Search

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., (© 2023. Controlled Release Society.) more...

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., Competing Interests: The authors have declared that no competing interests exist. more...

Purpose: We investigated the adverse events (AEs) and treatment completion rates of a 3 month course of once-weekly isoniazid and rifapentine (3H 1 P 1 ) 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 3H 1 P 1 regimen (3H 1 P 1 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 3H 1 P 1 group, and 180 (44.3%) in the 3HR group. The number of subjects with AEs was significantly greater in the 3H 1 P 1 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 3H 1 P 1 (7.5% vs. 20.0%, P < 0.001). Per protocol definition, anaphylaxis developed in 1.8% of the 3H 1 P 1 group. The overall treatment completion rate was greater in the 3H 1 P 1 group (92.9% vs 86.7%, P = 0.036)., Conclusions: The 3H 1 P 1 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.) more...

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 AβPP-PS1 (Alzheimer's disease, AD) and wild-type C57BL/6 mice. In this report, we further substantiate the approach through an extended use in AβPP-PS1 mice, which upon treatment with seven doses of β-secretase AβPP 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. more...

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., (Copyright © 2018. Published by Elsevier B.V.) more...

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. more...

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., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.) more...

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.) more...

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., (©2016 American Association for Cancer Research.) more...

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., (Copyright © 2015 by the Research Society on Alcoholism.) more...

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. more...

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., (Copyright © 2015 Elsevier B.V. All rights reserved.) more...

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. more...

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. more...

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. more...

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., (Copyright © 2014 Elsevier B.V. All rights reserved.) more...

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 β-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., (Copyright © 2013 Elsevier B.V. All rights reserved.) more...

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. more...

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. more...

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., (© 2012 The Authors APMIS © 2012 APMIS.) more...

Current influenza vaccines mainly induce strain-specific neutralizing antibodies and need to be updated each year, resulting in significant burdens on vaccine manufacturers and regulatory agencies. Genetic immunization strategies based on the highly conserved nucleoprotein (NP) of influenza have attracted great attention as NP could induce heterosubtypic immunity. It is unclear, however, whether different forms of vectors and/or vaccination regimens could have contributed to the previously reported discrepancies in the magnitude of protection of NP-based genetic vaccinations. Here, we evaluated a plasmid DNA vector (pNP) and a recombinant adenovirus vector (rAd-NP) containing the NP gene through various combinations of immunization regimens in mice. We found that pNP afforded only partial protection even after 4 injections, with full protection against lethal challenge achieved only with the fourth boost using rAd-NP. Alternatively, only two doses of rAd-NP delivered subcutaneously were needed to induce an enhanced immune response and completely protect the animals, a finding which, to our knowledge, has not been reported before. more...

Gemcitabine has limited clinical benefits for pancreatic ductal adenocarcinoma (PDAC). The phosphatidylinositol-3-kinase (PI3K)/AKT and mammalian target of rapamycin (mTOR) signaling pathways are frequently dysregulated in PDAC. We investigated the effects of NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, in combination with gemcitabine and endothelial monocyte activating polypeptide II (EMAP) in experimental PDAC. Cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting. Animal survival experiments were performed in murine xenografts. BEZ235 caused a decrease in phospho-AKT and phospho-mTOR expression in PDAC (AsPC-1), endothelial (HUVECs), and fibroblast (WI-38) cells. BEZ235 inhibited in vitro proliferation of all four PDAC cell lines tested. Additive effects on proliferation inhibition were observed in the BEZ235-gemcitabine combination in PDAC cells and in combination of BEZ235 or EMAP with gemcitabine in HUVECs and WI-38 cells. BEZ235, alone or in combination with gemcitabine and EMAP, induced apoptosis in AsPC-1, HUVECs, and WI-38 cells as observed by increased expression of cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and caspase-3 proteins. Compared to controls (median survival: 16 days), animal survival increased after BEZ235 and EMAP therapy alone (both 21 days) and gemcitabine monotherapy (28 days). Further increases in survival occurred in combination therapy groups BEZ235 + gemcitabine (30 days, P = 0.007), BEZ235 + EMAP (27 days, P = 0.02), gemcitabine + EMAP (31 days, P = 0.001), and BEZ235 + gemcitabine + EMAP (33 days, P = 0.004). BEZ235 has experimental PDAC antitumor activity in vitro and in vivo that is further enhanced by combination of gemcitabine and EMAP. These findings demonstrate advantages of combination therapy strategies targeting multiple pathways in pancreatic cancer treatment., (Copyright © 2011 Wiley Periodicals, Inc.) more...

Unlabelled: Recombinant cytokine-like endothelial monocyte-activating polypeptide II (EMAP II) and antiandrogen flutamide target different mechanisms of growth of androgen-dependent prostate cancer (PC). The aim of this study was to clarify whether combined treatment with EMAP II and flutamide is more effective than monotherapy with regard to retardation of PC progression., Materials and Methods: Antitumor effects of EMAP II (10 µg/kg b.w./d, s.c., 3d), or flutamide (10 mg/kg b.w./d, per os, 3d), or their combination were studied in CBA male mice bearing human androgen-dependent PC xenografts for 7 days. Androgen-dependent phenotype of the tumors was verified in preliminary castrated mice. The xenografts were weighed and underwent a histopathologic examination. The results were compared with those of non-treated mice., Results: EMAP II and flutamide used separately inhibited growth of the xenografts by 74% and 53% respectively. Both drugs caused destructive changes in malignant epithelial cells along with leukocyte infiltration of the tumor. Combined treatment inhibited tumor growth by 85%, and was more effective than monotherapy with regard to morphological changes., Conclusions: This study demonstrates cooperative inhibitory effect of EMAP II and flutamide on growth and morphology of human PC xenografts that could represent a new modality of palliative treatment of this disease. more...

The efficacy of recombinant Lactococcus lactis as a delivery vehicle for a rotavirus antigen was evaluated in a mouse model. The rotavirus VP8* protein was expressed intracellularly and extracellularly in L. lactis wild type and in an alr mutant deficient in alanine racemase activity, necessary for the synthesis of the cell-wall component D: -alanine. When the mucosal immune response was evaluated by measuring VP8*-specific IgA antibody in faeces, wild-type L. lactis triggered a low IgA synthesis only when the secreting strain was used. In contrast, VP8*-specific IgA was detected in faeces of both groups of mice orally given the alr mutant expressing extracellular VP8* and intracellular VP8*, which reached levels similar to that obtained with the wild type secreting strain. However, oral administration of the recombinant strains did not induce serum IgG or IgA responses. L. lactis cell-wall mutants may therefore provide certain advantages when low-antigenic proteins are expressed intracellularly. However, the low immune response obtained by using this antigen-bacterial host combination prompts to the use of new strains and vaccination protocols in order to develop acceptable rotavirus immunization levels. more...

Vaccines can greatly reduce the spread of and deaths from many infectious diseases. However, many infections have no successful vaccines. Better understanding of the generation of protective CD8 memory T cells by vaccination is essential for the rational design of new vaccines that aim to prime cellular immune responses. Here we demonstrate that the combination of two adjuvants that are currently licensed for use in humans can be used to prime long-lived memory CD8 T cells that protect mice from viral challenge. The universally used adjuvant, aluminum salts, primed long-lived memory CD8 T cells; however, effective cytotoxic T-cell differentiation occurred only in the presence of an additional adjuvant, monophosphoryl lipid A (MPL). MPL-induced IL-6 was required for cytotoxic differentiation. The IL-6 acted by inducing granzyme B production and reducing expression of inhibitory molecule PD1 on the surface of the primed CD8 T cells. CD8 memory T cells generated by antigen delivered with both aluminum salts and MPL provided significant protection from influenza A challenge. These adjuvants could be used in human vaccines to prime protective memory CD8 T cells. more...

Seasonal influenza epidemics recur due to antigenic drift of envelope glycoprotein antigens and immune evasion of circulating viruses. Additionally, antigenic shift can lead to influenza pandemics. Thus, a universal vaccine that protects against multiple influenza virus strains could alleviate the continuing impact of this virus on human health. In mice, accelerated clearance of a new viral strain (cross-protection) can be elicited by prior infection (heterosubtypic immunity) or by immunization with the highly conserved internal nucleoprotein (NP). Both heterosubtypic immunity and NP-immune protection require antibody production. Here, we show that systemic immunization with NP readily accelerated clearance of a 2009 pandemic H1N1 influenza virus isolate in an antibody-dependent manner. However, human immunization with trivalent inactivated influenza virus vaccine (TIV) only rarely and modestly boosted existing levels of anti-NP IgG. Similar results were observed in mice, although the reaction could be enhanced with adjuvants, by adjusting the stoichiometry among NP and other vaccine components, and by increasing the interval between TIV prime and boost. Importantly, mouse heterosubtypic immunity that had waned over several months could be enhanced by injecting purified anti-NP IgG or by boosting with NP protein, correlating with a long-lived increase in anti-NP antibody titers. Thus, current immunization strategies poorly induce NP-immune antibody that is nonetheless capable of contributing to long-lived cross-protection. The high conservation of NP antigen and the known longevity of antibody responses suggest that the antiviral activity of anti-NP IgG may provide a critically needed component of a universal influenza vaccine. more...

Background: Programmed cell death 4 (PDCD4), a protein that binds to eukaryotic initiation factor 4A (eIF4A), inhibits the initiation of translation. Although a number of tumor suppressors target transcription, Pdcd4 is the first suppressor targeting protein translation, and has also been suggested to function as a tumor suppressor gene in human cancer. The majority of tumor suppressors are mutationally inactivated, but the expression of Pdcd4 is downregulated with progression in a number of human cancer sites, including the lung., Methods: An aerosol of lentivirus-shRNA Pdcd4 was delivered into A/J mice, through a nose-only inhalation system twice a week for 1 month., Results and Conclusions: Downregulated Pdcd4 resulted in increase levels of antiapoptotic and uPA-regulated proteins. We also found that downregulated Pdcd4 induced the mTOR/p70S6K pathway and cell-cycle proteins. Our results suggest that Pdcd4 may perform a critical function in the regulation of lung cancer cell proliferation. more...

Background: Highly pathogenic avian influenza virus subtype H5N1 infects humans with a high fatality rate and has pandemic potential. Vaccination is the preferred approach for prevention of H5N1 infection. Seasonal influenza virus infection has been reported to provide heterosubtypic immunity against influenza A virus infection to some extend. In this study, we used a mouse model pre-exposed to an H1N1 influenza virus and evaluated the protective ability provided by a single dose of DNA vaccines encoding conserved H5N1 proteins., Results: SPF BALB/c mice were intranasally infected with A/PR8 (H1N1) virus beforehand. Six weeks later, the mice were immunized with plasmid DNA expressing H5N1 virus NP or M1, or with combination of the two plasmids. Both serum specific Ab titers and IFN-gamma secretion by spleen cells in vitro were determined. Six weeks after the vaccination, the mice were challenged with a lethal dose of H5N1 influenza virus. The protective efficacy was judged by survival rate, body weight loss and residue virus titer in lungs after the challenge. The results showed that pre-exposure to H1N1 virus could offer mice partial protection against lethal H5N1 challenge and that single-dose injection with NP DNA or NP + M1 DNAs provided significantly improved protection against lethal H5N1 challenge in mice pre-exposed to H1N1 virus, as compared with those in unexposed mice., Conclusions: Pre-existing immunity against seasonal influenza viruses is useful in offering protection against H5N1 infection. DNA vaccination may be a quick and effective strategy for persons innaive to influenza A virus during H5N1 pandemic. more...

The proteasome inhibitor bortezomib (B) has been shown to enhance gemcitabine (G) effects against pancreatic ductal adenocarcinoma (PDAC). Endothelial monocyte activating polypeptide II (EMAP, E) is an antiendothelial and antiangiogenic cytokine. We tested the combination effects of bortezomib, gemcitabine and EMAP in experimental PDAC. Bortezomib inhibited the in vitro proliferation of PDAC and endothelial cells, with additive effects in combination with gemcitabine or EMAP. Bortezomib induced apoptosis as observed by PARP-1 cleavage; it also increased the expression of p21 (>27-fold) and p27 (>2.5-fold), with additive effects in combination with gemcitabine and EMAP. Bortezomib caused a decrease in the expression of the antiapoptotic protein Bcl-2, and an increase in the proapoptotic protein Bax and in p53. Bortezomib had no effect on the intracellular levels of full length or mature EMAP. An in vivo murine xenograft model showed extended survival in all combination groups except B + E compared with control or monotherapy, but no benefit of B + E + G over E + G. The relative local tumor growth compared to controls after bortezomib, EMAP, gemcitabine, B + G, E + G or B + E + G was 92, 52, 48, 36, 18 and 35%, respectively. Our results show that in vitro bortezomib had an antiproliferative and proapoptotic effect, and it's combination with gemcitabine and EMAP increased these effects. In vivo, bortezomib had no antitumor effect by itself, enhanced gemcitabine effects in combination, but failed to further significantly improve the E + G combination benefit. The potential value of proteasome inhibition in experimental therapy approaches for PDAC appears to relate primarily to the combination with the cytotoxic drug rather than with the antiendothelial agent. more...

Background: Pancreatic ductal adenocarcinoma (PDAC) frequently resists conventional cytotoxic therapy. The antitumor effects of endothelial monocyte-activating polypeptide II (EMAP) have been attributed to its antiendothelial and antiangiogenic activities. We tested the hypothesis that a combination of EMAP with bevacizumab (Bev) and gemcitabine (Gem) targets different pathways of PDAC progression and represents more effective treatment., Methods: Proliferation of PDAC and endothelial cell lines was evaluated in vitro. In vivo tumor growth and survival PDAC xenograft experiments were performed with EMAP, Bev, and Gem, either alone or in combination. Intratumoral microvessel density and proliferative activity were analyzed by immunostaining with PECAM-1 and proliferating cell nuclear antigen antibodies, and apoptotic activity was measured by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) procedure., Results: Compared with controls, net reduction in tumor growth in EMAP, Bev, Gem, EMAP + Bev, EMAP + Gem, Bev + Gem, and EMAP + Bev + Gem groups was 58, 40, 40, 67, 68, 69, and 96%, respectively. Addition of EMAP to the Bev + Gem group statistically significantly improved survival at a median of >8 days while inducing long-term survival in some animals after maintenance therapy. Combination treatment of EMAP with Bev and Gem reduced proliferation of endothelial but not of PDAC cells. Addition of EMAP to Bev and Gem statistically significantly decreased proliferative activity while maintaining a comparable rate of microvessel density and apoptosis., Conclusions: Addition of antiendothelial EMAP to a Bev and Gem regimen improves antitumor effects in a xenograft model of PDAC. This multitargeting strategy to prevent PDAC progression shows therapeutic promise and may overcome limitations by combinations of Gem with anti-vascular endothelial growth factor agents alone. more...

Although AIMP1 was identified as a component of the macromolecular aminoacyl tRNA synthetase complex involved in the cellular translation process, it was also found to be secreted as a cytokine having complex physiological functions. Among these, AIMP1's angiostatic and immune stimulating activities suggest its potential use as a novel antitumor therapeutic protein. Here we evaluated its antitumor efficacy in a mouse xenograft model bearing human stomach cancer cells. Intravenous injection of recombinant AIMP1 for 6 days resulted in significant decreases in both tumor volume and weight. Tumor volume decreased 31.1% and 54.0% when treated with AIMP1 at a concentration of 2mg/kg and 10mg/kg, respectively. Tumor weight decreased 29.1% and 52.2% when treated with AIMP1 at a concentration of 2mg/kg and 10mg/kg, respectively. Proliferating cell nuclear antigen (PCNA) staining of tumor tissues from AIMP1-treated mice (at both 2mg/kg and 10mg/kg) showed a 53% reduction of cells exhibiting an active cell cycle progression. Blood levels of tumor-suppressing cytokines such as TNF-alpha and IL-1beta increased in AIMP1-treated mice, whereas IL-12p40 and IFN-gamma levels remained unaltered. Thus, this work suggests that AIMP1 may exert its antitumor activity by inducing tumor-suppressing cytokines. In a pharmacokinetic study in rats after a single intravenous injection, AMP1 exhibited a low clearance showing a one-compartmental disposition. However, due to a low volume of distribution, AIMP1 had a short half-life of 0.1h. In a serum stability test, AIMP1 showed a half life of >60 min in human serum, 52 min in dog serum and 32 min in rat serum., (Crown Copyright 2009. Published by Elsevier Ireland Ltd. All rights reserved.) more...

Background: Combination treatments in addition to gemcitabine have failed to improve outcomes in pancreatic cancer. We tested gemcitabine in combination with the antiendothelial agent endothelial monocyte-activating polypeptide II (EMAP II)., Methods: Human pancreatic cancer cell line murine xenografts were treated with recombinant EMAP II (80 mug/kg), gemcitabine (100 mg/kg), or a combination, and survival and local tumor outcomes were studied., Results: Both EMAP II and gemcitabine inhibited tumor growth, but the combination of both was always more effective. EMAP II and gemcitabine also inhibited microvessel density, with the combination being more effective. Apoptotic activity was increased by factors of 3.2-, 2.7-, and 4.2-fold in EMAP II, gemcitabine, or their combination, respectively. There was a significant extension of survival after EMAP II and gemcitabine combination therapy compared with controls in 2 different pancreatic cancer cell line models at P = .0001 and P = .006, respectively. The median EMAP II survival contribution over gemcitabine was 16 days, from 35 to 51 days (P = .017). EMAP II had no impact on gemcitabine-induced antiproliferative effects against pancreatic cancer cells in vitro., Conclusion: The antiendothelial agent EMAP II enhanced gemcitabine-mediated tumor inhibition, pointing toward a promising strategy for improved combination treatment of pancreatic cancer. more...

Current influenza vaccines elicit Abs to the hemagglutinin and neuraminidase envelope proteins. Due to antigenic drift, these vaccines must be reformulated annually to include the envelope proteins predicted to dominate in the following season. By contrast, vaccination with the conserved nucleoprotein (NP) elicits immunity against multiple serotypes (heterosubtypic immunity). NP vaccination is generally thought to convey protection primarily via CD8 effector mechanisms. However, significant titers of anti-NP Abs are also induced, yet the involvement of Abs in protection has largely been disregarded. To investigate how Ab responses might contribute to heterosubtypic immunity, we vaccinated C57BL/6 mice with soluble rNP. This approach induced high titers of NP-specific serum Ab, but only poorly detectable NP-specific T cell responses. Nevertheless, rNP immunization significantly reduced morbidity and viral titers after influenza challenge. Importantly, Ab-deficient mice were not protected by this vaccination strategy. Furthermore, rNP-immune serum could transfer protection to naive hosts in an Ab-dependent manner. Therefore, Ab to conserved, internal viral proteins, such as NP, provides an unexpected, yet important mechanism of protection against influenza. These results suggest that vaccines designed to elicit optimal heterosubtypic immunity to influenza should promote both Ab and T cell responses to conserved internal proteins. more...

High-dose endothelial-monocyte activating polypeptide II (EMAP-II), a tumor-derived antiangiogenic cytokine, can sensitize tumor vasculature to the damaging activity of high-dose tumor necrosis factor (TNF)-alpha. However, this combination cannot be used for systemic treatment of patients because of prohibitive toxicity. We have found that this limitation can be overcome by combining a TNF-targeting strategy with the use of ultra low-dose EMAP-II. Coadministration of 0.1 ng of EMAP-II and 0.1 ng of CNGRCG-TNF (NGR-TNF), a peptide-TNF conjugate able to target tumor blood vessels, inhibited lymphoma and melanoma growth in mice, with no evidence of toxicity. This drug combination induced endothelial cell apoptosis in vivo and, at later time points, caused reduction of vessel density and massive apoptosis of tumor cells. Ligand-directed targeting of TNF was critical because the combination of nontargeted TNF with EMAP-II was inactive in these murine models. The synergism was progressively lost when the dose of EMAP-II was increased in the nanogram to microgram range, supporting the concept that the use of low-dose EMAP-II is critical. Studies on the mechanism of this paradoxical behavior showed that EMAP-II doses >1 ng induce the release of soluble TNF receptor 1 in circulation, a strong counter-regulatory inhibitor of TNF. Tumor vascular targeting with extremely low amounts of these cytokines may represent a new strategy for cancer treatment. more...

Bone metastasis is a common sequelae of breast cancer and the interaction of alpha v beta3-integrin with osteopontin (OPN) found in the extracellular matrix of mineralized tissues is implicated in this process. The integrin-dependent proadhesive and promigratory functions of OPN are particularly attributed to the 40 kD N-terminal fragment that derives upon matrix metalloproteinase (MMP) cleavage. Based on the broad repertoire of interactions between Staphylococcus aureus extracellular adherence protein (Eap) and host components, we here characterized Eap to specifically interact with recombinant full-length OPN and the 40 kD N-terminal MMP cleavage fragment, but not with the 32 kD or the 25 kD C-terminal fragments of OPN. Eap thereby prevented the OPN/alpha v beta3-integrin interaction, as well as the alpha v beta3-integrin-dependent adhesion of MDA-MB-231 breast cancer cells to full-length OPN or to the 40 kD fragment and the migration of these cells towards OPN. Furthermore, Eap treatment markedly impaired the development of osseous metastasis of human MDA-MB-231 cells in vivo. Taken together, Eap may represent an attractive novel treatment for the prevention of breast cancer bone metastasis. more...

CD8(+) T lymphocytes recognize infected cells that display virus-derived antigenic peptides complexed with major histocompatibility complex class I molecules. Peptides are mainly byproducts of cellular protein turnover by cytosolic proteasomes. Cytosolic tripeptidyl-peptidase II (TPPII) also participates in protein degradation. Several peptidic epitopes unexpectedly do not require proteasomes, but it is unclear which proteases generate them. We studied antigen processing of influenza virus nucleoprotein epitope NP(147-155), an archetype epitope that is even destroyed by a proteasome-mediated mechanism. TPPII, with the assistance of endoplasmic reticulum trimming metallo-aminopeptidases, probably ERAAP (endoplasmic reticulum aminopeptidase associated with antigen processing), was crucial for nucleoprotein epitope generation both in the presence of functional proteasomes and when blocked by lactacystin, as shown with specific chemical inhibitors and gene silencing. Different protein contexts and subcellular targeting all allowed epitope processing by TPPII as well as trimming. The results show the plasticity of the cell's assortment of proteases for providing ligands for recognition by antiviral CD8(+) T cells. Our observations identify for the first time a set of proteases competent for antigen processing of an epitope that is susceptible to destruction by proteasomes. more...

The low efficiency of conventional therapies in achieving long-term survival of patients with lung cancer calls for development of novel treatment options. Although several genes have been investigated for their antitumor activities through gene delivery, problems surrounding the methods used, such as efficiency, specificity, and toxicity, hinder application of such therapies in clinical settings. Aerosol gene delivery as nonviral and noninvasive method for gene therapy may provide an alternative for a safer and more effective treatment for lung cancer. In this study, imidazole ring-containing urocanic acid-modified chitosan (UAC) designed in previous study was used as a gene carrier. The efficiency of UAC carrier in lungs was confirmed, and the potential effects of the programmed cell death protein 4 (PDCD4) tumor suppressor gene on three major pathways (apoptosis, cell cycle, and angiogenesis) were evaluated. Aerosol containing UAC/PDCD4 complexes was delivered into K-ras null lung cancer model mice through the nose-only inhalation system developed by our group. Delivered UAC/PDCD4 complex facilitated apoptosis, inhibited pathways important for cell proliferation, and efficiently suppressed pathways important for tumor angiogenesis. In summary, results obtained by Western blot analysis, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated nick end labeling assay suggest that our aerosol gene delivery technique is compatible with in vivo gene delivery and can be applied as a noninvasive gene therapy. more...

Purpose: The accumulation of T cells into the tumor site is crucial for the elicitation of in vivo antitumor effects after cancer vaccination. In this study, we investigated the antitumor effects and associated mechanisms of action that were induced by systemic and local immunization with a CTL-directed peptide in combination with a peritumoral injection of a streptococcal preparation, OK-432., Experimental Design and Results: The human SART3(315-323) peptide, which has the potential to induce human leukocyte antigen-A24-restricted CTLs, not only has the same amino acid sequence as the mouse SART3, but also has the capacity for binding to H-2K(d) molecules. Therefore, the SART3(315-323) peptide could be used as a tumor antigen-derived peptide in H-2(d) mice. Systemic immunization with the SART3(315-323) peptide and the subsequent peritumoral injection of both the SART3(315-323) peptide and OK-432 effectively induced peptide-specific and colon26 carcinoma-reactive CTLs in BALB/c mice. The combination therapy suppressed the growth of s.c. established colon26 carcinoma. The accumulation of both CD8(+) and CD4(+) T cells into the tumor site was more apparent in mice treated with the combination therapy than in those treated with other protocols. In addition, the level of IgG reactive to the administered SART3(315-323) peptide increased in mice that were treated with the combination therapy., Conclusion: These results indicate that antitumor effects could be efficiently induced by a combination therapy that included systemic and local immunization with a CTL-directed peptide together with a local injection of OK-432. more...

Improving DNA vaccination remains a fundamental goal in vaccine research. Theoretically, this could be achieved by molecules encoded by DNA capable of activating TLRs to mimic inflammatory responses generated by infection. Therefore, we constructed an expression vector that allows mammalian cells to express the TLR5 agonist flagellin (FliC) at the cell surface. In vitro, cell lines expressing FliC stimulated production of proinflammatory cytokines and the up-regulation of costimulatory molecules on monocytes. Mice given the FliC expression vector intradermally exhibited site-specific inflammation and, in combination with vectors expressing Ags, developed dramatic increases in Ag-specific IgG as well as IgA. Surprisingly, mice also developed strong Ag-specific MHC class I-restricted cellular immunity. To determine whether vaccination using FliC vectors could elicit protective immunity to an infectious agent, mice were given dermal injections of FliC expression vector together with a vector encoding the influenza A virus nucleoprotein. This vaccination strategy elicited protective immunity to lethal influenza A virus infection. These results demonstrate that expression of DNA-encoded TLR agonists by mammalian cells greatly enhance and broaden immune responses, imposing new possibilities on DNA vaccination to infectious agents and cancer. more...

Background: Continued growth of solid tumors beyond a critical diameter is thought to require active angiogenic mechanisms, the inhibition of which carries therapeutic potential. We tested this strategy in an in vivo model of local tumor progression utilizing an agent with previously identified direct antiendothelial properties., Materials and Methods: Nonmetastatic C(6) glioma cells (10(6) per animal) were injected subcutaneously into the flank of nude mice with 6 to 8 animals per treatment group. Nodules at the injection site were measured at day 3 and every fourth day thereafter until day 15. Endothelial-monocyte-activating polypeptide II (EMAP-II), a substance known to induce endothelial cell apoptosis, was injected intraperitoneally daily from day 3 to day 15 at doses of 8 microg/kg (low dose) and 80 microg/kg (high dose). Tumor growth kinetics, histologic parameters, and tissue expression of VEGF were studied., Results: Tumor growth was significantly decreased in the EMAP-II-treated animals. Median tumor volume (in mm(3)) at day 15 was 2311 (Control), 727 (low dose), and 454 (high dose, P = 0.003). Median tumor weight (g) measured 1.8 (Control, 0.95 low dose), and 0.9 (high dose, P = 0.06). Median 12-day specific tumor growth rate (in mm(3)/day) per group was 191, 60, and 36 (P = 0.003). Decreased tumor growth after EMAP-II treatment correlated with significantly reduced microvessel counts, higher vascular thrombosis rate, and reduced tumor cell proliferation indices within the neoplastic tissue. Tumor levels, but not serum levels, of VEGF were significantly reduced after EMAP-II treatment. At the doses administered, there was no obvious systemic toxicity. EMAP-II had no direct cytotoxic or antiproliferative effects on tumor cells in vitro., Conclusions: Daily administration of the antiendothelial agent EMAP-II led to a significant retardation of tumor growth, but no complete cancer abrogation. The findings support the hypothesis of an in vivo therapeutic benefit to antiangiogenic therapy with an agent that displays specific toxicity in vitro against endothelial cells. The mechanism of action remains unclear, but likely involves vascular thrombosis and leads to decreased VEGF expression. more...

Apoptotically modified forms of autoantigens have been hypothesized to participate in lupus immunopathogenesis. This study identifies a major B cell epitope present on the apoptotic but not the intact form of the U1-70-kDa ribonucleoprotein lupus autoantigen (70k). Human autoimmune sera with strong recognition of apoptotic 70k and minimal recognition of intact 70k were identified and tested for reactivity to truncated forms of 70k by immunoblot and ELISA. Patient sera that preferentially recognized apoptotic 70k were specific for an epitope dependent on residues 180-205 of the protein. This epitope was also recognized by 19 of 28 (68%) intact anti-70k-positive autoimmune human sera with Abs also recognizing apoptotic but not the intact form 70k, but only 1 of 9 (11%) intact 70k-positive sera without such Abs (Fisher's exact, p = 0.0055). Immunization of HLA-DR4-transgenic C57BL/6 mice with a peptide containing this epitope induced anti-70k immunity in 13 of 15 mice, including Abs recognizing apoptotic but not intact forms of autoantigens in 12 of 15 mice. Anti-70k responder mice also developed spreading of immunity to epitopes on the endogenous form of 70k, and proliferative lung lesions consistent with those described in patients with anti-70k autoimmunity. Thus, a major epitope in the B cell response to U1-70 kDa localizes to the RNA binding domain of the molecule, overlaps with the most common T cell epitope in the anti-70k response, and is not present on the intact form of the 70k molecule. Immunization of mice against this epitope induces an immune response with features seen in human anti-70k autoimmune disease. more...

Anti-Hu syndrome is a paraneoplastic neurologic disease seemingly associated with an efficient antitumoral immune response against HuD protein expressed by both small cell lung cancer (SCLC) and neurons. Since anti-Hu antibodies are not pathogenic, and oligoclonal CD8(+) T cells infiltrate neoplastic and nervous tissues, we examined MHC class I-restricted immunogenicity of human HuD. Among 14 HuD-derived peptides potentially immunogenic in HLA-A*0201 restriction, 10 had actual in vitro binding capacity to the HLA molecule, 8 elicited specific cytotoxic T lymphocytes (CTLs) in a humanized murine model after peptidic vaccination, 2 also elicited specific CTLs in healthy humans, and 1 was naturally processed and presented to the immune system. more...

The double-stranded RNA dependent protein kinase (PKR) is a negative regulator of cell proliferation and thus itself a target for modulation. We show that a cell-permeable peptide (PRI), containing a conserved double-stranded RNA binding motif found in PKR, inhibits activation of the kinase and activity to phosphorylate its substrate. Further, the PRI-peptide localizes to the cytoplasm of murine embryonic fibroblasts and ablates cellular PKR activation. The PRI-peptide enhances cell proliferation compared to treatment with a variant control peptide, resulting in cultures with increased cell density. We conclude that peptides that interfere with PKR may be useful tools for regulating cell proliferation. more...

Vigilin is a ubiquitous multi heterogeneous nuclear ribonucleoprotein (hnRNP) K homologous (KH)-domain protein. Here we demonstrate that purified recombinant human vigilin binds tRNA molecules with high affinity, although with limited specificity. Nuclear microinjection experiments revealed for the first time that the immuno-affinity-purified nuclear vigilin core complex (VCC(N)) as well as recombinant vigilin accelerate tRNA export from the nucleus in human cells. The nuclear tRNA receptor exportin-t is part of the VCC(N). Elongation factor (EF)-1alpha is enriched in VCC(N) and its cytoplasmic counterpart VCC(C), whereas EF-1beta, EF-1gamma and EF-1delta are basically confined to the VCC(C). Our results suggest further that vigilin and exportin-t might interact during tRNA export, provide evidence that the channeled tRNA cycle is already initiated in the nucleus, and illustrate that intracellular tRNA trafficking is associated with discrete changes in the composition of cellular cytoplasmic multi-protein complexes containing tRNA. more...

The outer capsid proteins VP4 and VP7 induce neutralizing antibody against rotavirus. We have investigated in a mouse model the protection mediated by immunization with VP8*, the amino-terminal tryptic fragment of VP4. BALB/c female mice immunized with simian rotavirus SA11 VP6 and VP8* proteins expressed in Escherichia coli were mated with seronegative males. Litters were orally challenged with the SA11 strain (P5B[2], G3) or with the murine rotavirus strain EDIM (P10[16], G3) to verify the degree of protection against diarrhea induced in the newborns. Only those pups born to dams immunized with VP8* did not develop diarrhea after having been orally challenged with the SA11 strain. Pups born to naive dams but foster nursed by VP8*-immunized dams did not develop diarrhea after having been orally infected with the SA11 strain, but they suffered diarrhea when challenged with the EDIM strain. These results support the concepts that (1) VP8* is a highly immunogenic polypeptide that induces effective homotypic protection against disease in pups born to dams immunized with this antigen and (2) in newborn mice the protection against disease is mediated by neutralizing secretory antibodies present in the milk rather than by serum antibodies transferred through the placenta to the offspring. more...

Using an in vivo assay system, nuclear import ability in individual cells was determined by examining the nuclear import rate. It was found that when a small (not excess) amount of SV40 T-NLS peptides was co-injected, the nuclear import rate of SV40 T-NLS-containing substrates apparently increased. This up-regulation was reproduced by the co-injection of peptides containing bipartite type NLS of CBP80, but not mutated non-functional NLS peptides, which suggests that these phenomena are specific for functional NLSs. It was further shown that although, in growth-arrested cells, the nuclear import rate was down-regulated compared to growing cells, the elevation of the functional import rate by co-injected NLS peptides reached the same level as in proliferating cells. This up-regulation was abolished by the addition of a protein kinase inhibitor, staurosporine. These results suggest that although potential nuclear import ability does not vary in each cell, the rate of nuclear import may be controlled by the amount of karyophilic proteins, which need to be carried into the nucleus from the cytoplasm, possibly via an NLS-dependent phosphorylation reaction. more...

Paraneoplastic encephalomyelitis/paraneoplastic sensory neuronopathy (PEM/PSN) associated with small cell lung cancer is characterized by high serum and CSF titers of anti-neuronal (anti-Hu) antibodies and by intrathecal synthesis of anti-Hu IgG. A pathologic role for the anti-Hu antibodies in PEM/PSN is further suggested by reported intraneuronal accumulation of the antibodies in the nervous system of PEM/PSN patients at autopsy. We immunized SJL/J mice, Lewis rats, and Hartley guinea pigs with purified recombinant HuD fusion protein. In spite of high-titer anti-HuD antibodies, neurologic and pathologic examination of the animals was normal. Apparent uptake of purified IgG by neurons in the brain proved to be artifactual. more...