A 3D fibrous scaffold inducing tumoroids: a platform for anticancer drug development.

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.

Natriuretic peptide receptor a as a novel target for prostate cancer.

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.

Plasmid-encoded NP73-102 modulates atrial natriuretic peptide receptor signaling and plays a critical role in inducing tolerogenic dendritic cells.

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-ß, but not IL-12. CONCLUSIONS: Modulation of NPRA signaling in DCs leads to immune tolerance and TLR2 and SOCS3 are involved in this induction.

Regulating the Regulators: microRNA and Asthma.

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 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, 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.

The infectious march: the complex interaction between microbes and the immune system in asthma.

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.

Immunotherapy for allergies and asthma: present and future.

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.

Modulation of lung inflammation by vessel dilator in a mouse model of allergic asthma.

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.

Natriuretic peptide receptor a as a novel anticancer target.

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.

Chitosan Interferon-gamma Nanogene Therapy for Lung Disease: Modulation of T-Cell and Dendritic Cell Immune Responses.

: 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-gamma (IFN-gamma) plasmid deoxyribonucleic acid (DNA) nanoparticles (chitosan interferon-gamma nanogene [CIN]) led to in situ production of IFN-gamma 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-gamma 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-gamma 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/DNA nanocomplexes exhibit enhanced and sustained gene delivery.

PURPOSE: 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. METHODS: Thiolated chitosan was prepared by the reaction with thioglycolic acid. Nanocomplexes of unmodified chitosan or thiolated chitosan with plasmid DNA encoding green fluorescenct protein (GFP) were characterized for their size, zeta potential, their ability to bind and protect plasmid DNA from degradation. The transfection efficiency of thiolated chitosan and sustained gene expression were evaluated in various cell lines in vitro and in Balb/c mice in vivo. RESULTS: Thiolated chitosan-DNA nanocomplexes ranged in size from 75 to 120 nm in diameter and from +2.3 to 19.7 mV in zeta potential, depending on the weight ratio of chitosan to DNA. Thiolated chitosan, CSH360, exhibited effective physical stability and protection against DNase I digestion at a weight ratio>or=2.5:1. CSH360/DNA nanocomplexes induced significantly (P<0.01) higher GFP expression in HEK293, MDCK and Hep-2 cell lines than unmodified chitosan. Nanocomplexes of disulphide-crosslinked CSH360/DNA showed a sustained DNA release and continuous expression in cultured cells lasting up to 60 h post transfection. Also, intranasal administration of crosslinked CSH360/DNA nanocomplexes to mice yielded gene expression that lasted for at least 14 days. CONCLUSIONS: Thiolated chitosans condense pDNA to form nanocomplexes, which exhibit a significantly higher gene transfer potential and sustained gene expression upon crosslinking, indicating their great potential for gene therapy and tissue engineering.

Combined fluticasone propionate and salmeterol reduces RSV infection more effectively than either of them alone in allergen-sensitized mice.

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.

An immunocompromised BALB/c mouse model for respiratory syncytial virus infection.

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.

Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene.

Respiratory syncytial virus (RSV) infection is one of the major causes of respiratory tract infection for which no vaccine or antiviral treatment is available. The RSV NS1 protein seems to antagonize the host interferon (IFN) response; however, its mechanism is unknown. Here, we used a plasmid-borne small interfering RNA targeting the NS1 gene (siNS1) to examine the role of NS1 in modulating RSV infection. RSV replication was reduced in A549 cells, but not IFN-deficient Vero cells, transfected with siNS1. siNS1 induced upregulated expression of IFN-beta and IFN-inducible genes in A549 cells. siNS1-transfected human dendritic cells, upon RSV infection, produced elevated type-1 IFN and induced differentiation of naive CD4+ T cells to T helper type 1 (TH1) cells. Mice treated intranasally with siNS1 nanoparticles before or after infection with RSV showed substantially decreased virus titers in the lung and decreased inflammation and airway reactivity compared to controls. Thus, siNS1 nanoparticles may provide an effective inhibition of RSV infection in humans.

Attenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery.

BACKGROUND: The need for safe and effective treatment of dengue virus (DEN), a class A agent that causes dengue hemorrhagic fever/dengue shock syndrome, has been a critical global priority. An effective vaccine for DEN is not yet available. In this study the possibility of attenuating DEN infection using adeno-associated virus (AAV)-encoded short interfering RNAs (siRNA) was examined in Vero cells and human dendritic cells (DCs). METHODS: A cassette encoding siRNA targeted to a 3' untranslated sequence common to all DEN serotypes was designed and tested for its ability to attenuate DEN infection by use of AAV delivery. RESULTS: Vero cells or DCs infected with AAV-siRNA showed a significant, dose-dependent reduction in DEN infection. Treatment of DCs with AAV-siRNA also decreased the DEN-induced apoptosis of DCs and did not induce significant inflammation. CONCLUSION: These results demonstrate that AAV-mediated siRNA delivery is capable of reducing DEN infection in cells and may be useful in decreasing DEN replication in humans.

Nanoparticle-mediated gene delivery: state of the art.

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.

Aging enhances vascular dysfunction induced by the Alzheimer's peptide beta-amyloid.

Aging is a major risk factor for Alzheimer's disease and the evidence suggests a role for cerebrovascular pathology in cognitive dysfunction. The hypothesis in this study is that aging is a significant risk factor in the effect of the Alzheimer peptide beta-amyloid on endothelium-dependent function of cerebral and peripheral vessels. The diameter response to acetylcholine, an endothelium-dependent vasodilator, was recorded in pressurized segments of rat posterior cerebral vessels from mature (3 months) and aged (20 months) rats. The threshold concentration of beta-amyloid for a significant decrease in the response to acetylcholine was lower in vessels from aged rats (10(-9) M) than in vessels from mature rats (10(-8) M). The threshold concentration of beta-amyloid for a significant decrease in the sensitivity to acetylcholine was lower for ring segments of aorta from aged rats (10(-10) M) than for aorta from mature rats (10(-8) M). Structural changes of the endothelium were first observed in electron micrographs of aorta from aged rats when the concentration of beta-amyloid was 10(-8) M, whereas structural changes in aorta from mature rats did not occur until the concentration of beta-amyloid was increased to 10(-7) M. The results suggest that aging increases the susceptibility of cerebral and peripheral blood vessels to beta-amyloid related dysfunction and that functional change precedes structural change.

Mechanism of bronchoprotective effects of a novel natriuretic hormone peptide.

BACKGROUND: The natriuretic hormone peptide (NHP)(99-126), a C-terminal peptide of pro-atrial natriuretic factor (proANF), induces bronchodilatory effects in people with asthma. Recently, another plasmid-encoded C-terminal peptide, pNHP(73-102), was shown to induce a long-lasting bronchoprotective effect in a mouse model of allergic asthma. OBJECTIVE: This study was carried out to determine the role of lung epithelial cells in the bronchoprotective and anti-inflammatory activity of these peptides. METHODS: Human type II alveolar epithelial cells (A549) and normal human bronchial epithelial (NHBE) cells were transfected with pNHP(73-102) to test the effect of this peptide on activation of these cells. After transfection, cells were analyzed for changes in Ca(++) and nitric oxide (NO) levels. Also, activation of NFkappaB and the extracellularly regulated kinase (ERK) 1, 2 signaling pathway was examined by luciferase reporter assay and phosphorylation studies respectively. RESULTS: Analysis of intracellular Ca(++) levels in pNHP(73-102) -transfected A549 or NHBE showed that the peptide increases release. This Ca(++) release was accompanied by an increase in the production of NO. Also, overexpression of pNHP(73-102), but not pVAX control, in phorbol myristate acetate-activated A549 cells resulted in a significant decrease in expression of a cotransfected nuclear factorkappaB (NFkappaB)-luciferase reporter. Similarly, pNHP(73-102) decreased TNF-alpha-induced NFkappaB activation in NHBE cells. Furthermore, NHP(73-102) but not atrial natriuretic peptide decreased phosphorylation of Erk-1, 2 in A549 cells. CONCLUSIONS: Overexpression of pNHP(73-102) in epithelial cells causes increased production of intracellular Ca(++) and NO, with a concomitant decrease in activation of NFkappaB and ERK1, 2. These results suggest a bronchodilatory and anti-inflammatory activity of this peptide.

Chitosan IFN-gamma-pDNA Nanoparticle (CIN) Therapy for Allergic Asthma.

BACKGROUND: Allergic subjects produce relatively low amounts of IFN-gamma, a pleiotropic Th-1 cytokine that downregulates Th2-associated airway inflammation and hyperresponsiveness (AHR), the hallmarks of allergic asthma. Adenovirus-mediated IFN-gamma gene transfer reduces AHR, Th2 cytokine levels and lung inflammation in mice, but its use would be limited by the frequency of gene delivery required; therefore, we tested chitosan/IFN-gamma pDNA nanoparticles (CIN) for in situ production of IFN-gamma and its in vivo effects. METHODS: CIN were administered to OVA-sensitized mice to investigate the possibility of using gene transfer to modulate ovalbumin (OVA)-induced inflammation and AHR. RESULTS: Mice treated with CIN exhibit significantly lower AHR to methacholine challenge and less lung histopathology. Production of IFN-gamma is increased after CIN treatment while the Th2-cytokines, IL-4 and IL-5, and OVA-specific serum IgE are reduced compared to control mice. AHR and eosinophilia are also significantly reduced by CIN therapy administered therapeutically in mice with established asthma. CIN was found to inhibit epithelial inflammation within 6 hours of delivery by inducing apoptosis of goblet cells. Experiments performed on STAT4-defective mice do not show reduction in AHR with CIN treatment, thus implicating STAT4 signaling in the mechanism of CIN action. CONCLUSION: These results demonstrate that mucosal CIN therapy can effectively reduce established allergen-induced airway inflammation and AHR.