Rv1860 gene of M. tuberculosis, can elicit both lymphoproliferative response and gamma interferon (IFN-gamma) production from peripheral blood mononuclear cells (PBMC) of purified protein derivative (PPD)-positive individuals, with significant differences recorded in the levels of responsiveness between PPD-positive healthy controls and pulmonary tuberculosis patients. Flow cytometric analysis of whole blood stimulated with the recombinant Apa protein revealed a sizeable proportion of CD8(+) T cells in addition to CD4(+) T cells contributing to IFN-gamma secretion. PBMC responding to the Apa protein produced no interleukin-4, revealing a Th1 phenotype. A DNA vaccine and a poxvirus recombinant expressing the Apa protein were constructed and tested for their ability to protect immunized guinea pigs against a challenge dose of virulent M. tuberculosis. Although the DNA vaccine afforded little protection, the poxvirus recombinant boost after DNA vaccine priming conferred a significant level of protective immunity, bringing about a considerable reduction in mycobacterial counts from the challenge bacilli in spleens of immunized guinea pigs, a result comparable to that achieved by BCG vaccination.

A situation analysis was undertaken to assess impediments to health impact assessment (HIA) in the South-East Asia Region of WHO (SEARO). The countries of the region were assessed on the policy framework and procedures for HIA, existing infrastructure required to support HIA, the capacity for undertaking HIA, and the potential for intersectoral collaboration. The findings show that environmental impact assessment (EIA) is being used implicitly as a substitute for HIA, which is not explicitly or routinely conducted in virtually all countries of the Region. Therefore, policy, infrastructure, capacity, and intersectoral collaboration need strengthening for the routine implementation of HIA.