Peptide: MHC-based DNA vaccination strategy to activate natural killer cells by targeting killer cell immunoglobulin-like receptors.

BACKGROUND: Natural killer (NK) cells are increasingly being recognized as agents for cancer immunotherapy. The killer cell immunoglobulin-like receptors (KIRs) are expressed by NK cells and are immunogenetic determinants of the outcome of cancer. In particular, KIR2DS2 is associated with protective responses to several cancers and also direct recognition of cancer targets in vitro. Due to the high homology between activating and inhibitory KIR genes to date, it has been challenging to target individual KIR for therapeutic benefit. METHODS: A novel KIR2DS2-targeting therapeutic peptide:MHC DNA vaccine was designed and used to immunize mice transgenic for KIR genes (KIR-Tg). NK cells were isolated from the livers and spleens of vaccinated mice and then analyzed for activation by flow cytometry, RNA profiling and cytotoxicity assays. In vivo assays of NK cell function using a syngeneic cancer model (B16 melanoma) and an adoptive transfer model for human hepatocellular carcinoma (Huh7) were performed. RESULTS: Injecting KIR-Tg mice with the vaccine construct activated NK cells in both liver and spleens of mice, with preferential activation of KIR2DS2-positive NK cells. KIR-specific activation was most marked on the CD11b+CD27+ mature subset of NK cells. RNA profiling indicated that the DNA vaccine upregulated genes associated with cellular metabolism and downregulated genes related to histone H3 methylation, which are associated with immune cell maturation and NK cell function. Vaccination led to canonical and cross-reactive peptide:MHC-specific NK cell responses. In vivo, DNA vaccination led to enhanced antitumor responses against B16F10 melanoma cells and also enhanced responses against a tumor model expressing the KIR2DS2 ligand HLA-C*0102. CONCLUSION: We show the feasibility of a peptide-based KIR-targeting vaccine strategy to activate NK cells and hence generate functional antitumor responses. This approach does not require detailed knowledge of the tumor peptidomes nor HLA matching with the patient. It therefore offers a novel opportunity for targeting NK cells for cancer immunotherapy.

Aerosol Delivery of siRNA to the Lungs. Part 2: Nanocarrier-based Delivery Systems.

In this article, applications of engineered nanoparticles containing siRNA for inhalation delivery are reviewed and discussed. Diseases with identified protein malfunctions may be mitigated through the use of well-designed siRNA therapeutics. The inhalation route of administration provides local delivery of siRNA therapeutics to the lungs for various pulmonary diseases. A siRNA delivery system can be used to overcome the barriers of pulmonary delivery, such as anatomical barriers, mucociliary clearance, cough clearance, and alveolar macrophage clearance. Apart from naked siRNA aerosol delivery, previously studied siRNA carrier systems include those of lipidic, polymeric, peptide, or inorganic origin. These delivery systems can achieve pulmonary delivery through the generation of an aerosol via an inhaler or nebulizer. The preparation methodologies for these siRNA nanocarrier systems will be discussed herein. The use of inhalable nanocarrier siRNA delivery systems have barriers to their effective delivery, but overcoming these constraints while formulating a safe and effective delivery system will offer unique advances to the field of inhaled medicine.

The Addiction-Related Protein ANKK1 is Differentially Expressed During the Cell Cycle in Neural Precursors.

TaqIA is a polymorphism associated with addictions and dopamine-related traits. It is located in the ankyrin repeat and kinase domain containing 1 gene (ANKK1) nearby the gene for the dopamine D2 receptor (D2R). Since ANKK1 function is unknown, TaqIA-associated traits have been explained only by differences in D2R. Here we report ANKK1 studies in mouse and human brain using quantitative real-time PCR, Western blot, immunohistochemistry, and flow cytometry. ANKK1 mRNA and protein isoforms vary along neurodevelopment in the human and mouse brain. In mouse adult brain ANKK1 is located in astrocytes, nuclei of postmitotic neurons and neural precursors from neurogenic niches. In both embryos and adults, nuclei of neural precursors show significant variation of ANKK1 intensity. We demonstrate a correlation between ANKK1 and the cell cycle. Cell synchronization experiments showed a significant increment of ANKK1-kinase in mitotic cells while ANKK1-kinase overexpression affects G1 and M phase that were found to be modulated by ANKK1 alleles and apomorphine treatment. Furthermore, during embryonic neurogenesis ANKK1 was expressed in slow-dividing neuroblasts and rapidly dividing precursors which are mitotic cells. These results suggest a role of ANKK1 during the cell cycle in neural precursors thus providing biological support to brain structure involvement in the TaqIA-associated phenotypes.

Enhanced Anticancer Activity of PF-04691502, a Dual PI3K/mTOR Inhibitor, in Combination With VEGF siRNA Against Non-small-cell Lung Cancer.

Lung cancer is the leading cause of cancer deaths in both men and women in the United States accounting for about 27% of all cancer deceases. In our effort to develop newer therapy for lung cancer, we evaluated the combinatory antitumor effect of siRNA targeting VEGF and the PI3K/mTOR dual inhibitor PF-04691502. We analyzed the anticancer effect of siRNA VEGF and PF-04691502 combination on proliferation, colony formation and migration of A549 and H460 lung cancer cells. Additionally, we assessed the combination treatment antiangiogenic effect on human umbilical vein endothelial cells. Here, we show for the first time that the antiangiogenic siRNA VEGF potentiates the PF-04691502 anticancer activity against non-small-cell lung cancer. We observed a significant (P < 0.05) decrease in cell viability, colony formation, and migration for the combination comparing with the single drug treatment. We also showed a significant (P < 0.05) enhanced effect of the combination treatment inhibiting angiogenesis progression and tube formation organization compared to the single drug treatment groups. Our findings demonstrated an enhanced synergistic anticancer effect of siRNA VEGF and PF-04691502 combination therapy by targeting two main pathways involved in lung cancer cell survival and angiogenesis which will be useful for future preclinical studies and potentially for lung cancer patient management.

Aerosol Delivery of siRNA to the Lungs. Part 1: Rationale for Gene Delivery Systems.

This article reviews the pulmonary route of administration, aerosol delivery devices, characterization of pulmonary drug delivery systems, and discusses the rationale for inhaled delivery of siRNA. Diseases with known protein malfunctions may be mitigated through the use of siRNA therapeutics. The inhalation route of administration provides local delivery of siRNA therapeutics for the treatment of various pulmonary diseases, however barriers to pulmonary delivery and intracellular delivery of siRNA exists. siRNA loaded nanocarriers can be used to overcome the barriers associated with the pulmonary route, such as anatomical barriers, mucociliary clearance, and alveolar macrophage clearance. Apart from naked siRNA aerosol delivery, previously studied siRNA carrier systems comprise of lipidic, polymeric, peptide, or inorganic origin. Such siRNA delivery systems formulated as aerosols can be successfully delivered via an inhaler or nebulizer to the pulmonary region. Preclinical animal investigations of inhaled siRNA therapeutics rely on intratracheal and intranasal siRNA and siRNA nanocarrier delivery. Aerosolized siRNA delivery systems may be characterized using in vitro techniques, such as dissolution test, inertial cascade impaction, delivered dose uniformity assay, laser diffraction, and laser Doppler velocimetry. The ex vivo techniques used to characterize pulmonary administered formulations include the isolated perfused lung model. In vivo techniques like gamma scintigraphy, 3D SPECT, PET, MRI, fluorescence imaging and pharmacokinetic/pharmacodynamics analysis may be used for evaluation of aerosolized siRNA delivery systems. The use of inhalable siRNA delivery systems encounters barriers to their delivery, however overcoming the barriers while formulating a safe and effective delivery system will offer unique advances to the field of inhaled medicine.

ßArrestin1 regulates the guanine nucleotide exchange factor RasGRF2 expression and the small GTPase Rac-mediated formation of membrane protrusion and cell motility.

ßArrestin proteins shuttle between the cytosol and nucleus and have been shown to regulate G protein-coupled receptor signaling, actin remodeling, and gene expression. Here, we tested the hypothesis that ßarrestin1 regulates actin remodeling and cell migration through the small GTPase Rac. Depletion of ßarrestin1 promotes Rac activation, leading to the formation of multipolar protrusions and increased cell circularity, and overexpression of a dominant negative form of Rac reverses these morphological changes. Small interfering RNA library screen identifies RasGRF2 as a target of ßarrestin1. RasGRF2 gene and protein expression levels are elevated following depletion of ßarrestin1, and the consequent activation of Rac results in dephosphorylation of cofilin that can promote actin polymerization and formation of multipolar protrusions, thereby retarding cell migration and invasion. Together, these results suggest that ßarrestin1 regulates rasgrf2 gene expression and Rac activation to affect membrane protrusion and cell migration and invasion.

The ANKK1 gene associated with addictions is expressed in astroglial cells and upregulated by apomorphine.

BACKGROUND: TaqIA, the most widely analyzed genetic polymorphism in addictions, has traditionally been considered a gene marker for association with D2 dopamine receptor gene (DRD2). TaqIA is located in the coding region of the ANKK1 gene that overlaps DRD2 and encodes a predicted kinase ANKK1. The ANKK1 protein nonetheless had yet to be identified. This study examined the ANKK1 expression pattern as a first step to uncover the biological bases of TaqIA-associated phenotypes. METHODS: Northern blot and quantitative reverse-transcriptase polymerase chain reaction analyses were performed to analyze the ANKK1 mRNA. To study ANKK1 protein expression, we developed two polyclonal antibodies to a synthetic peptides contained in the putative Ser/Thr kinase domain. RESULTS: We demonstrate that ANKK1 mRNA and protein were expressed in the adult central nervous system (CNS) in human and rodents, exclusively in astrocytes. Ankk1 mRNA level in mouse astrocyte cultures was upregulated by apomorphine, suggesting a potential relationship with the dopaminergic system. Developmental studies in mice showed that ANKK1 protein was ubiquitously located in radial glia in the CNS, with an mRNA expression pick around embryonic Day 15. This time expression pattern coincided with that of the Drd2 mRNA. On induction of differentiation by retinoic acid, a sequential expression was found in human neuroblastoma, where ANKK1 was expressed first, followed by that of DRD2. An opposite time expression pattern was found in rat glioma. CONCLUSIONS: Spatial and temporal regulation of the expression of ANKK1 suggest an involvement of astroglial cells in TaqIA-related neuropsychiatric phenotypes both during development and adult life.

Gender-specific COMT Val158Met polymorphism association in Spanish schizophrenic patients.

The functional Val158Met polymorphism (rs4680) located at the gene that codes for the catechol-O-methyltransferase (COMT) has been extensively investigated in schizophrenia although current data are still controversial. Since COMT activity is sexually dimorphic, we carried out two independent studies in homogeneous samples of male and female Spanish schizophrenic patients. In males, we found an association between the homozygous Val genotype and the disorder, which resembled a recessive model (P = 0.022; odds ratio [OR] = 1.67). This Val homozygotes overrepresentation is produced at the expense of the heterozygous individuals decrease, whilst the Met homozygotes showed no differences when compared controls and patients. As a consequence, the heterozygous genotype in this sample had a protective effect (P = 0.03; OR = 0.65) and a strong deviation from Hardy-Weinberg equilibrium in male cases was observed (P = 0.006). In addition, a 2-SNP haplotype analysis (rs4818-Val158Met) confirmed there is an overrepresentation of the different homozygous Val genotypes in the male schizophrenic sample. Regarding females, we did not find any statistically significant association between COMT SNP and schizophrenia. In the light of this we suggest that the Val158Met SNP is involved in risk and protective genotypes for the vulnerability to schizophrenia in Spanish male population.