Targeted co-delivery of FOXM1 aptamer and DOX by nucleolin aptamer-functionalized pH-responsive biocompatible nanodelivery system to enhance therapeutic efficacy against breast cancer: in vitro and in vivo.

Targeted nanodelivery systems offer a promising approach to cancer treatment, including the most common cancer in women, breast cancer. In this study, a targeted, pH-responsive, and biocompatible nanodelivery system based on nucleolin aptamer-functionalized biogenic titanium dioxide nanoparticles (TNP) was developed for targeted co-delivery of FOXM1 aptamer and doxorubicin (DOX) to improve breast cancer therapy. The developed targeted nanodelivery system exhibited almost spherical morphology with 124.89 ± 12.97 nm in diameter and zeta potential value of - 23.78 ± 3.66 mV. FOXM1 aptamer and DOX were loaded into the nanodelivery system with an efficiency of 100% and 97%, respectively. Moreover, the targeted nanodelivery system demonstrated excellent stability in serum and a pH-responsive sustained drug release profile over a period of 240 h following Higuchi kinetic and Fickian diffusion mechanism. The in vitro cytotoxicity experiments demonstrated that the targeted nanodelivery system provided selective internalization and strong growth inhibition effects of about 45 and 51% against nucleolin-positive 4T1 and MCF-7 breast cancer cell lines. It is noteworthy that these phenomena were not observed in nucleolin-negative cells (CHO). The preclinical studies revealed that a single-dose intravenous injection of the targeted nanodelivery system into 4T1-bearing mice inhibited tumor growth by 1.7- and 1.4-fold more efficiently than the free drug and the non-targeted nanodelivery system, respectively. Our results suggested that the developed innovative targeted pH-responsive biocompatible nanodelivery system could serve as a prospectively potential platform to improve breast cancer treatment.

Development of an adjuvanted nanoparticle vaccine against influenza virus, an in vitro study.

Influenza is an infectious respiratory illness caused by influenza viruses. Despite yearly updates, the efficacy of influenza vaccines is significantly curtailed by the virus antigenic drift and antigenic shift. These constant changes to the influenza virus make-up also challenge the development of a universal flu vaccine, which requires conserved antigenic regions shared by influenza viruses of different subtypes. We propose that it is possible to bypass these challenges by the development of an influenza vaccine based on conserved proteins delivered in an adjuvanted nanoparticle system. In this study, we generated influenza nanoparticle constructs using trimethyl chitosan nanoparticles (TMC nPs) as the carrier of recombinant influenza hemagglutinin subunit 2 (HA2) and nucleoprotein (NP). The purified HA2 and NP recombinant proteins were encapsulated into TMC nPs to form HA2-TMC nPs and NP-TMC nPs, respectively. Primary human intranasal epithelium cells (HNEpCs) were used as an in vitro model to measure immunity responses. HA2-TMC nPs, NP-TMC nPs, and HA2-NP-TMC nPs (influenza nanoparticle constructs) showed no toxicity in HNEpCs. The loading efficiency of HA2 and NP into the TMC nPs was 97.9% and 98.5%, respectively. HA2-TMC nPs and NP-TMC nPs more efficiently delivered HA2 and NP proteins to HNEpCs than soluble HA2 and NP proteins alone. The induction of various cytokines and chemokines was more evident in influenza nanoparticle construct-treated HNEpCs than in soluble protein-treated HNEpCs. In addition, soluble factors secreted by influenza nanoparticle construct-treated HNEpCs significantly induced MoDCs maturation markers (CD80, CD83, CD86 and HLA-DR), as compared to soluble factors secreted by protein-treated HNEpCs. HNEpCs treated with the influenza nanoparticle constructs significantly reduced influenza virus replication in an in vitro challenge assay. The results indicate that TMC nPs can be used as influenza vaccine adjuvants and carriers capable of delivering HA2 and NP proteins to HNEpCs.

Adverse event and treatment completion rates of a 12-dose weekly isoniazid and rifapentine course for South Korean healthcare workers.

PURPOSE: We investigated the adverse events (AEs) and treatment completion rates of a 3 month course of once-weekly isoniazid and rifapentine (3H1P1) in South Korean health care workers (HCWs) with latent tuberculosis infection (LTBI). METHODS: HCWs who were candidates for LTBI treatment were enrolled from two tertiary referral centers between December 2016 and October 2017. From December 2016 through March 2017, HCWs who agreed were treated with the 3H1P1 regimen (3H1P1 group). Their compliance and AEs were prospectively collected. From April 2017 onward, HCWs who required LTBI treatment received 3 months of isoniazid plus rifampin (3HR group), and their medical records were retrospectively reviewed. RESULTS: During the study period, 406 HCWs were treated, 226 (55.7%) in the 3H1P1 group, and 180 (44.3%) in the 3HR group. The number of subjects with AEs was significantly greater in the 3H1P1 group (75.2% vs 56.7%, P < 0.001), in particular a flu-like syndrome (19.0% vs. 0%, P < 0.001). However, hepatotoxicity occurred less frequently in those receiving 3H1P1 (7.5% vs. 20.0%, P < 0.001). Per protocol definition, anaphylaxis developed in 1.8% of the 3H1P1 group. The overall treatment completion rate was greater in the 3H1P1 group (92.9% vs 86.7%, P = 0.036). CONCLUSIONS: The 3H1P1 regimen had a higher treatment completion rate and lower hepatotoxicity compared with the 3HR regimen. However, it resulted in a higher rate of flu-like syndromes. Additionally, a few subjects had anaphylaxis, although there were no fatalities.

Delivery of BACE1 siRNA mediated by TARBP-BTP fusion protein reduces ß-amyloid deposits in a transgenic mouse model of Alzheimer's disease.

Systemic delivery of nucleic acids to the central nervous system (CNS) is a major challenge for the development of RNA interference-based therapeutics due to lack of stability, target specificity, non-permeability to the blood-brain barrier (BBB), and lack of suitable carriers. Using a designed bi-functional fusion protein TARBP-BTP in a complex with siRNA, we earlier demonstrated knockdown of target genes in the brain of both AßPP-PS1 (Alzheimer's disease, AD) and wild-type C57BL/6 mice. In this report, we further substantiate the approach through an extended use in AßPP-PS1 mice, which upon treatment with seven doses of ß-secretase AßPP cleaving Enzyme 1 (BACE1) TARBP-BTP:siRNA, led to target-specific effect in the mouse brain. Concomitant gene silencing of BACE1, and consequent reduction in plaque load in the cerebral cortex and hippocampus (greater than 60%) in mice treated with TARBP-BTP:siRNA complex, led to improvement in spatial learning and memory. The study validates the efficiency of TARBP-BTP fusion protein as an efficient mediator of RNAi, giving considerable scope for future intervention in neurodegenerative disorders through the use of short nucleic acids as gene specific inhibitors.

Design and testing of a highly conserved human rotavirus VP8* immunogenic peptide with potential for vaccine development.

Rotavirus infection of young children particularly below five years of age resulting in severe diarhoea, is the cause of a large number of infant deaths all over the world, more so in developing countries like India. Vaccines developed against this infection in the last two decades have shown mixed results with some of them leading to complications. Oral vaccines have not been very effective in India. Significant diversity has been found in circulating virus strains in India. Development of a vaccine against diverse genetic variants of the different strains would go a long way in reducing the incidence of infection in developing countries. Success of such a vaccine would depend to a large extent on the antigenic peptide to be used in antibody production. The non-glycosylated protein VP4 on the surface capsid of the virus is important in rota viral immunogenicity and the major antigenic site(s) responsible for neutralization of the virus via VP4 is in the VP8* subunit of VP4. It is necessary that the peptide should be very specific and a peptide sequence which would stimulate both the T and B immunogenic cells would provide maximum protection against the virus. Advanced computational techniques and existing databases of sequences of the VP4 protein of rotavirus help in identification of such specific sequences. Using an in silico approach we have identified a highly conserved VP8* subunit of the VP4 surface protein of rotavirus which shows both T and B cell processivity and is also non-allergenic. This sub-unit could be used in in vivo models for induction of antibodies.

Cold-inducible RNA-binding protein activates splenic T cells during sepsis in a TLR4-dependent manner.

Cold-inducible RNA-binding protein (CIRP) is a novel inflammatory mediator that stimulates the release of proinflammatory cytokines from macrophages in sepsis. Given the immune dysregulation that characterizes sepsis, the effect of CIRP on other immune cells is an area of increasing interest that has not yet been studied. In the present study, we hypothesized that extracellular CIRP promotes activation of T lymphocytes in the spleen during sepsis. We observed that mice subjected to sepsis by cecal ligation and puncture showed significantly higher expression of the early activation markers CD69 and CD25 at 20 h on CD4+ splenic T cells, and significantly higher CD69 expression on CD8+ splenic T cells compared with sham-operated controls. Furthermore, at 20 h after receiving intravenous injection of recombinant murine CIRP (rmCIRP, 5 mg/kg body weight (BW)) or PBS (vehicle), those mice receiving rmCIRP showed significantly increased expression of CD69 and CD25 on both CD4+ and CD8+ splenic T cells. This effect, however, was not seen in TLR4-deficient mice after rmCIRP injection. In addition, treatment with CIRP predisposed CD4+ T cells to a Th1 hyperinflammatory response profile, and influenced CD8+ T cells toward a cytotoxic profile. Taken together, our findings indicate that CIRP is a proinflammatory mediator that plays an important role in T-cell dysregulation during sepsis in a TLR4-dependent manner.

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins.

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

A SEMA3E mutant resistant to cleavage by furins (UNCL-SEMA3E) inhibits choroidal neovascularization.

Abnormal subretinal choroidal neovascularization (CNV) is a major cause of blindness in exudative age-related macular degeneration (AMD). Current anti-angiogenic treatments by VEGF sequestering agents have been successful, but a significant proportion of patients do not respond well to these treatments, and the response of others diminishes over time, suggesting that additional anti-angiogenic agents that function by separate mechanisms may be of use to such patients. We have previously found that a point mutated form of semaphorin-3E resistant to cleavage by furin like pro-protein convertases (UNCL-Sema3E) displays potent anti-angiogenic properties. We therefore determined if UNCL-Sema3E has potential as an inhibitor of CNV formation. We chose to study UNCL-Sema3E rather than wild type sema3E because unlike full length sema3E, the major p61-Sema3E peptide that is produced by cleavage of sema3E with furin like pro-protein convertases activates signal transduction mediated by the ErbB2 receptor and can promote tumor metastasis in addition to its anti-angiogenic activity. UNCL-Sema3E inhibited efficiently vascular endothelial growth factor-A (VEGF), platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) signaling in human umbilical vein derived endothelial cells (HUVEC) and to a lesser extent hepatocyte growth factor (HGF) signal transduction. CNV that was induced in the eyes of C57 black mice by laser photocoagulation was inhibited by 65% (P < 0.01) following a single bolus intra-vitreal injection of 5 µg UNCL-Sema3E. This inhibitory effect was similar to the inhibition produced by a single bolus intra-vitreal injection of 5 µg aflibercept. A similar inhibition of CNV was observed following the injection of UNCL-Sema3E into the eyes of Long-Evans rats. However, a higher dose of UNCL-Sema3E (125 µg), partially due to the larger volume of the vitreous cavity of rats, was required to achieve maximal inhibition of CNV. Injection of UNCL-Sema3E into eyes of healthy mice did not have any adverse effect on retinal function as assessed by optic kinetic reflex (OKR) or by electroretinogram (ERG) assays nor did UNCL-Sema3E injection affect the structure of the retina as determined using histology. To conclude, our results suggest that UNCL-Sema3E may be useful for the treatment of exudative AMD, which does not respond well to conventional anti-VEGF therapy.

Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth.

Growing evidence shows that cancer cells use mRNA-binding proteins and miRNAs to posttranscriptionally regulate signaling pathways to adapt to harsh tumor microenvironments. In ovarian cancer, cytoplasmic accumulation of mRNA-binding protein HuR (ELAVL1) is associated with poor prognosis. In this study, we observed high HuR expression in ovarian cancer cells compared with ovarian primary cells, providing a rationale for targeting HuR. RNAi-mediated silencing of HuR in ovarian cancer cells significantly decreased cell proliferation and anchorage-independent growth, and impaired migration and invasion. In addition, HuR-depleted human ovarian xenografts were smaller than control tumors. A biodistribution study showed effective tumor-targeting by a novel Cy3-labeled folic acid (FA)-derivatized DNA dendrimer nanocarrier (3DNA). We combined siRNAs against HuR with FA-3DNA and found that systemic administration of the resultant FA-3DNA-siHuR conjugates to ovarian tumor-bearing mice suppressed tumor growth and ascites development, significantly prolonging lifespan. NanoString gene expression analysis identified multiple HuR-regulated genes that function in many essential cellular and molecular pathways, an attractive feature of candidate therapeutic targets. Taken together, these results are the first to demonstrate the versatility of the 3DNA nanocarrier for in vivo-targeted delivery of a cancer therapeutic and support further preclinical investigation of this system adapted to siHuR-targeted therapy for ovarian cancer.

Differential Effects of Alcohol on Excitatory and Inhibitory Puberty-Related Peptides in the Basal Hypothalamus of the Female Rat.

BACKGROUND: An increase in development of excitatory inputs along with a decline in inhibitory inputs ultimately govern the timely increased secretion of hypothalamic luteinizing hormone-releasing hormone (LHRH) at the time of puberty. As chronic alcohol (ALC) exposure acts at the hypothalamic level to suppress LHRH secretion and delay puberty, we assessed its ability to differentially affect the expression of key puberty-related proteins. METHODS: ALC was administered to female rats from days 27 to 33, at which time animals were killed and tissues collected for protein expression. In the medial basal hypothalamus (MBH), we assessed kisspeptin (Kp) 10, an excitatory peptide critical for prepubertal LHRH secretion, and Lin28b, a peptide with an inhibitory influence on puberty. As a direct mechanism of action of Lin28b was not known, we determined whether its central administration could induce dynorphin (DYN), a peptide that is inhibitory on LHRH secretion. Also, ALC's effect on DYN protein expression was assessed, as well as its effect on DYN release in vitro. RESULTS: ALC markedly suppressed (p < 0.01) the expression of the excitatory Kp protein, while at the same time increased (p < 0.001) the expression of inhibitory Lin28b protein. Subsequently, we showed for the first time that the central administration of Lin28b stimulated (p < 0.01) the synthesis of DYN. Finally, ALC also induced (p < 0.01) the protein expression and stimulated (p < 0.01) the in vitro release of DYN from the MBH. CONCLUSIONS: These results indicate that ALC can simultaneously and differentially alter both excitatory and inhibitory influences governing pubertal development, show for the first time a mechanism of action by which Lin28b exerts its prepubertal inhibitory tone, and further demonstrate the negative influences of ALC on the pubertal process.

[ENDOTHELIAL MONOCYTEACTIVATING FACTOR II CANCELS OXIDATIVE STRESS, CONSTITUTIVE NOS UNCOUPLING AND INDUCED VIOLATIONS OF CARDIAC HEMODYNAMICS IN HYPERTENSION (PART II)].

The purpose of this study was to investigate the effect of EMAP II on free radical state of the heart and blood vessels, to restore cNOS coupling and cardiac hemodynamics in spontaneously hypertensive rats. It was found that, due to the combined inhibition of oxidative and nitrosative stress, EMAP I quickly restores impaired in hypertension constitutive de novo synthesis of NO by restoring cNOS coupling. Restoration by EMAP II of constitutive de novo synthesis NO abolished cardiac and endothelial dysfunction in spontaneously hypertensive rats. In hypertension, the introduction of EMAP II helped to improve the performance of the pumping function of the heart (stroke volume increased by 18.2 %, cardiac output -22 %), an arterial stiffness decreased by 23.2 %, process of relaxation of the left ventricle improved, due to decreased in 4,7 times myocardial end-diastolic stiffness.

Broadly protective immunity against divergent influenza viruses by oral co-administration of Lactococcus lactis expressing nucleoprotein adjuvanted with cholera toxin B subunit in mice.

BACKGROUND: Current influenza vaccines need to be annually reformulated to well match the predicated circulating strains. Thus, it is critical for developing a novel universal influenza vaccine that would be able to confer cross-protection against constantly emerging divergent influenza virus strains. Influenza virus A is a genus of the Orthomyxoviridae family of viruses. Influenza virus nucleoprotein (NP) is a structural protein which encapsidates the negative strand viral RNA, and anti-NP antibodies play role in cross-protective immunity. Lactococcus lactis (L. lactis) is an ideal vaccine delivery vehicle via oral administration route. However, L. lactis vectored vaccine exhibits poor immunogenicity without the use of mucosal adjuvant. To enhance the immunogenicity of L. lactis vectored vaccine, cholera toxin B (CTB) subunit, one of mucosal adjuvants, is a safe adjuvant for oral route, when combined with L. lactis vectored vaccine. In this study, we hypothesized that pNZ8008, a L. lactis expression plasmid, encoding NP antigen, would be able to elicit cross-protection with the use of CTB via oral administration route. RESULTS: To construct L. lactis vectored vaccine, nucleoprotein (NP) gene of A/California/04/2009(H1N1) was sub-cloned into a L. lactis expression plasmid, pNZ8008. The expression of recombinant L. lactis/pNZ8008-NP was confirmed by Western blot, immunofluorescence assay and flow cytometric analysis. Further, immunogenicity of L. lactis/pNZ8008-NP alone or adjuvanted with cholera toxin B (CTB) subunit was evaluated in a mouse model via oral administration route. Antibodies responses were detected by ELISA. The result indicated that oral administration of L. lactis/pNZ8008-NP adjuvanted with CTB could elicit significant humoral and mucosal immune responses, as well as cellular immune response, compared with L. lactis/pNZ8008-NP alone. To further assess the cross-protective immunity of L. lactis/pNZ8008-NP adjuvanted with CTB, we used L. lactis/pNZ8110-pgsA-HA1 alone or adjuvanted with CTB as controls. Mice that received L. lactis/pNZ8008-NP adjuvanted with CTB were completely protected from homologous H1N1 virus and showed 80% protection against heterologous H3N2 or H5N1 virus, respectively. By contrast, L. lactis/pNZ8110-pgsA-HA1 adjuvanted with CTB also conferred 100% protection against H5N1 virus infection, but indicated no cross-protection against H1N1 or H5N1 virus challenge. As controls, mice vaccinated orally with L. lactis/pNZ8008-NP alone or L. lactis/pNZ8110-pgsA-HA1 alone could not survive. CONCLUSION: This study is the first to report the construction of recombinant L. lactis/pNZ8008-NP and investigate its immunogenicity with the use of CTB. Compared with L. lactis/pNZ8110-pgsA-HA1 adjuvanted with CTB, our data support 5 × 10(11) CFU of L. lactis/pNZ8008-NP adjuvanted with 1 µg of CTB is a better combination for universal influenza vaccines development that would provide cross-protective immunity against divergent influenza A viruses.

Cross-protection against influenza virus infection by intranasal administration of nucleoprotein-based vaccine with compound 48/80 adjuvant.

The nucleoprotein (NP) of influenza viruses is highly conserved and therefore has become one of the major targets of current universal influenza vaccine (UIV) studies. In this study, the recombinant nucleoprotein (NP) of the A/PR/8/34 (H1N1) influenza virus strain was expressed using an Escherichia coli (E. coli) expression system and then purified as a candidate UIV. The NP protein was administered intranasally or intraperitoneally twice at 3-week intervals to female BALB/c mice in combination with C48/80 adjuvant. Then, the mice were challenged with homologous or heterologous influenza viruses at a lethal dose 3 weeks after the last immunization. The results showed that the serum IgG titers of all of the mice immunized with NP reached a higher level and the protection provided by NP vaccine against the homologous virus depended on the administered dosage and adjuvant. In addition, immunization with 100 µg NP in combination with C48/80 adjuvant could provide good cross-protection against heterologous H9N2 avian influenza viruses. This study indicated that NP as a candidate antigen of UIV immunized intranasally could effectively induce mucosal and cell-mediated immunity, with the potential to control epidemics caused by the appearance of new emerging influenza viruses.

Cloning, expression, and purification of cold inducible RNA-binding protein and its neuroprotective mechanism of action.

BACKGROUND: Cold-inducible RNA-binding protein (CIRP) is induced in response to hypothermia, where it exerts neuroprotective effects. Our preliminary studies revealed that it inhibits H2O2-induced apoptosis in rat neurons. In the current study, we report effective expression and purification approaches for the synthesis of CIRP, and assess its potential protective effects against oxidative stress. METHODS: CIRP-encoding was expressed using the prokaryotic expression system pGEX-4T-1, and SP-Sepharose and Sephacryl S-200 columns were used to purify rCIRP. To mimic ischemia/reperfusion injury-associated oxidative stress, neuro2a cells (N2a) were pre-treated with rCIRP for 2h, followed by hydrogen peroxide (H2O2 60 µmol/ml) for 24h. Cell viability was then quantified using an MTT assay. In addition, western blotting was performed to measure the cell cycle related signal transduction pathways. RESULTS: N2a cells exhibited decreased viability following H2O2 treatment, whereas rCIRP significantly improved viability following H2O2 treatment. CIRP also accelerated cell cycle progression from S to G2/M phase in cultured mouse neuroblastoma cells. In addition, CIRP increased levels of p-ERK and p-Akt, and also re-activated the cell cycle-related protein cyclin D1 and c-Myc. These results suggest that CIRP activated the Akt and ERK signal transduction pathways in N2a cells. CONCLUSIONS: Our findings suggest that CIRP could exert protective effects against oxidative stress, and that it might be a novel neuroprotective agent.

Protection against influenza H7N9 virus challenge with a recombinant NP-M1-HSP60 protein vaccine construct in BALB/c mice.

A novel influenza virus of H7N9 subtype circulated throughout China in 2013. The high fatality rate, appearance of several family clusters, and transmission in animal models observed during this outbreak accelerated efforts to identify effective strategies to prevent the spread of this influenza subtype. In this study, the recombinant protein NP-M1-HSP60, a fusion of the nucleoprotein and M1 matrix protein of the A/PR/8/34 (H1N1) influenza virus strain and HSP60, was effectively expressed in Escherichia coli and purified as a candidate component for an influenza vaccine. Intranasal immunization of female BALB/c mice with NP-M1-HSP60 in combination with an oil-in-water adjuvant twice at a 2-week interval induced robust humoral, mucosal, and cell-mediated immune responses. Moreover, this immunization strategy completely protected mice from lethal influenza H7N9 virus challenge and significantly inhibited viral replication in the challenged mouse lung. These data suggest that this vaccine construct has great potential for the basic development of an influenza H7N9 vaccine.

Aerosol delivery of programmed cell death protein 4 using polysorbitol-based gene delivery system for lung cancer therapy.

The development of a safe and effective gene delivery system is the most challenging obstacle to the broad application of gene therapy in the clinic. In this study, we report the development of a polysorbitol-based gene delivery system as an alternative gene carrier for lung cancer therapy. The copolymer was prepared by a Michael addition reaction between sorbitol diacrylate (SD) and spermine (SPE); the SD-SPE copolymer effectively condenses with DNA on the nanoscale and protects it from nucleases. SD-SPE/DNA complexes showed excellent transfection with low toxicity both in vitro and in vivo, and aerosol delivery of SD-SPE complexes with programmed cell death protein 4 DNA significantly suppressed lung tumorigenesis in K-ras(LA1) lung cancer model mice. These results demonstrate that SD-SPE has great potential as a gene delivery system based on its excellent biocompatibility and high gene delivery efficiency for lung cancer gene therapy.

Adenoviral vector expressing murine ß-defensin 2 enhances immunogenicity of an adenoviral vector based H5N1 influenza vaccine in aged mice.

The ability to resist infections and respond to vaccinations is greatly reduced in the older adult population owing to a general decline in innate and adaptive immune functions with aging. Over the years several strategies such as increasing the vaccine dose, number of immunizations and using adjuvants have been evaluated to improve the immunogenicity and efficacy of vaccines in the older adult population. Murine ß-defensin 2 (Mbd2) has been shown to function as a molecular adjuvant by recruiting and activating immature dendritic cells (DCs), professional antigen-presenting cells (APC), to the site of the immunization. In this study, we evaluated the potential utility of Mbd2 to enhance the efficacy of an adenoviral vector-based H5N1 influenza vaccine expressing hemagglutinin (HA) and nucleoprotein (NP) (HAd-HA-NP) in an aged mouse model. Our results indicated that immunostimulation with an adenoviral vector expressing Mbd2 (HAd-Mbd2) activated DCs and significantly enhanced the humoral and cellular immune responses induced by HAd-HA-NP. Furthermore, immunostimulation with HAd-Mbd2 followed by immunization with HAd-HA-NP resulted in significantly lower virus titers in the lungs following challenge with a H5N1 influenza virus compared to the group immunized with HAd-HA-NP without immunostimulation. Overall, our results highlight the potential utility of Mbd2 as a molecular adjuvant to enhance the immunogenicity and protective efficacy of vaccines for the elderly.

Enhancement of Th1 immune responses to recombinant influenza nucleoprotein by Ribi adjuvant.

A broad coverage influenza vaccine against multiple viral strains based on the viral nucleoprotein (NP) is a goal pursued by many laboratories. If the goal is to formulate the vaccine with recombinant NP it is essential to count on adjuvants capable of inducing cellular immunity. This work have studied the effect of the monophosphoryl lipid A and trehalose dimycolate, known as the Ribi Adjuvant System (RAS), in the immune response induced in mice immunized with recombinant NP. The NP was formulated with RAS and used to immunize BALB/c mice. Immunizations with NP-RAS increased the humoral and cellular immune responses compared to unadjuvanted NP. The predominant antibody isotype was IgG2a, suggesting the development of a Th1 response. Analysis of the cytokines from mice immunized with NP-RAS showed a significant increase in the production of IFN-g and a decreased production of IL-10 and IL-4 compared to controls without RAS. These results are similar to those usually obtained using Freund's adjuvant, known to induce Th1 and CTL responses when co-administered with purified proteins, and suggest that a similar approach may be possible to enhance the performance of a T-cell vaccine containing NP.

Enhancing sorafenib-mediated sensitization to gemcitabine in experimental pancreatic cancer through EMAP II.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human malignancies and tends to be relatively resistant to conventional therapies. Activated Ras oncogene mutations are found in up to 90% of PDAC, leading to activation of the Ras/Raf/MEK/ERK signaling pathway. Sorafenib is a multikinase inhibitor of the Ras/Raf/MEK/ERK pathway and of tumor angiogenesis. Endothelial monocyte activating polypeptide II (EMAP) enhances gemcitabine effects in PDAC. Antitumor activity of sorafenib was evaluated in combination with gemcitabine (Gem) and the antiangiogenic agent EMAP in experimental PDAC. METHODS: Cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting. Animal survival studies were performed in murine PDAC xenografts. RESULTS: Sorafenib decreased phospho-MEK, phospho-ERK1/2, phospho-p70S6K and phospho-4EBP-1 expression in PDAC cells. Sorafenib inhibited in vitro proliferation of all four PDAC cell lines tested. Additive effects on cell proliferation inhibition were observed in the gemcitabine-sorafenib combination in PDAC cells, and in combinations of sorafenib or EMAP with gemcitabine in endothelial (HUVEC) and fibroblast (WI-38) cells. Sorafenib, alone or in combination with gemcitabine and EMAP, induced apoptosis in HUVECs and WI-38 cells as observed via increased expression of cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and caspase-3 proteins. Compared to controls (median survival: 22 days), animal survival increased after Gem therapy (29 days) but not in sorafenib (23 days) or EMAP therapy alone (25 days). Further increases in survival occurred in combination therapy groups Gem+sorafenib (30 days, p=0.004), Gem+EMAP (33 days, p=0.002), and Gem+sorafenib+EMAP (36 days, p=0.004), but not after the sorafenib+EMAP combination (24 days). CONCLUSIONS: These findings demonstrate that the addition of a polymechanistic antiangiogenic agent such as EMAP can enhance the combination treatment effects of sorafenib and cytotoxic PDAC therapy.

Protective immunization against Staphylococcus aureus infection in a novel experimental wound model in mice.

A novel murine experimental wound infection model was used to assess the efficacy of multi-component immunization against Staphylococcus aureus infection. Necrotic lesions were induced in mice with venom from Bothrops asper and infected with a low inoculum, 1 × 10(2) CFU. The wound infection model therefore more resembles a clinical case of S. aureus infection compared with conventional infection models where far more bacteria are required. Before infection, mice were immunized with four recombinant S.aureus proteins expressed from Escherichia coli: (i) domains 1-3 of Extracellular adherence protein (Eap), (ii) Efb - D (fusion protein combining Extracellular fibrinogen binding protein (Efb) and a fibronectin binding domain (D) of the fibronectin binding protein (FnBP) and (iii) clumping factor A (ClfA). In the immunized group, lower bacterial colonization, undisturbed crust formation and significantly faster wound healing were found compared with the unimmunized control group. Efb and Eap have previously been found to impair wound healing and neutralization of these proteins by antibodies restores a more natural wound healing process. This effect is further also enhanced by the proposed opsonic activity of antibodies against ClfA and FnBP.