The development of a suitable three dimensional (3D) culture system for anticancer drug development remains an unmet need. Despite progress, a simple, rapid, scalable and inexpensive 3D-tumor model that recapitulates in vivo tumorigenesis is lacking. Herein, we report on the development and characterization of a 3D nanofibrous scaffold produced by electrospinning a mixture of poly(lactic-co-glycolic acid) (PLGA) and a block copolymer of polylactic acid (PLA) and mono-methoxypolyethylene glycol (mPEG) designated as 3P. Cancer cells cultured on the 3P scaffold formed tight irregular aggregates similar to in vivo tumors, referred to as tumoroids that depended on the topography and net charge of the scaffold. 3P scaffolds induced tumor cells to undergo the epithelial-to-mesenchymal transition (EMT) as demonstrated by up-regulation of vimentin and loss of E-cadherin expression. 3P tumoroids showed higher resistance to anticancer drugs than the same tumor cells grown as monolayers. Inhibition of ERK and PI3K signal pathways prevented EMT and reduced tumoroid formation, diameter and number. Fine needle aspirates, collected from tumor cells implanted in mice when cultured on 3P scaffolds formed tumoroids, but showed decreased sensitivity to anticancer drugs, compared to tumoroids formed by direct seeding. These results show that 3P scaffolds provide an excellent platform for producing tumoroids from tumor cell lines and from biopsies and that the platform can be used to culture patient biopsies, test for anticancer compounds and tailor a personalized cancer treatment.

A vast array of technological advances in both nanotechnology and nanoscience has been achieved in the last decade, including advances in drug targeting and delivery applications. A diverse array of polymeric biomaterials, including chitosan, has been used to develop multifunctional nanoparticles, which carry not only the drug payload, but also targeting and imaging moieties. Chitosan represents a cationic polymer that is biodegradable and biocompatible and can be readily used for gene therapy. However, the safety of these nanogene particles has not been reported. This article reviews the properties of chitosan that make it one of the best nanocarriers for gene therapy, as well as addresses safety issues and recent developments of using chitosan as a theranostic.

Background Atrial natriuretic peptide (ANP) is an important endogenous hormone that controls inflammation and immunity by acting on dendritic cells (DCs); however, the mechanism remains unclear. Objective We analyzed the downstream signaling events resulting from the binding of ANP to its receptor, NPRA, and sought to determine what aspects of this signaling modulate DC function. Methods We utilized the inhibitory peptide, NP73-102, to block NPRA signaling in human monocyte-derived DCs (hmDCs) and examined the effect on DC maturation and induced immune responses. The potential downstream molecules and interactions among these molecules involved in NPRA signaling were identified by immunoprecipitation and immunoblotting. Changes in T cell phenotype and function were determined by flow cytometry and BrdU proliferation ELISA. To determine if adoptively transferred DCs could alter the in vivo immune response, bone marrow-derived DCs from wild-type C57BL/6 mice were incubated with ovalbumin (OVA) and injected i.v. into C57BL/6 NPRA-/- knockout mice sensitized and challenged with OVA. Lung sections were stained and examined for inflammation and cytokines were measured in bronchoalveolar lavage fluid collected from parallel groups of mice. Results Inhibition of NPRA signaling in DCs primes them to induce regulatory T cells. Adoptive transfer of wild type DCs into NPRA-/- mice reverses the attenuation of lung inflammation seen in the NPRA-knockout model. NPRA is associated with TLR-2, SOCS3 and STAT3, and inhibiting NPRA alters expression of IL-6, IL-10 and TGF-β, but not IL-12. Conclusions Modulation of NPRA signaling in DCs leads to immune tolerance and TLR2 and SOCS3 are involved in this induction.

One obstacle to developing an effective therapeutic strategy to treat or prevent asthma is that the fundamental causes of asthma are not totally understood. Asthma is thought to be a chronic T H 2 immune-mediated inflammatory disease. Epigenetic changes are recognized to play a role in the initiation and maintenance of a T H 2 response. MicroRNAs (miRNAs) are key epigenetic regulators of gene expression, and their expression is highly regulated, therefore, deregulation of miRNAs may play an important role in the pathogenesis of asthma. Profiling circulating miRNA might provide the highest specificity and sensitivity to diagnose asthma; similarly, correcting potential defects in the miRNA regulation network may lead to new therapeutic modalities to treat this disease.

One obstacle to developing an effective therapeutic strat- egy to treat or prevent asthma is that the fundamental causes of asthma are not totally understood. Asthma is thought to be a chronic TH2 immune-mediated inflammatory disease. Epigenetic changes are recognized to play a role in the initiation and maintenance of a TH2 response. MicroRNAs (miRNAs) are key epigenetic regulators of gene expression, and their expression is highly regulated, there- fore, deregulation of miRNAs may play an important role in the pathogenesis of asthma. Profiling circulating miRNA might provide the highest specificity and sensitivity to diagnose asthma; similarly, correcting potential defects in the miRNA regulation network may lead to new therapeutic modalities to treat this disease.

Background The receptor for the cardiac hormone atrial natriuretic peptide (ANP), natriuretic peptide receptor A (NPRA), is expressed in cancer cells, and natriuretic peptides have been implicated in cancers. However, the direct role of NPRA signaling in prostate cancer remains unclear. Results NPRA expression was examined by western blotting, RT-PCR and immunohistochemistry. NPRA was downregulated by transfection of siRNA, shRNA and NPRA inhibitor (iNPRA). Antitumor efficacy of iNPRA was tested in mice using a TRAMP-C1 xenograft. Here, we demonstrated that NPRA is abundantly expressed on tumorigenic mouse and human prostate cells, but not in nontumorigenic prostate epithelial cells. NPRA expression showed positive correlation with clinical staging in a human PCa tissue microarray. Down-regulation of NPRA by siNPRA or iNPRA induced apoptosis in PCa cells. The mechanism of iNPRA-induced anti-PCa effects was linked to NPRA-induced expression of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine over-expressed in PCa and significantly reduced by siNPRA. Prostate tumor cells implanted in mice deficient in atrial natriuretic peptide receptor A (NPRA-KO) failed to grow, and treatment of TRAMP-C1 xenografts with iNPRA reduced tumor burden and MIF expression. Using the TRAMP spontaneous PCa model, we found that NPRA expression correlated with MIF expression during PCa progression. Conclusions Collectively, these results suggest that NPRA promotes PCa development in part by regulating MIF. Our findings also suggest that NPRA is a potential prognostic marker and a target for PCa therapy.

There has been significant progress in our knowledge about the relationship between infectious disease and the immune system in relation to asthma, but many unanswered questions still remain. Respiratory tract infections such as those caused by respiratory syncytial virus and rhinovirus during the first 2 years of life are still clearly associated with later wheezing and asthma, but the mechanism has not been completely worked out. Is there an "infectious march" triggered by infection in infancy that progresses to disease pathology or are infants who contract respiratory infections predisposed to developing asthma? This review focuses on the common themes in the interaction between microbes and the immune system, and presents a critical appraisal of the evidence to date. The various mechanisms whereby microbes alter the immune response and how this might influence asthma are discussed along with new and promising clinical practices for prevention and therapy. Recent advances in using sensitive polymerase chain reaction detection methods have allowed more rigorous testing of the causality hypothesis of virus infection leading to asthma, but the evidence is still equivocal. Various exceptions and inconsistencies in the clinical trials are discussed in light of new guidelines for subject inclusion/exclusion in hopes of providing some standardization. Despite past failures in vaccination and disappointing results of some clinical trials, the new strategies for prophylaxis including RNA interference and targeted delivery of microbicides offer a large dose of hope to a world suffering from an increasing incidence of asthma as well as a huge burden of health care cost and loss of quality of life.

Allergen immunotherapy (IT) is a proven approach for treating allergic rhinitis and allergic asthma that has been practiced since 1911 and has undergone significant development in the past two decades. As currently practiced, IT involves subcutaneous or sublingual administration of allergens, both methods of which have been extensively investigated. In addition to allergen IT, a number of additional nonspecific IT approaches are being used or are in phase II/phase III clinical trials, which may be available in clinics within the next one to three years. Such therapies include anti-IgE antibodies and the soluble IL-4 receptor. Other experimental IT approaches are at the preclinical research stage and may proceed to clinical trials and the clinic within the next five to ten years. This review discusses the pros and cons of recent developments in both currently practiced and experimental IT approaches.

Background Atrial natriuretic peptide (ANP) and its receptor, NPRA, have been extensively studied in terms of cardiovascular effects. We have found that the ANP-NPRA signaling pathway is also involved in airway allergic inflammation and asthma. ANP, a C-terminal peptide (amino acid 99–126) of pro-atrial natriuretic factor (proANF) and a recombinant peptide, NP73-102 (amino acid 73–102 of proANF) have been reported to induce bronchoprotective effects in a mouse model of allergic asthma. In this report, we evaluated the effects of vessel dilator (VD), another N-terminal natriuretic peptide covering amino acids 31–67 of proANF, on acute lung inflammation in a mouse model of allergic asthma. Methods A549 cells were transfected with pVD or the pVAX1 control plasmid and cells were collected 24 hrs after transfection to analyze the effect of VD on inactivation of the extracellular-signal regulated receptor kinase (ERK1/2) through western blot. Luciferase assay, western blot and RT-PCR were also performed to analyze the effect of VD on NPRA expression. For determination of VD's attenuation of lung inflammation, BALB/c mice were sensitized and challenged with ovalbumin and then treated intranasally with chitosan nanoparticles containing pVD. Parameters of airway inflammation, such as airway hyperreactivity, proinflammatory cytokine levels, eosinophil recruitment and lung histopathology were compared with control mice receiving nanoparticles containing pVAX1 control plasmid. Results pVD nanoparticles inactivated ERK1/2 and downregulated NPRA expression in vitro, and intranasal treatment with pVD nanoparticles protected mice from airway inflammation. Conclusion VD's modulation of airway inflammation may result from its inactivation of ERK1/2 and downregulation of NPRA expression. Chitosan nanoparticles containing pVD may be therapeutically effective in preventing allergic airway inflammation.

The receptor for atrial natriuretic peptide (ANP), natriuretic peptide receptor A (NPRA), is expressed in cancer cells, and natriuretic peptides have been implicated in cancers. However, the direct role of NPRA signaling in tumorigenesis remains elusive. Here, we report that NPRA expression and signaling is important for tumor growth. NPRA-deficient mice showed significantly reduced antigen-induced pulmonary inflammation. NPRA deficiency also substantially protected C57BL/6 mice from lung, skin, and ovarian cancers. Furthermore, a nanoparticle-formulated interfering RNA for NPRA attenuated B16 melanoma tumors in mice. Ectopic expression of a plasmid encoding NP73-102, the NH(2)-terminal peptide of the ANP prohormone, which down-regulates NPRA expression, also suppressed lung metastasis of A549 cells in nude mice and tumorigenesis of Line 1 cells in immunocompetent BALB/c mice. The antitumor activity of NP73-102 was in part attributed to apoptosis of tumor cells. Western blot and immunohistochemistry staining indicated that the transcription factor, nuclear factor-kappaB, was inactivated, whereas the level of tumor suppressor retinoblastoma protein was up-regulated in the lungs of NPRA-deficient mice. Furthermore, expression of vascular endothelial growth factor was down-regulated in the lungs of NPRA-deficient mice compared with that in wild-type mice. These results suggest that NPRA is involved in tumor angiogenesis and represents a new target for cancer therapy.

The use of chitosan nanoparticles as carriers for expression plasmids represents a major improvement in gene expression technology. We demonstrated previously that treatment with chitosan interferon-γ (IFN-γ) plasmid deoxyribonucleic acid (DNA) nanoparticles (chitosan interferon-γ nanogene [CIN]) led to in situ production of IFN-γ and a reduction in inflammation and airway reactivity in mice, but the mechanism underlying the immunomodulatory effects of CIN remains unclear. In this report, the effect of CIN treatment on the immune responses of CD8+ T cells and dendritic cells was examined in a BALB/c mouse model of ovalbumin (OVA)-induced allergic asthma. OT1 mice (OVA-T cell receptor [TCR] transgenic) were also used to test the effects of CIN on OVA-specific CD8+ T cells. CIN treatment caused a reduction in IFN-γ production in a subpopulation of OVA-specific CD8+ T cells cultured in vitro in the presence of OVA. CIN also reduced apoptosis of the CD8+ T cells. Examination of dendritic cells from lung and lymph nodes indicated that CIN treatment decreased their antigen-presenting activity, as evident from the reduction in CD80 and CD86 expression. Furthermore, CIN treatment significantly decreased the number of CD11c+b+ dendritic cells in lymph nodes, suggesting that endogenous IFN-γ expression may immunomodulate dendritic cell migration and activation. CIN therapy results in a reduction in proinflammatory CD8+ T cells and decreases the number and antigen-presenting activity of dendritic cells.

Thiolated chitosan appears to possess enhanced mucoadhesiveness and cell penetration properties, however, its potential in gene-drug delivery remains unknown. Herein, we report on a highly effective gene delivery system utilizing a 33-kDa thiol-modified chitosan derivative.

Background Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in infants and is a risk factor for the development of asthma. Allergic asthmatics are more susceptible to RSV infection and viral exacerbation. Methods Since the effectiveness of corticosteroids in treating RSV infection has been controversial, we tested fluticasone propionate (FP) and salmeterol (Sal) alone versus FP plus Sal (FPS) on RSV-induced airway inflammation. Mice were sensitized and challenged with ovalbumin (OVA) and infected with RSV. Following infection they were treated with FP, Sal, or FPS intranasally and airway hyperreactivity (AHR), inflammation and RSV titers were examined. Results The group treated with FPS showed significantly lower AHR compared to the group treated with FP or Sal alone. The group treated with FP alone showed slightly decreased (non-significant) AHR compared to controls. Treatment with FPS resulted in significant decreases in the percentage of eosinophils and neutrophils in bronchoalveolar lavage fluid and in lung pathology compared to FP or Sal. FP alone decreased eosinophils but not neutrophils or lymphocytes, while Sal alone decreased eosinophils and neutrophils but not lymphocytes. FPS treatment of mice infected with RSV in the absence of allergen sensitization resulted in a 50% decrease of RSV titer in the lung and a reduction in neutrophils compared to FP or Sal. Conclusion Together, these results indicate that fluticasone in combination with salmeterol is a more effective treatment for decreasing airway hyperreactivity and inflammation than either of them alone in allergen-sensitized, RSV-infected mice.

With the development of genomic and proteomic technologies, the prospect for gene therapy has progressed rapidly. This has been partly possible due to the emergence of a diverse array of polymeric and non-polymeric nanoparticles that are being investigated for their ability to deliver genes and drugs. in this review, particles have been pragmatically divided as chitosan-related and chitosan-unrelated nanomaterials. The state of the art in terms of the development, characterisation and evaluation of their in vitro and/or in vivo potential is discussed for each of these various particles. Although substantial progress has been made, the potential of these particles in the clinical arena and human responses remain to be evaluated. it is hoped that this review will provide an impetus for further studies of these particles, with the ultimate intent that one or more of these diverse nanoparticle-based non-viral approaches for gene transfer will translate from 'bench to bedside' in the future.

Background: Respiratory syncytial virus (RSV) infection causes bronchiolitis in infants and children, which can be fatal, especially in immunocompromised patients. The BALB/c mouse, currently used as a model for studying RSV immunopathology, is semi-permissive to the virus. A mouse model that more closely mimics human RSV infection is needed. Since immunocompromised conditions increase risk of RSV infection, the possibility of enhancing RSV infection in the BALB/c mouse by pretreatment with cyclophosphamide was examined in this study. BALB/c mice were treated with cyclophosphamide (CYP) and five days later, they were infected with RSV intranasally. Pulmonary RSV titers, inflammation and airway hyperresponsiveness were measured five days after infection.Results: CYP-treated mice show higher RSV titers in their lungs of than the untreated mice. Also, a decreased percentage of macrophages and an increased number of lymphocytes and neutrophils were present in the BAL of CYP-treated mice compared to controls. The CYP-treated group also exhibited augmented bronchoalveolar and interstitial pulmonary inflammation. The increased RSV infection in CYP-treated mice was accompanied by elevated expression of IL-10, IL-12 and IFN-gamma mRNAs and proteins compared to controls. Examination of CYP-treated mice before RSV infection showed that CYP treatment significantly decreased both IFN-gamma and IL-12 expression.Conclusions: These results demonstrate that CYP-treated BALB/c mice provide a better model for studying RSV immunopathology and that decreased production of IL-12 and IFN-gamma are important determinants of susceptibility to RSV infection.