In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine

<p>Abstract</p> <p>Background</p> <p>Coccidioidomycosis or Valley fever is caused by a highly virulent fungal pathogen: <it>Coccidioides posadasii </it>or <it>immitis</it>. Vaccine development against <it>Coccidioides </it>is of contemporary interest because a large number of relapses and clinical failures are reported with antifungal agents. An efficient Th1 response engenders protection. Thus, we have focused on developing a dendritic cell (DC)-based vaccine for coccidioidomycosis. In this study, we investigated the immunostimulatory characteristics of an intranasal primary DC-vaccine in BALB/c mouse strain that is most susceptible to coccidioidomycosis. The DCs were transfected nonvirally with <it>Coccidioides-</it>Ag2/PRA-cDNA. Expression of DC-markers, Ag2/PRA and cytokines were studied by flow cytometry, dot-immunoblotting and cytometric bead array methods, respectively. The T cell activation was studied by assessing the upregulation of activation markers in a DC-T cell co-culture assay. For trafficking, the DCs were co-transfected with a plasmid DNA encoding HSV1 thymidine kinase (TK) and administered intranasally into syngeneic mice. The trafficking and homing of TK-expressing DCs were monitored with positron emission tomography (PET) using ¹⁸F-FIAU probe. Based on the PET-probe accumulation in vaccinated mice, selected tissues were studied for antigen-specific response and T cell phenotypes using ELISPOT and flow cytometry, respectively.</p> <p>Results</p> <p>We found that the primary DCs transfected with <it>Coccidioides</it>-Ag2/PRA-cDNA were of immature immunophenotype, expressed Ag2/PRA and activated naïve T cells. In PET images and subsequent biodistribution, intranasally-administered DCs were found to migrate in blood, lung and thymus; lymphocytes showed generation of T effector memory cell population (T_EM) and IFN-γ release.</p> <p>Conclusions</p> <p>In conclusion, our results demonstrate that the intranasally-administered primary DC vaccine is capable of inducing Ag2/PRA-specific T cell response. Unique approaches utilized in our study represent an attractive and novel means of producing and evaluating an autologous DC-based vaccine.</p

Ubiquitin Receptor RPN13 Mediates the Inhibitory Interaction of Diphenyldihaloketones CLEFMA and EF24 With the 26S Proteasome

The proteasome is a validated target in drug discovery for diseases associated with unusual proteasomal activity. Here we report that two diphenyldihaloketones, CLEFMA and EF24, inhibit the peptidase activity of the 26S proteasome. The objective of this study was to investigate interaction of these compounds with the proteasome and identify a putative target within the protein components of the 26S proteasome. We employed standard fluorogenic peptide-based proteasome activity assay for trypsin-like, chymotrypsin-like, and caspase-like activities of human purified 26S proteasome in cell-free conditions. GFPu-1 and HUVEC cells were used as proteasome reporter cells. Direct binding studies used purified 19S, 20S, 26S, and recombinant RPN13-Pru for interaction with biotinylated analogs of CLEFMA and EF24. The reaction mixtures were subjected to horizontal gel electrophoresis, streptavidin-blotting, pull-down assays, and immunoblotting. The identity of the interacting protein was determined by 2D gel electrophoresis and LC-MS/MS. Drug affinity responsive target stability technique was utilized to examine if CLEFMA binding confers protection to RPN13 against thermolysin-catalyzed proteolysis. We found that trypsin-and chymotrypsin-like activities of the 26S proteasome were reduced significantly by both compounds. The compounds also reduced the proteolytic activity in GFPu-1 and HUVEC cells, resulting in accumulation of ubiquitinated proteins without affecting the autophagy process. From direct binding assays a 43 kDa protein in the 26S proteasome was found to be the interacting partner. This protein was identified by tandem mass spectroscopy as regulatory particle subunit 13 (RPN13), a ubiquitin receptor in the 19S regulatory particle. Furthermore, binding of CLEFMA to RPN13 did not protect latter from thermolysin-mediated proteolysis. Together, this study showed diphenyldihaloketones as potential proteasome inhibitors for treatment of diseases with perturbed proteasome function. The results also unraveled RPN13 as a unique target of CLEFMA and EF24. As a result, these compounds inhibit both trypsin-like and chymotrypsin-like proteasome activities

Surface Engineering of Liposomes for Stealth Behavior

Liposomes are used as a delivery vehicle for drug molecules and imaging agents. The major impetus in their biomedical applications comes from the ability to prolong their circulation half-life after administration. Conventional liposomes are easily recognized by the mononuclear phagocyte system and are rapidly cleared from the blood stream. Modification of the liposomal surface with hydrophilic polymers delays the elimination process by endowing them with stealth properties. In recent times, the development of various materials for surface engineering of liposomes and other nanomaterials has made remarkable progress. Poly(ethylene glycol)-linked phospholipids (PEG-PLs) are the best representatives of such materials. Although PEG-PLs have served the formulation scientists amazingly well, closer scrutiny has uncovered a few shortcomings, especially pertaining to immunogenicity and pharmaceutical characteristics (drug loading, targeting, etc.) of PEG. On the other hand, researchers have also begun questioning the biological behavior of the phospholipid portion in PEG-PLs. Consequently, stealth lipopolymers consisting of non-phospholipids and PEG-alternatives are being developed. These novel lipopolymers offer the potential advantages of structural versatility, reduced complement activation, greater stability, flexible handling and storage procedures and low cost. In this article, we review the materials available as alternatives to PEG and PEG-lipopolymers for effective surface modification of liposomes

Surface Modification of Liposomes by a Lipopolymer Targeting Prostate Specific Membrane Antigen for Theranostic Delivery in Prostate Cancer

Prostate specific membrane antigen (PSMA) is a marker for diagnosis and targeted delivery of therapeutics to advanced/metastasized prostate cancer. We report a liposome-based system for theranostic delivery to PSMA-expressing (PSMA+) LNCaP cells. A lipopolymer (P3) comprising of PSMA ligand (PSMAL), polyethylene glycol (PEG2000), and palmitate was synthesized and post-inserted into the surface of preformed liposomes. These P3-liposomes were loaded with doxorubicin and radiolabeled with 99mTc radionuclide to study their theranostic characteristics. Differential expression of PSMA on LNCaP and PC3 cells was confirmed by immunoblotting as well as by uptake of PSMAL labeled with 18F radionuclide. We found that the uptake of 99mTc-labeled P3-liposomes by LNCaP cells was &gt;3-fold higher than 99mTc-labeled Plain-liposomes; the amount of doxorubicin delivered to LNCaP cells was also found to be &gt;3-fold higher by P3-liposomes. Cell-based cytotoxicity assay results showed that doxorubicin-loaded P3-liposomes were significantly more toxic to LNCaP cells (p &lt; 0.05), but not to PSMA-negative PC3 cells. Compared to doxorubicin-loaded Plain-liposomes, the IC50 value of doxorubicin-loaded P3-liposomes was reduced by ~5-fold in LNCaP cells. Together, these results suggest that surface functionalization of liposomes with small PSMA-binding motifs, such as PSMAL, can provide a viable platform for specific delivery of theranostics to PSMA+ prostate cancer

2-[3,5-Bis-(2-fluorobenzylidene)-4-piperidon-1-yl]-N-(4-fluorobenzyl)-acetamide and Its Evaluation as an Anticancer Agent

Synthesis of 2-[3,5-bis-(2-fluorobenzylidene)-4-piperidon-1-yl]-N-(4-fluorobenzyl)-acetamide, a derivative of 3,5-bis-(2-fluorobenzylidene)-4-piperidone (EF24), as an antiproliferative and imageable compound is described. The radioactive derivative was synthesized in 40–45% radiochemical yield using N-[4-fluoro(18F)benzyl]-2-bromoacetamide (NFLOBA) as a radiolabeled synthon for coupling with EF24. Cell proliferation assays showed that 2-[3,5-bis-(2-fluorobenzylidene)-4-piperidon-1-yl]-N-(4-fluorobenzyl)-acetamide (NFLOBA-EF24) had antiproliferative efficacy similar to that of EF24 in lung adenocarcinoma H441 cells. 18F-NFLOBA-EF24 was investigated in normal rats for whole-body PET imaging and biodistribution. At necropsy after 1 h of injection, about 12% of injected compound was still circulating in blood; liver, kidney, and muscle were other tissues with moderate amounts of accumulation. In order to assess the tumor-suppressive activity, nonradioactive NFLOBA-EF24 was administered in nude rats carrying xenograft H441 tumor. After 15 days of treatment, the tumor size decreased by approximately 83% compared to the tumors in control rats. The tumor regression was also confirmed by molecular imaging of glucose metabolism with 18F- fluorodeoxyglucose. The results suggest that EF24 could be efficiently modified with 18F-labeled synthon NFLOBA for convenient PET imaging without altering the antitumor efficacy of the original compound. This study provides visual kinetics of synthetic curcuminoid EF24 by positron emission tomography for the first time