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DNA Nanoparticle-Mediated Thymulin Gene Therapy Prevents Airway Remodeling in Experimental Allergic Asthma
Abstract
Thymulin has been shown to present anti-inflammatory and anti-fibrotic properties in experimental lung diseases. We hypothesized that a biologically active thymulin analog gene, methionine serum thymus factor, delivered by highly compacted DNA nanoparticles composed of single molecule of plasmid DNA compacted with block copolymers of poly-l-lysine and polyethylene glycol (CK30PEG) that have been found safe in a human phase I clinical trial, may prevent lung inflammation and remodeling in a mouse model of allergic asthma. Thymulin plasmids were detected in the lungs of ovalbumin-challenged asthmatic mice up to 27days after administration of DNA nanoparticles carrying thymulin plasmids. A single dose of DNA nanoparticles carrying thymulin plasmids prevented lung inflammation, collagen deposition and smooth muscle hypertrophy in the lungs of a murine model of ovalbumin-challenged allergic asthma, leading to improved lung mechanics. In the present model of chronic allergic asthma, highly compacted DNA nanoparticles using thymulin analog gene modulated the inflammatory and remodeling processes improving lung mechanics.
... FTS is associated with antiinflammatory and anti-fibrotic properties in various disease models [161]. In 2014, da Silva and collaborators demonstrated that DNA nanoparticle-mediated Thymulin gene therapy prevented airway remodeling in an experimental model of allergic asthma [162]. Nanoparticle-based technologies relying on highly compacted DNA nanoparticles with CK 30 PEG have shown impressive results. ...
... These DNA nanoparticles were stable for at least 2 years at 4 • C and were non-toxic, non-inflammatory, and non-immunogenic. Previous studies demonstrated that nanoparticles carrying thymulin plasmid provided prolonged retention of upwards of 27 days post-administration in mice lungs [162]. A single dose of DNA nanoparticles effectively prevented lung remodeling processes in a rodent model of chronic allergic asthma, as demonstrated by decreased pulmonary inflammation, collagen deposition, and smooth muscle hypertrophy, and improved lung mechanics such as lung static elastance and airway hyperresponsiveness [162]. ...
... Previous studies demonstrated that nanoparticles carrying thymulin plasmid provided prolonged retention of upwards of 27 days post-administration in mice lungs [162]. A single dose of DNA nanoparticles effectively prevented lung remodeling processes in a rodent model of chronic allergic asthma, as demonstrated by decreased pulmonary inflammation, collagen deposition, and smooth muscle hypertrophy, and improved lung mechanics such as lung static elastance and airway hyperresponsiveness [162]. This study also provides a new platform on which to further develop nanoparticle-based gene therapies for asthma [162]. ...
Over the past decades, a better understanding of the genetic and molecular alterations underlying several respiratory diseases has encouraged the development of new therapeutic strategies. Gene therapy offers new therapeutic alternatives for inherited and acquired diseases by delivering exogenous genetic materials into cells or tissues to restore physiological protein expression and/or activity. In this review, we review (1) different types of viral and non-viral vectors as well as gene-editing techniques; and (2) the application of gene therapy for the treatment of respiratory diseases and disorders, including pulmonary arterial hypertension, idiopathic pulmonary fibrosis, cystic fibrosis, asthma, alpha-1 antitrypsin deficiency, chronic obstructive pulmonary disease, non-small-cell lung cancer, and COVID-19. Further, we also provide specific examples of lung-targeted therapies and discuss the major limitations of gene therapy.
... Then, antigen challenge was performed in OVA mice by intratracheal administration of 20 µg of OVA in 20 µl saline. This procedure was performed 3 times at 3-day intervals between applications (Da Silva et al., 2014;Xisto et al., 2005). Twentyfour hours after the last challenge, the ETR formulation containing roflumilast 600 μg/kg (OVA-ETR group) and ETRF formulation containing roflumilast 600 µg/kg and formoterol 288 µg/kg (OVA-ETRF group) were instilled intratracheally (i.t.) using a micro sprayer (Penn Century-Micro Sprayer, Model IA-1C, Philadelphia, PA, USA). ...
... However, given that roflumilast is better known for its anti-inflammatory rather than bronchodilatory effects, what is surprising is that the formulation containing roflumilast alone provided as good a bronchodilatory effect as that containing roflumilast and formoterol. As expected by the well-established nature of OVA-induced inflammation (Da Silva et al., 2014;Xisto et al., 2005), the total number of leukocytes in BALF was higher in the OVA-SALINE group compared to the CTRL group (Fig. 5). Although, total cellularity did not significantly differ between the OVA-SALINE group and the other groups, the percentage of macrophages, eosinophils, neutrophils and lymphocytes differed. ...
... Overproduction and deposition of extracellular matrix material, particularly collagen, is the most notable pathological alteration of airway remodelling (Da Silva et al., 2014). As expected, the collagen ...
In this study we aimed to develop a roflumilast (R) and formoterol fumarate (F) dry powder inhaler formulation (DPI) incorporating HPβCD by spray drying and evaluated if it attenuates the inflammatory process and improves lung function in a murine model of ovalbumin induced allergic asthma. The DPI was characterized by powder X-ray diffraction, thermal analysis, scanning electron microscopy, particle size, density, specific surface area and dynamic vapor sorption analyses. In vitro deposition studies were performed using a NGI, while transepithelial permeability and in vivo effects on lung mechanics and inflammation in a model of allergic asthma were also assessed. The R:F formulation was amorphous with high glass transition temperatures, comprised of wrinkled particles, had low bulk and tapped densities, high surface area, suitable particle size for pulmonary delivery and exhibited no recrystallization even at high relative humidities. MMAD were statistically similar of 4.22 ± 0.19 and 4.32 ± 0.13 µm for F and R, respectively. Fine particle fractions (< 5 µm) were of more than 50% of the emitted dose. The R:F formulation led to reduced eosinophil infiltration and airway collagen fiber content, yielding decreased airway hyperresponsiveness. In the current asthma model, the R:F formulation combination decreased inflammation and remodeling, thus improving lung mechanics.
... Thus, in addition to the therapies for attenuating airway inflammation, intervention of asthma-associated structural changes in the lung, including smooth muscle hypertrophy, wall thickening and collagen deposition, could reduce the rate of loss of lung function. Recently, da Silva et al. reported a proof-of-concept study where they evaluated effect of the gene encoding an active form of thymulin peptide in a mouse model of asthma [77]. Thymulin peptide has been shown to mediate anti-inflammatory and anti-fibrotic effects in several disease models by modulating T cell differentiation [78]. ...
... Numerous groups using this model have reported that chronic asthmatic symptoms do not exacerbate over time; the symptoms are actually ameliorated by natural development of tolerance and/or termination of OVA challenge [404,405]. More recently, the development of tolerance to repeated challenges has been addressed by optimizing amount and frequency of OVA administration [77,400]. Although not as frequently used, rats and guinea pigs are also utilized as background species of OVA models. ...
... (A-B) Reproduced from[351] with permission from AAAS. (C-D) Reproduced from[374] with permission from APS. (E) Reproduced from[77] with permission from Elsevier. ...
Knowledge of genetic origins of obstructive lung diseases has made inhaled gene therapy an attractive alternative to the current standards of care that are limited to managing disease symptoms. Initial lung gene therapy clinical trials occurred in the early 1990s following the discovery of the genetic defect responsible for cystic fibrosis (CF), a monogenic disorder. However, despite over two decades of intensive effort, gene therapy has yet to help patients with CF or any other obstructive lung disease. The slow progress is due in part to poor understanding of the biological barriers to inhaled gene therapy. Encouragingly, clinical trials have shown that inhaled gene therapy with various viral vectors and non-viral gene vectors is well tolerated by patients, and continued research has provided valuable lessons and resources that may lead to future success of this therapeutic strategy. In this review, we first introduce representative obstructive lung diseases and examine limitations of currently available therapeutic options. We then review key components for successful execution of inhaled gene therapy, including gene delivery systems, primary physiological barriers and strategies to overcome them, and advances in preclinical disease models with which the most promising systems may be identified for human clinical trials.
... The previous description indicates that thymic hormones can mediate anti-inflammatory effects, and future clinical trials are needed to translate them against inflammatory disorders and viral diseases [48]. In an experimental model of allergic asthma, a dose of DNA nanoparticles, including thymuline plasmids, could protect the lungs from some injurious inflammation and muscular hypertrophy, which recovered respiratory mechanical functions [49]. Mice were treated through intratracheal administration with a dose of thymuline-expressing plasmids administrated with nanoparticles to enable the thymuline to infiltrate the mucus barrier of the respiratory system [50]. ...
... them against inflammatory disorders and viral diseases [48]. In an experimental model of allergic asthma, a dose of DNA nanoparticles, including thymuline plasmids, could protect the lungs from some injurious inflammation and muscular hypertrophy, which recovered respiratory mechanical functions [49]. Mice were treated through intratracheal administration with a dose of thymuline-expressing plasmids administrated with nanoparticles to enable the thymuline to infiltrate the mucus barrier of the respiratory system [50]. ...
The thymus is the main lymphoid organ that regulates the immune and endocrine systems by controlling thymic cell proliferation and differentiation. The gland is a primary lymphoid organ responsible for generating mature T cells into CD4+ or CD8+ single-positive (SP) T cells, contributing to cellular immunity. Regarding humoral immunity, the thymic plasma cells almost exclusively secrete IgG1 and IgG3, the two main complement-fixing effector IgG subclasses. Deformity in the thymus can lead to inflammatory diseases. Hassall’s corpuscles’ epithelial lining produces thymic stromal lymphopoietin, which induces differentiation of CDs thymocytes into regulatory T cells within the thymus medulla. Thymic B lymphocytes produce immunoglobulins and immunoregulating hormones, including thymosin. Modulation in T cell and naive T cells decrement due to thymus deformity induce alteration in the secretion of various inflammatory factors, resulting in multiple diseases. Influenza virus activates thymic CD4+ CD8+ thymocytes and a large amount of IFNγ. IFNs limit virus spread, enhance macrophages’ phagocytosis, and promote the natural killer cell restriction activity against infected cells. Th2 lymphocytes-produced cytokine IL-4 can bind to antiviral INFγ, decreasing the cell susceptibility and downregulating viral receptors. COVID-19 epitopes (S, M, and N proteins) with ≥90% identity to the SARS-CoV sequence have been predicted. These epitopes trigger immunity for antibodies production. Boosting the immune system by improving thymus function can be a therapeutic strategy for preventing virus-related diseases. This review aims to summarize the endocrine-immunoregulatory functions of the thymus and the underlying mechanisms in the prevention of COVID-19.
... K. Patel et al., 2019). In an attempt to improve patient compliance by lowering dosing frequency, Hanes and coworkers developed an inhalable long-lasting gene therapy composed of DNA-thymulin polymeric particles that were capable of inducing anti-inflammatory effects in asthma (da Silva et al., 2014). The biologically active thymulin-analog gene, methionine serum thymus factor (MSTF), prevents lung inflammation and remodeling in an allergic asthma model. ...
... Moreover, the CK 30 PEG-MSTF NPs did not elevate lung static elastance nor airway resistance, as depicted in Figure 5f and g, respectively. These findings showcase a long-acting system for asthma treatment through pulmonary delivery of therapeutic genes for CPDs (da Silva et al., 2014). ...
Respiratory illnesses are prevalent around the world, and inhalation‐based therapies provide an attractive, noninvasive means of directly delivering therapeutic agents to their site of action to improve treatment efficacy and limit adverse systemic side effects. Recent trends in medicine and nanoscience have prompted the development of inhalable nanomedicines to further enhance effectiveness, patient compliance, and quality of life for people suffering from lung cancer, chronic pulmonary diseases, and tuberculosis. Herein, we discuss recent advancements in the development of inhalable nanomaterial‐based drug delivery systems and analyze several representative systems to illustrate their key design principles that can translate to improved therapeutic efficacy for prevalent respiratory diseases.
This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Respiratory Disease
... BALB/c mice have been used to induce asthma [84][85][86][87][88] due to their ability to develop airway hyperresponsiveness after chronic sensitization and challenge with ovalbumin [80]. These animals also present increased airway inflammation [89][90][91][92], epithelial cell damage, eosinophil infiltration [93], airway hyperreactivity [94][95][96] and remodeling, characterized by higher collagen fiber content [94,[97][98][99][100][101][102][103]. These structural changes in the ECM lead to impairment of lung mechanics, Vol. 29, No. 2019 Drug Discovery Today: Disease Models | www.drugdiscoverytoday.com characterized by increased airway resistance [93], static lung elastance [98,104], and viscoelastic pressure [99,105]. ...
... These animals also present increased airway inflammation [89][90][91][92], epithelial cell damage, eosinophil infiltration [93], airway hyperreactivity [94][95][96] and remodeling, characterized by higher collagen fiber content [94,[97][98][99][100][101][102][103]. These structural changes in the ECM lead to impairment of lung mechanics, Vol. 29, No. 2019 Drug Discovery Today: Disease Models | www.drugdiscoverytoday.com characterized by increased airway resistance [93], static lung elastance [98,104], and viscoelastic pressure [99,105]. In C57BL/6 mice, ovalbumin-induced allergic asthma is associated with similar lung functional and morphological changes [16,82,[106][107][108]. ...
Mice are frequently used in experimental models of respiratory diseases due to their ease of manipulation, genetic homogeneity within inbred populations, and possibility of standardizing environmental exposures. However, it is well established that genetic strain variations in mice may exist, which imply changes in extracellular matrix (ECM) composition and degree of ECM remodeling, with potential for major impacts on respiratory mechanics. The lung ECM is mainly composed of fibrous proteins (collagen and elastin), glycoproteins (fibronectin and laminin), proteoglycans (PGs), and glycosaminoglycans (GAGs). The functions of many ECM components are well described, but their role in the pathogenesis of respiratory diseases, such as emphysema and asthma, requires further elucidation. The aim of this review is to address ECM composition, function, and remodeling as well as demonstrate its relationship with the mechanical profile of the lung in different strains of mice subjected to experimental emphysema and allergic asthma.
... In the latter model, thymulin effect was related to the inhibition of the expression of the proinflammatory cytokine IL-6 and to the suppression of the p38 MAPK pathway. In a more recent report, DaSilva et al. (2014)implemented thymulin gene therapy in a mouse model of allergic asthma. In this study, intratracheal injection of a nanoparticle-complexed plasmid expressing our synthetic gene for thymulin decreased lung eosinophil infiltration, IL-13, eotaxin, transforming growth factor-β, vascular endothelial growth factor, and increased lung interferon-γ. ...
... This study revealed that DNA-nanoparticle-mediated thymulin gene therapy can modulate the inflammatory and remodeling processes in experimental allergic asthma, therefore improving lung mechanics. The thymulin plasmid used in the above study (Da Silva et al., 2014) revealed the suitability of thymulin vectors for the treatment of inflammatory pathologies. We reasoned that a viral vector expressing thymulin and a reporter gene both under the control of a bidirectional regulatable promoter would allow experimenters, and eventually clinical gene therapists, to manipulate the expression of these genes according to treatment needs. ...
Thymulin is a thymic peptide possessing anti-inflammatory effects. In order to manipulate thymulin expression in gene therapy studies, we built a bidirectional regulatable two-vector Tet-Off system and the corresponding control system. The experimental two-vector system, ETV, consists of a recombinant adenovector (RAd) harboring an expression cassette centered on a Tet-Off bidirectional promoter flanked by a synthetic gene for thymulin and the gene for humanized Green Fluorescent Protein (hGFP). The second adenovector of this system, RAd-tTA, constitutively expresses the regulatory protein tTA. When cells are co-transduced by the two adenovector components, tTA activates the bidirectional promoter and both transgenes are expressed. In the presence of the antibiotic doxycycline (DOX) transgene expression is deactivated. The control two-vector system, termed CTV, is similar to ETV but only expresses hGFP. In CHO-K1, BHK, and C2C12 cells, ETV and CTV induced a dose-dependent hGFP expression. In CHO-K1 cells, transgene expression was almost completely inhibited by DOX (1 mg/ml). After intracerebroventricular injection of ETV in rats, thymulin levels increased significantly in the cerebrospinal fluid and there was high hGFP expression in the ependymal cell layer. When injected intramuscularly the ETV system induced a progressive increase in serum thymulin levels, which were inhibited when DOX was added to the drinking water. We conclude that our regulatable two-adenovector system is an effective molecular tool for implementing short and long-term anti-inflammatory thymulin gene therapy in animal models of acute or chronic inflammation.
... These results suggest that the secretion of IFN-g is more beneficial for the treatment and recovery of AR mice. Some studies have found that IFN-g secretion may not change significantly or may even decrease significantly after effective AR therapy in vivo and in vitro (26,29,67,(77)(78)(79)(80)(81)(82)(83). The level of IFN-g was significantly lower in the conditioned media from the T-MSCs group than in the AR group (58). ...
Background
Human adipose tissue-derived stem cells (hADSCs) exert potent immunosuppressive effects in the allogeneic transplantation treatment. In mouse model of allergic rhinitis (AR), ADSCs partially ameliorated AR. However, no study has evaluated the potential therapeutic effects of hADSC-derived extracellular vesicles (hADSC-EVs) on AR.
Methods
Female BALB/c mice were sensitized and challenged with ovalbumin (OVA) to induce AR. One day after the last nasal drop, each group received phosphate buffered saline (PBS) or hADSC-EVs treatment. Associated symptoms and biological changes were then assessed.
Results
hADSC-EV treatment significantly alleviated nasal symptoms, and reduced inflammatory infiltration. Serum levels of OVA-specific IgE, interleukin (IL)-4 and interferon (IFN)-γ were all significantly reduced. The mRNA levels of IL-4 and IFN-γ in the spleen also changed accordingly. The T helper (Th)1/Th2 cell ratio increased. The treatment efficacy index of hADSC-EV was higher than that of all human-derived MSCs in published reports on MSC treatment of AR. ADSC-EVs exhibited a greater therapeutic index in most measures when compared to our previous treatment involving ADSCs.
Conclusion
These results demonstrated that hADSC-EVs could ameliorate the symptoms of AR by modulating cytokine secretion and Th1/Th2 cell balance. hADSC-EVs could potentially be a viable therapeutic strategy for AR. Further animal studies are needed to elucidate the underlying mechanisms and to optimize potential clinical protocols.
... Adding polyethylene glycol (PEG), a neutrally charged hydrophilic biocompatible polymer, to the rAAV formulation seemed to prevent the vector from binding to the mucous layer as well as protecting it from neutralizing antibodies [54]. Likewise, the construction of PEG-conjugated nanoparticles (muco-penetrating particles, MPPs) based on polymers has shown efficiency in the treatment of diseases that affect the lung, such as asthma [55,56]. However, these methods all present challenges to persistent AAT expression from the dividing cell populations targeted by airway delivery. ...
Introduction:
Altering the human genetic code has been explored since the early 1990s as a definitive answer for the treatment of monogenic and acquired diseases which do not respond to conventional therapies. In Alpha-1 antitrypsin deficiency (AATD) the proper synthesis and secretion of alpha-1 antitrypsin (AAT) protein is impaired, leading to its toxic hepatic accumulation along with its pulmonary insufficiency, which is associated with parenchymal proteolytic destruction. Because AATD is caused by mutations in a single gene whose correction alone would normalize the mutant phenotype, it has become a popular target for both augmentation gene therapy and gene editing. Although gene therapy products are already a reality for the treatment of some pathologies, such as inherited retinal dystrophy and spinal muscular atrophy, AATD-related pulmonary and, especially, liver diseases still lack effective therapeutic options.
Areas covered:
Here, we review the course, challenges, and achievements of AATD gene therapy as well as update on new strategies being developed.
Expert opinion:
Reaching safe and clinically effective expression of the AAT is currently the greatest challenge for AATD gene therapy. The improvement and emergence of technologies that use gene introduction, silencing and correction hold promise for the treatment of AATD.
... Additionally, it has been shown that CIN inhibits the generation of Th2 cytokines and reduces inflammatory cytokines, CD8 T lymphocytes, and dendritic cell downregulation. Human lungs may tolerate CK30PEG NPs, which are immune-compatible and made of poly-L-lysine and polyethylene glycol connected by cysteine residue [206]. H.-Y. Y. Li et al. created a unique platform wherein W/O microemulsions were spray-dried to make salbutamol sulfate and insulin NPs. ...
Pulmonary diseases pose an immense threat to global health. Several nanotherapeutics-based approaches aimed to tackle pulmonary ailments in recent years. The current innovation includes developing nanomedicine for pulmonary delivery of active pharmaceutical ingredients (API), repurposed drugs, host-directed therapies, or molecules with unconventional mechanisms (such as antimicrobial peptides). But pulmonary delivery of nanomedicines is troublesome due to poor aerodynamic properties for lung deposition and expulsion during inhalation. Nanomedicine can deliver drugs directly into lung tissue via aerosol or dry powder for inhalation to target deep lung deposition and subsequent therapeutic efficacy. Particulate-based drug delivery systems are potentially complementary to conventional inhaled drugs. There are several ways to produce an aerosol or DPI-based therapeutics, and we reviewed some standard methods used to develop nanotherapeutics for inhalation. The developed inhalable formulations can be delivered using inhalation devices, and advanced inhaler device technology has allowed the efficacious delivery of therapeutic compounds via inhalation. In addition, particle engineering is a crucial criterion in developing inhalable formulations to improve drug delivery, enhance therapeutic effects, and perform superior targeting. Technological advancements in inhalation devices have improved the efficiency of lung-based drug delivery mechanisms. This review also enumerated the assessment of pulmonary drug delivery systems, i.e., in vitro, ex vivo, in vivo, or in silico models, which have made significant strides in recent years.
... Thymic nonapeptide (thymulin or serum thymus factor) is capable of preventing the cascades of inflammatory and fibrotic responses in a mouse model of allergic asthma (121). In this regard, PBAE NPs (Poly (ethylene oxide)-modified poly (beta-amino ester), consisted of thymulin expressing plasmids (50 μl of NP that contained 50 μg of plasmid per mouse) were applied for the treatment of asthmatic female BALB/c mice (117). ...
Immunotherapy is a novel preference for the treatment of various complex diseases. Considering the application of varying agents for suppression or activation of the immune system, immunogene therapy was confirmed to stand as a proper alternative for other immunotherapeutic strategies due to its capability in targeting cells with more specificity that leads to controlling the expression of therapeutic genes. This method facilitates the local and single-dose application of most gene therapies that result in the usage of high therapeutic doses with a low risk of systemic side effects while being cost-efficient in long-term administrations. However, the existing barriers between the administration site and cell nucleus limited the clinical uses of genetic materials. These challenges can be overcome through the promising method of exerting non-carriers with high stability, low toxicity/immunogenicity, and simple modifications. In this study, we attempted to review the potential of nanoparticle application throughout the immunogene therapy of different diseases including cancer, microbial diseases, allergies, inflammatory bowel disease, rheumatoid arthritis, and respiratory infections. We included the outline of some challenges and opportunities in regards to the delivery of genetic materials that are based on nano-systems through immunotherapy of these disorders. Next to the promising future of these vectors, more detailed analyses are required to overcome the current limitations in clinical approaches. © 2022 Mashhad University of Medical Sciences. All rights reserved.
... Non Commercial Use by lowering dose frequency (Kaczmarek et al., 2016;Patel et al., 2019). Similarly, thymulin-analog gene methionine serum thymus factor (MSTF) inhibits inflammation, collagen deposition, and smooth muscle hypertrophy in the allergic asthma murine model (da Silva et al., 2014) to minimize inflammation in asthma patients. ...
Nanotechnology has potential to bridge the gaps of biological and physical sciences by applying nanostructures and nanophases at various applications of biomedical science, more explicitly in nanomedicine. Nanomedicine is of major concern for biomedical application owing to fascinating applications in the field of drug delivery and disease diagnosis. Nanomedicine and nano-delivery systems are moderately latest fields but quickly emerging science where substances in the nanorange are exploited to offer a means for diagnostic tools or to carry therapeutically active substances to specific targeted sites in a controlled manner. In this chapter, the benefits of nanotechnology in drug-delivery applications are examined. The transport of therapeutic agents to their desired sites occurs by employing different nanosystems. Over the several years, drug-delivery systems more precisely the nanosystems have emerged into diverse forms of second-generation drug-delivery systems, including nanobots, injectable nanoparticle generator, nanoghosts, niosomes, nanoclews, carbon nanotubes, nanocrystals. A detailed description of these systems has been also included. As nanotechnology offers multiple benefits in treating various human diseases, we have provided an updated review of current advances in the field of nanomedicines and nano-based drug-delivery systems through inclusive inspection of the discovery and application of nanomaterials in improving the efficacy of both new and old drugs.
... Non Commercial Use by lowering dose frequency (Kaczmarek et al., 2016;Patel et al., 2019). Similarly, thymulin-analog gene methionine serum thymus factor (MSTF) inhibits inflammation, collagen deposition, and smooth muscle hypertrophy in the allergic asthma murine model (da Silva et al., 2014) to minimize inflammation in asthma patients. ...
... Non Commercial Use by lowering dose frequency (Kaczmarek et al., 2016;Patel et al., 2019). Similarly, thymulin-analog gene methionine serum thymus factor (MSTF) inhibits inflammation, collagen deposition, and smooth muscle hypertrophy in the allergic asthma murine model (da Silva et al., 2014) to minimize inflammation in asthma patients. ...
... Non Commercial Use by lowering dose frequency (Kaczmarek et al., 2016;Patel et al., 2019). Similarly, thymulin-analog gene methionine serum thymus factor (MSTF) inhibits inflammation, collagen deposition, and smooth muscle hypertrophy in the allergic asthma murine model (da Silva et al., 2014) to minimize inflammation in asthma patients. ...
... This treatment reduced inflammation, collagen deposition, and smooth muscle hypertrophy. Researchers were encouraged by the results to continue working on nanoparticle-based gene treatments to deliver therapeutic genes for asthma safely and effectively [50,51]. Chitosan-hyaluronic acid nanoparticles coated with heparin were produced by Oyarzun-Ampuero, et al. for the treatment of asthma. ...
With a high prevalence globally, asthma is a severe hazard to human health, as well as an economic and social burden. There are now novel therapies available for asthma with the use of nanotechnology. Recent developments in nanoscience and medicine have encouraged the creation of inhalable nanomedicines that can enhance the efficacy, patient compliance, and life quality for sufferers of asthma. Nanocarriers for asthma therapy, including liposomes, micelles, polymers, dendrimers, and inorganics, are presented in depth in this study as well as the current research status of these nanocarriers. Aerosolized nanomaterial-based drug transport systems are currently being developed, and some examples of these systems, as well as prospective future paths, are discussed. New research subjects include nano-modification of medicines and the development of innovative nano-drugs. Clinical experiments have proven that nanocarriers are both safe and effective. Before nanotherapy can be applied in clinical practice, several obstacles must be addressed. We look at some of the most recent research discoveries in the subject of nanotechnology and asthma therapy in this article.
... These NPs inhibit proliferation of lung fibroblasts and significantly decrease the release of IL-8 in alveolar macrophages [144]. A highly compacted DNA nanoparticle produced from poly-lysin and polyethylene glycol copolymers can trigger the release of thymulin analog genes and significantly decrease the inflammatory and remodeling process in a mouse model of allergic airway diseases [145]. Moreover, Yoo et al. developed PLGA-based polymeric nanoparticles containing antiinflammatory compound (hydroxybenzyl-alcohol-conjugated polyoxalate), which was administered in an ovalbumin-induced asthma mice model intratracheally. ...
Chronic respiratory disorders (CRDs) affect millions of people worldwide. Pathophysiological changes to the normal airway wall structure, including changes in the composition and organization of its cellular and molecular constituents, are referred to as airway remodeling. The inadequacy of effective treatment strategies and scarcity of novel therapies available for the treatment and management of chronic respiratory diseases have given rise to a serious impediment in the clinical management of such diseases. The progress made in advanced drug delivery, has offered additional advantages to fight against the emerging complications of airway remodeling. This review aims to address the gaps in current knowledge about airway remodeling, the relationships between remodeling, inflammation, clinical phenotypes and the significance of using novel drug delivery methods.
... A number of nanoparticles have been evaluated in the retina [6,7]. One of the most well-characterized examples has been polyethylene glycol-linked polylysine (CK30PEG) nanoparticles which do not cause adverse effects in the retina and have a large carrying capacity (tested up to 14 kbp in the retina and 20 kbp in the lung [8][9][10][11]). These nanoparticles exhibited persistent gene expression and phenotypic rescue in animal models [9,12,13]; however, in common with other gene therapy approaches, phenotypic rescue was incomplete, and efficient outer retinal targeting required subretinal injections. ...
Gene and drug delivery to the retina is a critical therapeutic goal. While the majority of inherited forms of retinal degeneration affect the outer retina, specifically the photoreceptors and retinal pigment epithelium, effective targeted delivery to this region requires invasive subretinal delivery. Our goal in this work was to evaluate two innovative approaches for increasing both the persistence of delivered nanospheres and their penetration into the outer retina while using the much less invasive intravitreal delivery method. We formulated novel hyaluronic acid nanospheres (HA-NS, 250 nm and 500 nm in diameter) conjugated to fluorescent reporters and delivered them intravitreally to the adult Balb/C mouse retina. They exhibited persistence in the vitreous and along the inner limiting membrane (ILM) for up to 30 days (longest timepoint examined) but little retinal penetration. We thus evaluated the ability of the small molecule, sulfotyrosine, to disrupt the ILM, and found that 3.2 µg/µL sulfotyrosine led to significant improvement in delivery to the outer retina following intravitreal injections without causing retinal inflammation, degeneration, or loss of function. Co-delivery of sulfotyrosine and HA-NS led to robust improvements in penetration of HA-NS into the retina and accumulation along the interface between the photoreceptors and the retinal pigment epithelium. These exciting findings suggest that sulfotyrosine and HA-NS may be an effective strategy for outer retinal targeting after intravitreal injection.
... In its research, Silva et al., 2014 found that a single administration of thymulin plasmid-containing DNA nanoparticles, but not plasma alone, significantly enhanced IFN-γ which is known to inject the role of Th2 mediated effectors by stimulating Th1 cells [217]. ...
Both communicable and non-communicable chronic respiratory conditions have accorded for suffering of millions of people of all ages and stated to be leading cause of death, morbidity, economic and social pressures, and disability-adjusted life-years (DALYs) worldwide. These illnesses impair patient's health and negatively impacts families and society, particularly in low and middle-income countries. Chronic respiratory diseases (CRDs) affect different organs of respiratory system, involving airways, parenchyma, and pulmonary vasculature. As the number of respiratory diseases are exponentially escalating but still the stakeholders are not paying attention towards its serious complications. Currently, the treatment being used primarily focusses only on alleviating symptoms of these illness rather delivering the therapeutic agent at target site for optimal care and/or prevention. Lately, extensive research is being conducted on airways and systemic inflammation, oxidative stress, airway, or parenchymal rehabilitation. From which macrophages, neutrophils, and T cells, as well as structural cells as fibroblasts, epithelial, endothelial, and smooth muscle cells have been found to be active participants that are involved in these chronic respiratory diseases. The pathogenesis of all these chronic respiratory diseases gets caused differently via mediators and proteins, including cytokines, chemokines, growth factors and oxidants. Presently, the target of prescription therapies is to reduce the inflammation of airways and relieve the airway contraction. In all studies, cytokines have been found to play an imperative role in fostering chronic airway inflammation and remodelling. Owing to the limitations of conventional treatments, the current review aims to summarize the current knowledge about the chronic respiratory disease and discuss further about the various conventional methods that can be used for treating this ailment. Additionally, it also highlights and discusses about the advanced drug delivery system that are being used for targeting the interleukins for the treatment of CRDs.
... In its research, Silva et al., 2014 found that a single administration of thymulin plasmid-containing DNA nanoparticles, but not plasma alone, significantly enhanced IFN-γ which is known to inject the role of Th2 mediated effectors by stimulating Th1 cells [217]. ...
Nowadays, respiratory diseases have become the major cause of health care burded and death worldwide. According to the WHO, pulmonary disease holds the third position for mortality and supplement the development of antimicrobial resistance among immune-compromised patience are increasing the complication in health. Approximately, 65 million people are suffering from chronic obstructive pulmonary disease, 334 million are suffering from asthma, 10 million are suffering from tuberculosis, and 2.3 million are suffering from lung cancer. Different conventional methods have been used to treat these diseases, but certain limitations of conventional therapy limit their efficacy and efficiency. Hence, this leads to the exploitation of new therapeutic approaches as conventional approaches are not that much effective. Among the available alternatives, the targeted nanodrug delivery system (NDDS) has gained significant attention concerning their ability, activity, and tolerability to control these pulmonary diseases. Additionally, NDDS has associated advantages like a controlled deposition, controlled release of drugs and appropriate size for systemic and local treatment. This chapter plans to highlight the efficacy and physiological aspects of the targeted drug delivery, mechanism of pulmonary drug administration. Moreover, it will also discuss the different nanocarriers for drug delivery. As a better insight into the challenges and complications associated with developing the pulmonary disease will propose new opportunities to pharmacists to minimize the medical and technical gaps.
... Lunin and colleagues considered this when analyzing its possible suitability in controlling severe experimental autoimmune encephalomyelitis (sEAE) [85]. Previous studies had demonstrated additional beneficial actions of this molecule, such as its ability in preventing LPS-induced pancreatic cells damage and attenuation of experimental allergic asthma [85][86][87]. In mechanistic terms, the suppression of NF-κB signalling was demonstrated in LPS-stimulated lymphocytes, since similar effects were also obtained by inhibitors of the NF-κB pathway [85,88,89]. ...
For years the thymus gland (TG) and the pineal gland (PG) have been subject of increasingly in-depth studies, but only recently a link that can associate the activities of the two organs has been identified. Considering, on the one hand, the well-known immune activity of thymus and, on the other, the increasingly emerging immunological roles of circadian oscillators and the rhythmically secreted main pineal product, melatonin, many studies aimed to analyse the possible existence of an interaction between these two systems. Moreover, data confirmed that the immune system is functionally associated with the nervous and endocrine systems determining an integrated dynamic network. In addition, recent researches showed a similar, characteristic involution process both in TG and PG. Since the second half of the 20th century, evidence led to the definition of an effectively interacting thymus-pineal axis (TG-PG axis), but much has to be done. In this sense, the aim of this review is to summarize what is actually known about this topic, focusing on the impact of the TG-PG axis on human life and ageing. We would like to give more emphasis to the implications of this dynamical interaction in a possible therapeutic strategy for human health. Moreover, we focused on all the products of TG and PG in order to collect what is known about the role of peptides other than melatonin. The results available today are often unclear and not linear. These peptides have not been well studied and defined over the years. In this review we hope to awake the interest of the scientific community in them and in their future pharmacological applications.
... Red, caspase 3; blue, DAPI. potential application in the treatment of a number of lung diseases, such as infections, allergic asthma, and lung injury repair(14)(15)(16). Furthermore, the potent vector expression in lung ECs also holds the promise of making lung an important source of therapeutic secretory factors that are released into the circulation for the treatment of systemic diseases(13).The GAd-mediated GFP reporter gene expression in lung lasted for at least colored mCherry Pseudo-colored CD31/endomucin DAPIFIG 9 Replacement of GAd fiber knob (knobless) with the T4 fibritin domain tethered with the MBP targeting motif caused reduced GAd.MBP vector expression in lung and retargeting of expression to vascular beds in heart. ...
Human adenoviruses have many attractive features for gene therapy applications. However, the high prevalence of preexisting immunity against these viruses in general populations worldwide has greatly limited their clinical utility. In addition, the most commonly used human adenovirus subgroup C serotype 5 (HAd5), when systemically administered, triggers systemic inflammation and toxicity, with liver being the most severely affected organ. Here, we evaluated the utility and safety of a new low seroprevalence gorilla adenovirus (GC46) as a gene transfer vector in mice. Biodistribution studies revealed that systemically administered GAd had a selective and robust lung endothelial cell (EC) tropism with minimal vector expression throughout many other organs and tissues. Administration of a high dose of GAd accomplished extensive transgene expression in the lung and yet elicited no detectable inflammatory histopathology in this organ. Furthermore, GAd, unlike HAd5, did not exhibit heptotropism or induce liver inflammatory toxicity in mice, demonstrating the exceptional safety profile of the vector vis-à-vis systemic utility. We further demonstrated that GAd capsid fiber shared the flexibility of HAd5 equivalent for permitting genetic modification; GAd with the pan EC-targeting ligand MBP incorporated in the capsid displayed a reduced lung tropism and efficiently retargeted gene expression to vascular beds in other organs.
Importance
In the aggregate, our mouse studies suggest that GAd is a promising gene therapy vector that utilizes lung ECs as a source of therapeutic payload production and highly-desirable toxicity profile. Further genetic engineering of GAd capsid holds the promise of in vivo vector tropism modification and targeting.
... Corticosteroid-loaded NPs accumulated at the site of airway inflammation and exhibited anti-inflammatory activity. Highly compacted DNA NPs composed of a block copolymer of poly-L-lysine and polyethylene glycol can mediate thymulin analog gene delivery and effectively reduced the inflammatory and remodeling process in a mouse model of allergic airways disease (16). ...
Asthma is a common chronic inflammatory disease associated with intermittent airflow obstruction caused by airway inflammation, mucus overproduction and bronchial hyperresponsiveness. Despite current treatment and management options, a large number of patients with asthma still have poorly controlled disease and are susceptible to acute exacerbations, usually caused by a respiratory virus infection. As a result, there remains a need for novel therapies to achieve better control and prevent / treat exacerbations. Nanoparticles (NPs), including extracellular vesicles (EV) and their synthetic counterparts, have been developed for drug delivery in respiratory diseases. In the case of asthma, where airway epithelium dysfunction including dysregulated differentiation of epithelial cells, impaired barrier and immune response, is a driver of disease, targeting airway epithelial cells with NPs may offer opportunities to repair or reverse these dysfunctions with therapeutical interventions. EVs possess multiple advantages for airway epithelial targeting, such as their natural intrinsic cell targeting properties and low immunogenicity. Synthetic NPs can be coated with muco-inert polymers to overcome biological barriers such as mucus and the phagocytic response of immune cells. Targeting ligands could be also added to enhance targeting specificity to epithelial cells. The review presents current understanding and advances in NP-mediated drug delivery to airway epithelium for asthma therapy. Future perspectives in this therapeutic strategy will also be discussed including the development of novel formulations and physiologically relevant pre-clinical models.
... A single instillation of pDNA NP-mediated thymulin improved lung function for up to 27 days by preventing lung inflammation, muscle hypertrophy, and collagen deposition. 182 In recent times, biodegradable DNA NPs have shown potential for inhaled lung gene therapy, which is capable of overcoming the mucus barrier of lung airways. 183 Chronic obstructive pulmonary disease. ...
The application of nanomedicines is increasing rapidly with the promise of targeted and efficient drug delivery. Nanomedicines address the shortcomings of conventional therapy, as evidenced by several preclinical and clinical investigations indicating sitespecific
drug delivery, reduced side effects, and better treatment outcome. The development of suitable and biocompatible drug delivery vehicles is a prerequisite that has been successfully achieved by using simple and functionalized liposomes, nanoparticles,
hydrogels, micelles, dendrimers, and mesoporous particles. A variety of drug delivery vehicles have been established for the targeted and controlled delivery of therapeutic agents in a wide range of chronic diseases, such as diabetes, cancer, atherosclerosis,
myocardial ischemia, asthma, pulmonary tuberculosis, Parkinson’s disease, and Alzheimer’s disease. After successful outcomes in preclinical and clinical trials, many of these drugs have been marketed for human use, such as Abraxane®, Caelyx®, Mepact®,
Myocet®, Emend®, and Rapamune®. Apart from drugs/compounds, novel therapeutic agents, such as peptides, nucleic acids (DNA and RNA), and genes have also shown potential to be used as nanomedicines for the treatment of several chronic ailments.
However, a large number of extensive clinical trials are still needed to ensure the short-term and long-term effects of nanomedicines in humans. This review discusses the advantages of various drug delivery vehicles for better understanding of their utility in terms of current medical needs. Furthermore, the application of a wide range of nanomedicines is also described in the context of major chronic diseases.
... Alternatively, lungs were fixed in paraffin, sectioned (CM1950 cryostat, Leica Biosystems) and stained with haematoxylin and eosin. Inflammatory scores were calculated by enumerating neutrophil infiltration, as previously described [62,63]. Morphometric analysis was carried out double-blind in 10 random fields of view per slide and then summed (≥ 5 slides per mouse). ...
The lung remains an attractive target for the gene therapy of monogenetic diseases such as cystic fibrosis (CF). Despite over 27 clinical trials, there are still very few gene therapy vectors that have shown any improvement in lung function; highlighting the need to develop formulations with improved gene transfer potency and the desirable physiochemical characteristics for efficacious therapy. Herein, we introduce a novel cell penetrating peptide (CPP)-based non-viral vector that utilises glycosaminoglycan (GAG)-binding enhanced transduction (GET) for highly efficient gene transfer. GET peptides couple directly with DNA through electrostatic interactions to form nanoparticles (NPs). In order to adapt the GET peptide for efficient in vivo delivery, we engineered PEGylated versions of the peptide and employed a strategy to form DNA NPs with different densities of PEG coatings. We were able to identify candidate formulations (PEGylation rates ≥40%) that shielded the positively charged surface of particles, maintained colloidal stability in bronchoalveolar lavage fluid (BALF) and retained gene transfer activity in human bronchial epithelial cell lines and precision cut lung slices (PCLS) in vitro. Using multiple particle tracking (MPT) technology, we demonstrated that PEG-GET complexes were able to navigate the mucus mesh and diffuse rapidly through patient CF sputum samples ex vivo. When tested in mouse lung models in vivo, PEGylated particles demonstrated superior biodistribution, improved safety profiles and efficient gene transfer of a reporter luciferase plasmid compared to non-PEGylated complexes. Furthermore, gene expression was significantly enhanced in comparison to polyethylenimine (PEI), a non-viral gene carrier that has been widely tested in pre-clinical settings. This work describes an innovative approach that combines novel GET peptides for enhanced transfection with a tuneable PEG coating for efficacious lung gene therapy.
... DNA nanoparticles have also been investigated for use in asthma. Nanoparticles loaded with a thymulin plasmid demonstrated the ability to prevent inflammation in a mouse model of asthma (da Silva et al., 2014). The particles, which were around 90 nm in size, were administered intratracheally and demonstrated an antiinflammatory effect up to 27 days postadministration. ...
... More recently, researchers used highly compacted DNA NPs composed of pDNA compacted with block copolymers of poly-L-lysine and polyethylene glycol linked by a cysteine residue (CK30PEG), which have been shown to be nontoxic and nonimmunogenic in the lungs of mice and humans. Indeed, da Silva et al. (2014) used this NP system to deliver thymulin, a nonapeptide known for its anti-inflammatory and anti-fibrotic effects in the lung, to OVA-challenged allergic asthma in BALB/c mice. A single intratracheal instillation of DNA NPs carrying thymulin plasmids prevented lung inflammation, collagen deposition, and smooth muscle hypertrophy in the murine lungs up to 27 days after administration, leading to improved lung mechanics. ...
According to the World Health Organization (WHO), hundreds of millions of people of all ages and in all countries suffer from chronic respiratory diseases, with particular negative consequences such as poor health-related quality of life, impaired work productivity, and limitations in the activities of daily living. Chronic obstructive pulmonary disease, asthma, occupational lung diseases (such as silicosis), cystic fibrosis, and pulmonary arterial hypertension are the most common of these diseases, and none of them are curable with current therapies. The advent of nanotechnology holds great therapeutic promise for respiratory conditions, because non-viral vectors are able to overcome the mucus and lung remodeling barriers, increasing pharmacologic and therapeutic potency. It has been demonstrated that the extent of pulmonary nanoparticle uptake depends not only on the physical and chemical features of nanoparticles themselves, but also on the health status of the organism; thus, the huge diversity in nanotechnology could revolutionize medicine, but safety assessment is a challenging task. Within this context, the present review discusses some of the major new perspectives in nanotherapeutics for lung disease and highlights some of the most recent studies in the field.
... IL-10 can be released by many cell types, such as Th2 cells, Treg cells, mast cells, eosinophils, and macrophages[39]; in this study, no significant changes were observed between groups despite the increased number of eosinophils and Treg cells in BALF. IFN-γ and IL-10 levels did not increase after ovalbumininduced allergic asthma in accordance with previous studies[25,40]. The absence of changes in IFN-γ and IL-10 may be related to the timing of analysis (7 days after the last challenge), since such modifications have been described during asthma resolution[41].Human bone marrow-derived MSCs injected in mice migrate to the thymus, inhibiting maturation of naive lymphocytes into CD3 + CD4 + T cells[42,43]. ...
Background
Asthma is a chronic inflammatory disease that can be difficult to treat due to its complex pathophysiology. Most current drugs focus on controlling the inflammatory process, but are unable to revert the changes of tissue remodeling. Human mesenchymal stromal cells (MSCs) are effective at reducing inflammation and tissue remodeling; nevertheless, no study has evaluated the therapeutic effects of extracellular vesicles (EVs) obtained from human adipose tissue-derived MSCs (AD-MSC) on established airway remodeling in experimental allergic asthma.
Methods
C57BL/6 female mice were sensitized and challenged with ovalbumin (OVA). Control (CTRL) animals received saline solution using the same protocol. One day after the last challenge, each group received saline, 10⁵ human AD-MSCs, or EVs (released by 10⁵ AD-MSCs). Seven days after treatment, animals were anesthetized for lung function assessment and subsequently euthanized. Bronchoalveolar lavage fluid (BALF), lungs, thymus, and mediastinal lymph nodes were harvested for analysis of inflammation. Collagen fiber content of airways and lung parenchyma were also evaluated.
Results
In OVA animals, AD-MSCs and EVs acted differently on static lung elastance and on BALF regulatory T cells, CD3⁺CD4⁺ T cells, and pro-inflammatory mediators (interleukin [IL]-4, IL-5, IL-13, and eotaxin), but similarly reduced eosinophils in lung tissue, collagen fiber content in airways and lung parenchyma, levels of transforming growth factor-β in lung tissue, and CD3⁺CD4⁺ T cell counts in the thymus. No significant changes were observed in total cell count or percentage of CD3⁺CD4⁺ T cells in the mediastinal lymph nodes.
Conclusions
In this immunocompetent mouse model of allergic asthma, human AD-MSCs and EVs effectively reduced eosinophil counts in lung tissue and BALF and modulated airway remodeling, but their effects on T cells differed in lung and thymus. EVs may hold promise for asthma; however, further studies are required to elucidate the different mechanisms of action of AD-MSCs versus their EVs.
... Lung morphometry analysis was performed using an integrating eyepiece with a coherent system consisting of a grid with 100 points and 50 lines of known length coupled to a conventional light microscope (Olympus BX51, Olympus Latin America-Inc., Brazil). The volume fraction of collapsed and normal pulmonary areas, magnitude of bronchoconstriction, and number of mononuclear (MN) and polymorphonuclear (PMN) cells in pulmonary tissue were determined by the point-counting technique [18,19] across 10 random, noncoincident microscopic fields per mouse [15,20,21]. ...
Mesenchymal stromal cells (MSCs) from different sources have differential effects on lung injury. To compare the effects of murine MSCs from bone marrow (BM), adipose tissue (AD), and lung tissue (LUNG) on inflammatory and remodeling processes in experimental allergic asthma, female C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) or saline (C). Twenty-four hours after the last challenge, mice received either saline (50 µl, SAL), BM-MSCs, AD-MSCs, or LUNG-MSCs (10(5) cells per mouse in 50 µl total volume) intratracheally. At 1 week, BM-MSCs produced significantly greater reductions in resistive and viscoelastic pressures, bronchoconstriction index, collagen fiber content in lung parenchyma (but not airways), eosinophil infiltration, and levels of interleukin (IL)-4, IL-13, transforming growth factor (TGF)-β, and vascular endothelial growth factor (VEGF) in lung homogenates compared to AD-MSCs and LUNG-MSCs. Only BM-MSCs increased IL-10 and interferon (IFN)-γ in lung tissue. In parallel in vitro experiments, BM-MSCs increased M2 macrophage polarization, whereas AD-MSCs and LUNG-MSCs had higher baseline levels of IL-4, insulin-like growth factor (IGF), and VEGF secretion. Exposure of MSCs to serum specimens obtained from asthmatic mice promoted reductions in secretion of these mediators, particularly in BM-MSCs. Intratracheally administered BM-MSCs, AD-MSCs, and LUNG-MSCs were differentially effective at reducing airway inflammation and remodeling and improving lung function in the current model of allergic asthma. In conclusion, intratracheal administration of MSCs from BM, AD, and LUNG were differentially effective at reducing airway inflammation and remodeling and improving lung function comparably reduced inflammation and fibrogenesis in this asthma model. However, altered lung mechanics and lung remodeling responded better to BM-MSCs than to AD-MSCs or LUNG-MSCs. Moreover, each type of MSC was differentially affected in a surrogate in vitro model of the in vivo lung environment. Stem Cells Translational Medicine 2017.
... In other words, particles that diffused rapidly in mucus ex vivo also distributed more uniformly and persisted longer upon administration locally to a mucosal surface. Further, we have observed that improved NP distribution provides more efficacious treatment and prevention of diseases at mucosal sites, including prevention of herpes (HSV-2) infection in the cervicovaginal tract, prevention of asthma-induced lung inflammation, and treatment of cervical cancer [6,8,30,31]. Here, we found that PS-PEG NPs densely coated with as high as 40 kDa PEG also distributed uniformly in the cervicovaginal tract and colorectum of mice (Figure 3). ...
Aim:
We previously reported that nanoparticles (NPs) coated with 10 kDa PEG were mucoadhesive. Here, we demonstrate that by increasing the surface density, PEG with molecular weight (MW) as high as 40 kDa can be used as a mucoinert NP surface coating.
Materials & methods:
We compared two sets of reaction conditions for coating model polystyrene NPs with 10 kDa PEG and used optimized conditions to coat NPs with PEG as high as 40 kDa in MW. We then characterized NP transport in human cervicovaginal mucus ex vivo. We further administered PEG-coated NPs to the mouse cervicovaginal tract and colorectum to assess mucosal distribution in vivo.
Results & conclusion:
We demonstrate here that PEG with MW as high as 40 kDa can be densely grafted to the surface of NP to prevent interactions with mucus. NP coated with 10-40 kDa PEG rapidly diffused through human cervicovaginal mucus ex vivo, and uniformly lined the mouse colorectal and vaginal epithelium in vivo.
... While Dg-cSCK has the capacity for successful in vivo gene delivery, it is also possible that Dg-cSCK carriage of new generation minicircle DNA plasmids lacking prokaryotic sequences or plasmids with eukaryotic promoters would enhance both levels and maintenance of gene expression [ 59 , 60]. Due to its relative biocompatibility, gene transfer potential, and degradability, therapeutic plasmids such as those for modulating infection, inflammation or fibrosis could be delivered in an identical manner as pLuc in this study to provide therapy for acute (e.g., respiratory virus infection) and chronic (e.g., pulmonary fibrosis) lung diseases with minimal adverse effects from the nanocarrier [10,11]. ...
Nanoparticles (NPs) play expanding roles in biomedical applications including imaging and therapy, however, their long-term fate and clearance profiles have yet to be fully characterized in vivo. NP delivery via the airway is particularly challenging, as the clearance may be inefficient and lung immune responses complex. Thus, specific material design is required for cargo delivery and quantitative, noninvasive methods are needed to characterize NP pharmacokinetics. Here, biocompatible poly(acrylamidoethylamine)-b-poly(DL-lactide) block copolymer-based degradable, cationic, shell-cross-linked knedel-like NPs (Dg-cSCKs) were employed to transfect plasmid DNA. Radioactive and optical beacons were attached to monitor biodistribution and imaging. The preferential release of cargo in acidic conditions provided enhanced transfection efficiency compared to non-degradable counterparts. In vivo gene transfer to the lung was correlated with NP pharmacokinetics by radiolabeling Dg-cSCKs and performing quantitative biodistribution with parallel positron emission tomography and Čerenkov imaging. Quantitation of imaging over 14 days corresponded with the pharmacokinetics of NP movement from the lung to gastrointestinal and renal routes, consistent with predicted degradation and excretion. This ability to noninvasively and accurately track NP fate highlights the advantage of incorporating multifunctionality into particle design.
Immunoglobulin E, complement and eosinophils play an important role in host defense, but dysfunction of each of these components can lead to a variety of human disorders. In this review, we summarize how investigators have adapted gene therapy and anti-sense technology to modulate immunoglobin E, complement and/or eosinophils levels to treat these disorders.
The thymus gland is a central lymphoid organ in which developing T cell precursors, known as thymocytes, undergo differentiation into distinct type of mature T cells, ultimately migrating to the periphery where they exert specialized effector functions and orchestrate the immune responses against tumor cells, pathogens and self-antigens. The mechanisms supporting intrathymic T cell differentiation are pleiotropically regulated by thymic peptide hormones and cytokines produced by stromal cells in the thymic microenvironment and developing thymocytes. Interestingly, in the same way as T cells, thymic hormones (herein exemplified by thymosin, thymulin and thymopoietin), can circulate to impact immune cells and other cellular components in the periphery. Evidence on how thymic function influences tumor cell biology and response of patients with cancer to therapies remains unsatisfactory, although there has been some improvement in the knowledge provided by recent studies. Herein, we summarize research progression in the field of thymus-mediated immunoendocrine control of cancer, providing insights into how manipulation of the thymic microenvironment can influence treatment outcomes, including clinical responses and adverse effects of therapies. We review data obtained from clinical and preclinical cancer research to evidence the complexity of immunoendocrine interactions underpinning anti-tumor immunity.
Asthma is a chronic lung disease affecting millions worldwide. While classically acknowledged to result from allergen-driven type 2 inflammatory responses leading to IgE and cytokine production and the influx of immune cells such as mast cells and eosinophils, the wide range in asthmatic pathobiological subtypes lead to highly variable responses to anti-inflammatory therapies. Thus, there is a need to develop patient-specific therapies capable of addressing the full spectrum of asthmatic lung disease. Moreover, delivery of targeted treatments for asthma directly to the lung may help to maximize therapeutic benefit, but challenges remain in design of effective formulations for the inhaled route. In this review, we discuss the current understanding of asthmatic disease progression as well as genetic and epigenetic disease modifiers associated with asthma severity and exacerbation of disease. We also overview the limitations of clinically available treatments for asthma and discuss pre-clinical models of asthma used to evaluate new therapies. Based on the shortcomings of existing treatments, we highlight recent advances and new approaches to treat asthma via inhalation for monoclonal antibody delivery, mucolytic therapy to target airway mucus hypersecretion and gene therapies to address underlying drivers of disease. Finally, we conclude with discussion on the prospects for an inhaled vaccine to prevent asthma.
Diagnosis and treatment of lung diseases pose serious challenges. Currently, diagnostic as well as therapeutic methods show poor efficacy toward drug-resistant bacterial infections, while chemotherapy causes toxicity and nonspecific delivery of drugs. Advanced treatment methods that cure lung-related diseases, by enabling drug bioavailability via nasal passages during mucosal formation, which interferes with drug penetration to targeted sites, are in demand. Nanotechnology confers several advantages. Currently, different nanoparticles, or their combinations, are being used to enhance targeted drug delivery. Nanomedicine, a combination of nanoparticles and therapeutic agents, that delivers drugs to targeted sites increases the bioavailability of drugs at these sites. Thus, nanotechnology is superior to conventional chemotherapeutic strategies. Here, the authors review the latest advancements in nanomedicine-based drug-delivery methods for managing acute and chronic inflammatory lung diseases.
The lack of effective treatments for pulmonary diseases poses a global health burden. The direct local gene therapy serves as one of the alternative administrations to treat pulmonary diseases. Compared with the conventional viral/nonviral system, the peptide–vector‐mediated in vivo lung gene therapies exhibit various benefits. However, the related clinical trials are still in their infancy. The major obstacle to the pulmonary delivery of gene cargoes may be the barriers from various extracellular mucosal layers and intracellular membranes. This review highlights the recent development of peptide‐based gene delivery systems and their applications. The peptide designing rules for the barrier‐permeable pulmonary gene delivery are described first. After briefly summarizing how oligopeptides facilitate the local gene therapy in lung tissue with the focus on cell‐penetrating peptides, the local delivery system of the polypeptide and several alternative hybridizing systems of the peptide with other types of materials are discussed. Finally, the blueprint and the remaining challenges in peptide designations are discussed before they enter into the real translation process. Herein, the recent developments of peptide‐based gene delivery systems and their applications are described, which cover the peptide design principle, oligopeptides and polypeptides, and hybrid systems for gene delivery.
Respiratory illnesses are prevalent around the world, whereby hundreds and millions of people in all age groups are affected by chronic respiratory diseases. These include asthma, cystic fibrosis, and lung cancer, in which current conventional therapies are insufficient in controlling the progression of these diseases, leading to negative consequences such as impaired work productivity and poor quality of life. These have prompted medical researchers to design and develop novel respiratory nanotherapies, as they can provide targeted drug delivery specifically to lung tissues, to further enhance patient compliance and therapeutic effectiveness. Over the recent years, a wide range of polymers such as chitosan, alginate, gelatin, polylactic acid and poly(lactic-glycolic acid), as well as other inorganic nanomaterials including gold, silver and magnetic iron oxide based nanomaterials have been extensively studied for their potential as carriers for chemical and biological drugs. These nanosized drug carriers enable the delivery of therapeutic moieties with improved physiochemical properties, bioavailability, and biocompatibility. In this chapter, we will address the perspectives for the use of polymeric and inorganic nanoparticles for targeted drug delivery in chronic respiratory diseases and highlight some of the most recent studies performed in this field of research.
Allergic airway diseases, with incidence augmenting visibly as industrial development and environmental degradation, are characterized by sneezing, itching, wheezing, chest tightness, airway obstruction, and hyperresponsiveness. Current medical modalities attempt to combat these symptoms mostly by small molecule chemotherapeutants, such as corticosteroids, antihistamines, etc., via intranasal approach which is one of the most noninvasive, rapid-absorbed, and patient-friendly routes. Nevertheless, inherent defects for irritation to respiratory mucosa, drug inactivation and degradation, and rapid drug dispersal to off-target sites are inevitable. Lately, intratracheal micro/nano therapeutic systems are emerging as innovative alternatives for airway allergy interventions. This overview introduces several potential application directions of mic/nano-platform in the treatment of airway allergic diseases, including carriers, therapeutic agents, and immunomodulators. The improvement of the existing drug therapy of respiratory allergy management by micro/nano-platform is described in detail. The challenges of the micro/nano-platform nasal approach in the treatment of airway allergy are summarized and the development of micro/nano-platform is also prospected. Although still a burgeoning area, micro/nano therapeutic systems are gradually turning to be realistic orientations as crucial future alternative therapeutic options in allergic airway inflammation interventions.
Respiratory diseases are a major cause of concern throughout the world. Lung cancer, chronic obstructive pulmonary disease (COPD), and asthma deserve special mention among them. According to the WHO, 235 million people suffer from asthma, and above three million people die annually from COPD, which is about 6% of all deaths across the globe. These diseases do not have a full-proof cure, and the treatment strategies are only symptomatic medications for temporary relief. Moreover, the respiratory system has a number of potential barriers to drug delivery. Secretion of mucus, ciliary action, and presence of macrophages are few among them. This chapter focusses on chronic diseases like COPD, tuberculosis, emphysema, asthma, and lung cancer and acute disease like pneumonia. Tuberculosis and lung cancer are the deadliest form of respiratory diseases that cause high annual death rates in the world. Pulmonary oedema, a commonly observed clinical manifestation, and cystic fibrosis, an autosomal recessive disorder, have also been discussed. The pathophysiology of diseases at the molecular level, currently available therapies, and the recent research on improvement of clinical efficiency have been elaborated. Different novel drug delivery systems for pulmonary route and delivery device for pulmonary administration are discussed in detail. The mechanism of nanoparticle internalization by the epithelial cells and macrophages has been detailed together with the mechanism behind penetrating the mucus barrier. Finally, the future challenges in treating respiratory diseases are outlined.
Inhaled gene therapy poses a unique potential of curing chronic lung diseases, which are currently managed primarily by symptomatic treatments. However, it has been challenging to achieve therapeutically relevant gene transfer efficacy in the lung due to the presence of numerous biological delivery barriers. Here, we introduce a simple approach that overcomes both extracellular and cellular barriers to enhance gene transfer efficacy in the lung in vivo. We endowed tetra(piperazino)fullerene epoxide (TPFE)‐based nanoparticles with non‐adhesive surface polyethylene glycol (PEG) coatings, thereby enabling the nanoparticles to cross the airway mucus gel layer and avoid phagocytic uptake by alveolar macrophages. In parallel, we utilized a hypotonic vehicle to facilitate endocytic uptake of the PEGylated nanoparticles by lung parenchymal cells via the osmotically driven regulatory volume decrease (RVD) mechanism. We demonstrate that this two‐pronged delivery strategy provides safe, wide‐spread and high‐level transgene expression in the lungs of both healthy mice and mice with chronic lung diseases characterized by reinforced delivery barriers.
Tetra(piperazino)fullerene (TPFE)-based nanoparticles (NPs), surface-coated with polyethylene glycol (PEG), were engineered. The PEGylation allows the NPs to avoid interactions with mucin glycoproteins and phagocytic uptake by macrophages. After the NPs access the target lung cells, a hypotonic vehicle solution enhances their endocytosis via the osmotically driven regulatory volume effect, thereby providing highly efficient transgene expression.
Abstract
Inhaled gene therapy poses a unique potential of curing chronic lung diseases, which are currently managed primarily by symptomatic treatments. However, it has been challenging to achieve therapeutically relevant gene transfer efficacy in the lung due to the presence of numerous biological delivery barriers. Here, we introduce a simple approach that overcomes both extracellular and cellular barriers to enhance gene transfer efficacy in the lung in vivo. We endowed tetra(piperazino)fullerene epoxide (TPFE)-based nanoparticles with non-adhesive surface polyethylene glycol (PEG) coatings, thereby enabling the nanoparticles to cross the airway mucus gel layer and avoid phagocytic uptake by alveolar macrophages. In parallel, we utilized a hypotonic vehicle to facilitate endocytic uptake of the PEGylated nanoparticles by lung parenchymal cells via the osmotically driven regulatory volume decrease (RVD) mechanism. We demonstrate that this two-pronged delivery strategy provides safe, wide-spread and high-level transgene expression in the lungs of both healthy mice and mice with chronic lung diseases characterized by reinforced delivery barriers.
Despite long-standing efforts to enhance care for chronic asthma, symptomatic treatments remain the only option to manage this highly prevalent and debilitating disease. We demonstrate that key pathology of allergic asthma can be almost completely resolved in a therapeutic manner by inhaled gene therapy. After the disease was fully and stably established, we treated mice intratracheally with a single dose of thymulin-expressing plasmids delivered via nanoparticles engineered to have a unique ability to penetrate the airway mucus barrier. Twenty days after the treatment, we found that all key pathologic features found in the asthmatic lung, including chronic inflammation, pulmonary fibrosis, and mechanical dysregulation, were normalized. We conducted tissue- and cell-based analyses to confirm that the therapeutic intervention was mediated comprehensively by anti-inflammatory and antifibrotic effects of the therapy. We believe that our findings open a new avenue for clinical development of therapeutically effective gene therapy for chronic asthma.
Filamentous microorganisms traveling in aerosol particles display enhanced deposition and retention in the lungs. Inspired by this shape-related biological effect, we report here on the use of supramolecular filaments as potential inhalable drug carriers within aerosols via jet nebulization. We found that the peptide design and supramolecular stability play a crucial role in the interfacial stability and aerosolization properties of the supramolecular filaments. Monomeric units with a positively charged C-terminus produced filaments with reduced aerosol stability, promoting morphological changes after nebulization. Conversely, having a neutral or negatively charged terminus yielded filaments with enhanced stability, where supramolecular integrity is maintained with only reduced length. Our results suggest that molecular enrichment at the air-liquid interface during nebulization is the primary factor to deplete the monomeric peptide amphiphiles in solution, accounting for the observed morphological disruption/transitions. Importantly, encapsulation of drugs and dyes within filaments notably stabilize their supramolecular structure during nebulization, and the loaded filaments exhibit a linear release profile from a nebulizer device. We envision the use of this supramolecular carrier system as an effective platform for the inhalation-based treatment of many lung diseases.
Nanoparticle-based drug system is a powerful technique, which can be customized for targeting and causing a therapeutic effect. Numerous nanoparticle-based drug system has experimented, which includes different types of nanoparticles such as nanocrystals, dendrimers, polymeric micelles, protein-based, carbon nanotube, and liposome (Singh and Lillard Jr, 2009). When compared with conventional drug systems, these nanoparticlebased drug delivery systems offer many advantages, which include improving the pharmacokinetic behavior of drug molecule inside a biological entity such as rapid absorption of the nanodrug owing to its high surface area (Magenheim et al., 1993). The most common obstruction faced by conventional drug formulation is rapid immunoclearance by immune cells, which accounts for an inefficient drug system, but in case of this nanoparticle drug design, it seems to negotiate with this barrier, as there is the usage of biocompatible molecules, which prolong the circulation time of a drug molecule inside the target system. Some cases like doxorubicin and paclitaxel involved an increase in safety and morbidity of patients. If correct formulation has taken place, drug molecules have capabilities such as enhanced solubility and adhesion, which accounts for an early response (Muller et al., 2018). When the entire formulation is more effective, it obviously reduces the dosage of drug administered to achieve the same level of benefit as earlier, thereby making it safer and cost-effective. Nanocarrier systems can provide the advantage of sustained release in the lung tissue, resulting in reduced dosing frequency and improved …
Background/aims:
Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues.
Methods:
Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed.
Results:
No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response.
Conclusion:
Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy.
DNA can be compacted using polyethylene glycol-substituted poly-L-lysine into discrete unimolecular (with respect to DNA) nanoparticles with minor diameter <20 nm that are stable in normal saline for at least 23 months at 4°C. We compared the activity of firefly luciferase in lungs of C57BL/6 mice that received 100 g compacted plasmid in 25 l saline (shown to be the optimal dose) via intratracheal or intranasal instillation with levels in animals given 100 g naked plasmid or in untreated mice. Mice dosed with compacted DNA nanoparticles had peak activity of luciferase in lung at 2 days postinstillation, which declined in log-linear fashion with a half-life of 1.4 days. Luciferase activity in animals dosed with naked DNA was 200-fold less. Addition of polyethylene glycol to the complex was necessary for efficient gene transfer and animals that received DNA compacted with unmodified poly-L-lysine did not exhibit luciferase activity above background. Immunohistochemical staining for bacterial -galactosidase 2 days after administration of a compacted lacZ expression plasmid (n = 8) revealed expression predominantly in the dependent portions of the right lungs of mice, in alveolar and airway epithelial cells, though macrophages and sometimes endothelial cells also were transfected. No staining for -galactosidase was observed in uninjected animals (n = 4) or those dosed with naked lacZ plasmid (n = 7). Tissue survey for transgene expression shows expression only in lung and trachea following intranasal administration. Stable compacted DNA nanoparticles transfer exogenous genes to airway epithelium and show promise for lung gene therapy.
Asthma is an airway inflammatory disease with functional and structural changes, leading to bronchial hyperresponsiveness (BHR) and airflow obstruction. Airway structural changes or airway remodelling consist of epithelial injury, goblet cell hyperplasia, subepithelial layer thickening, airway smooth muscle hyperplasia and angiogenesis. These changes were previously considered as a consequence of chronic airway inflammation. However, several studies have demonstrated that inflammation and remodelling can occur as separate but parallel aspects of the asthmatic process. As such there is increasing evidence for the role of mechanocompressive forces within the asthmatic airway contributing to airway structural changes. Furthermore, it is unclear what is the best treatment to modify remodelling and which component to target. There is also a need to identify asthma phenotype that might specifically respond to novel therapies such as anti-IL5, anti-IL13 and tyrosine kinase inhibitors.
Background Eosinophilic inflammation is implicated in asthma. Eotaxin 1–3 regulate eosinophil trafficking into the airways along with other chemotactic factors. However, the epithelial and bronchoalveolar lavage (BAL) cell expression of these chemokines in relation to asthma severity and eosinophilic phenotypes has not been addressed.
Objective To measure the expression of the three eotaxin isoforms in bronchoscopically obtained samples and compare them with clinically relevant parameters between normal subjects and patients with asthma.
Methods Normal subjects and patients with asthma of varying severity recruited through the Severe Asthma Research Program underwent clinical assessment and bronchoscopy with airway brushing and BAL. Eotaxin 1–3 mRNA/protein were measured in epithelial and BAL cells and compared with asthma severity, control and eosinophilic inflammation.
Results Eotaxin-2 and eotaxin-3 mRNA and eotaxin-2 protein were increased in airway epithelial brushings from patients with asthma and were highest in cases of severe asthma (p values 0.0155, 0.0033 and 0.0006, respectively), with eotaxin-2 protein increased with age at onset. BAL cells normally expressed high levels of eotaxin-2 mRNA/protein but BAL fluid levels of eotaxin-2 were lowest in severe asthma. Epithelial eotaxin-2 and eotaxin-3 mRNA/protein was associated with sputum eosinophilia, lower forced expiratory volume in 1 s and more asthma exacerbations. Airway epithelial cell eotaxin-2 protein differed by asthma severity only in those with late onset disease, and tended to be highest in those with late onset eosinophilic asthma.
Conclusions Epithelial eotaxin-2 and 3 are increased in asthma and severe asthma. Their expression may contribute to luminal migration of eosinophils, especially in later onset disease, asthma control and severity.
Emerging evidence suggests that the T helper 17 (T(H)17) subset of αβ T cells contributes to the development of allergic asthma. In this study, we found that mice lacking the αvβ8 integrin on dendritic cells did not generate T(H)17 cells in the lung and were protected from airway hyper-responsiveness in response to house dust mite and ovalbumin sensitization and challenge. Because loss of T(H)17 cells inhibited airway narrowing without any obvious effects on airway inflammation or epithelial morphology, we examined the direct effects of T(H)17 cytokines on mouse and human airway smooth muscle function. Interleukin-17A (IL-17A), but not IL-17F or IL-22, enhanced contractile force generation of airway smooth muscle through an IL-17 receptor A (IL-17RA)-IL-17RC, nuclear factor κ light-chain enhancer of activated B cells (NF-κB)-ras homolog gene family, member A (RhoA)-Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling cascade. Mice lacking integrin αvβ8 on dendritic cells showed impaired activation of this pathway after ovalbumin sensitization and challenge, and the diminished contraction of the tracheal rings in these mice was reversed by IL-17A. These data indicate that the IL-17A produced by T(H)17 cells contributes to allergen-induced airway hyper-responsiveness through direct effects on airway smooth muscle.
Inhibiting allergic airway inflammation is the goal of therapy in persistent asthma. Administration of medication via the airways delivers drug directly to the site of inflammation and avoids systemic side effects but often fails to modulate systemic features of asthma. We have shown that Th1 cells, through production of IFN-γ, inhibit many Th2-induced effector functions that promote disease. Using a newly generated mouse that expresses IFN-γR only on airway epithelial cells, we show that the airway epithelium controls a range of pathological responses in asthma. IFN-γ acting only through the airway epithelium inhibits mucus, chitinases, and eosinophilia, independent of Th2 cell activation. IFN-γ signaling through the airway epithelium inhibits eosinophil generation in the bone marrow, indicating that signals on the airway mucosal surface can regulate distant functions to inhibit disease. IFN-γ actions through the airway epithelium will limit airway obstruction and inflammation and may be therapeutic in refractory asthma.
For effective airway gene therapy of cystic fibrosis (CF), inhaled gene carriers must first penetrate the hyperviscoelastic sputum covering the epithelium. Whether clinically studied gene carriers can penetrate CF sputum remains unknown. Here, we measured the diffusion of a clinically tested nonviral gene carrier, composed of poly-l-lysine conjugated with a 10 kDa polyethylene glycol segment (CK(30)PEG(10k)). We found that CK(30)PEG(10k)/DNA nanoparticles were trapped in CF sputum. To improve gene carrier diffusion across sputum, we tested adjuvant regimens consisting of N-acetylcysteine (NAC), recombinant human DNase (rhDNase) or NAC together with rhDNase. While rhDNase alone did not enhance gene carrier diffusion, NAC and NAC + rhDNase increased average effective diffusivities by 6-fold and 13-fold, respectively, leading to markedly greater fractions of gene carriers that may penetrate sputum layers. We further tested the adjuvant effects of NAC in the airways of mice with Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced mucus hypersecretion. Intranasal dosing of NAC prior to CK(30)PEG(10k)/DNA nanoparticles enhanced gene expression by up to ~12-fold compared to saline control, reaching levels observed in the lungs of mice without LPS challenge. Our findings suggest that a promising synthetic nanoparticle gene carrier may transfer genes substantially more effectively to lungs of CF patients if administered following adjuvant mucolytic therapy with NAC or NAC + rhDNase.
Integrity of the thymus during perinatal life is necessary for a proper maturation of the pituitary-gonadal axis in mice and other mammalian species. Thus congenitally athymic (nude) female mice show significantly reduced levels of circulating gonadotropins, a fact that seems to be causally related to a number of reproductive derangements described in these mutants. Interestingly, a number of in vitro studies suggest that the thymic peptide thymulin may be involved in thymus-pituitary communication. To determine the consequences of low serum thymulin in otherwise normal animals, we induced short (8 days)- and long (33 days)-term thymulin deficiency in C57BL/6 mice by neonatally injecting (intraperitoneally) an anti-thymulin serum and assessed their circulating gonadotropin levels at puberty and thereafter. Control mice received an irrelevant antiserum. Gonadotropins were measured by radioimmunoassay and thymulin by bioassay. Both long- and short-term serum thymulin immunoneutralization resulted in a significant reduction in the serum levels of gonadotropins at 33 and 45 days of age. Subsequently, we injected (intramuscularly) an adenoviral vector harboring a synthetic DNA sequence (5'-ATGCAAGCCAAATCTCAAGGTGGATCCAACTAGTAG-3') encoding a biologically active analog of thymulin, methionine-FTS, in newborn nude mice (which are thymulin deficient) and measured circulating gonadotropin levels when the animals reached 52 days of age. It was observed that neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels and prevented the reduction in circulating gonadotropin levels that typically emerges in these mutants after puberty. Our results indicate that thymulin plays a relevant physiological role in the thymus-pituitary-gonadal axis.
Extracellular nucleotides have recently been identified as proinflammatory mediators involved in asthma pathogenesis by signaling via purinergic receptors, but the role of the purinergic receptor type 6 (P2Y6R) has not been previously investigated.
To investigate the role of P2Y6R in asthma pathogenesis.
Acute and chronic OVA model and also HDM model of allergic inflammation in C57Bl/6 mice treated with specific P2Y6R antagonist and P2Y6R(-/-) mice were evaluated for classical features of asthmatic inflammation. In addition, primary epithelial cell culture from human and epithelial cell lines from mouse and human were stimulated with P2Y6R agonist and treated with P2Y6R antagonist and assessed for IL-6, IL-8/CXCL8 and KC levels. Experiments with P2Y6R(-/-) and P2Y6R(+/+) chimera were performed to discriminate the role of P2Y6R activation in structural lung cells and in cells from hematopoietic system.
We observed that the intratracheal application of a P2Y6R antagonist (MRS2578) and P2Y6R deficiency inhibited cardinal features of asthma, such as bronchoalveolar lavage eosinophilia, airway remodeling, Th2 cytokine production, and bronchial hyperresponsiveness in the ovalbumin-alum model. MRS2578 was also effective in reducing airway inflammation in a model using house dust mite extracts to induce allergic lung inflammation. Experiments with bone marrow chimeras revealed the importance of the P2Y6R expression on lung structural cells in airway inflammation. In accordance with this finding, we found a strong up-regulation of P2Y6 expression on airway epithelial cells of animals with experimental asthma. Concerning the underlying mechanism, we observed that MRS2578 inhibited the release of IL-6 and IL-8/KC by lung epithelial cells in vivo, whereas intrapulmonary application of the P2Y6R agonist uridine-5'-diphosphate increased the bronchoalveolar levels of IL-6 and KC. In addition, selective activation of P2Y6 receptors induced the release of IL-6 and KC/IL-8 by murine and human lung epithelial cells in vitro.
P2Y6R expression on airway epithelial cells is up-regulated during acute and chronic allergic airway inflammation, and selective blocking of P2Y6R or P2Y6R deficiency on the structural cells reduces cardinal features of experimental asthma. Thus, blocking pulmonary P2Y6R might be a target for the treatment of allergic airway inflammation.
The original concept of asthma being primarily a disease of airways smooth muscle drove the development of bronchodilator drugs. However when it was realised that airway inflammation underpinned the disordered airway function, this gave way to the development of controller therapies such as inhaled cromones and corticosteroids. More recently the discovery of complex interconnecting cytokine and chemokine networks has stimulated the development of biologics with varying success. With the recognition that airway wall "remodelling" is present early in asthma inception and is in part driven by aberrant epithelial-mesenchymal communication both genetic and environmental factors beyond allergen exposure such as virus infection and air pollution are being seen as being increasingly important not only in asthma exacerbations but in the origins of asthma and its evolution into different sub-phenotypes. This brings us round full circle to once again considering that the origins of asthma lie in defects in the formed elements of the airway; the epithelium, smooth muscle, and vasculature. Over the last 25 years Professor You Young Kim has engaged in the exciting discovery science of allergy and asthma and has made an enormous contribution in bringing Korea to the forefront of disease management and research, a position that both he and his colleagues can justly be proud of.
This study demonstrates proof of concept for delivery and expression of compacted plasmid DNA in the central nervous system. Plasmid DNA was compacted with polyethylene glycol substituted lysine 30-mer peptides, forming rod-like nanoparticles with diameters between 8 and 11 nm. Here we show that an intracerebral injection of compacted DNA can transfect both neurons and glia, and can produce transgene expression in the striatum for up to 8 weeks, which was at least 100-fold greater than intracerebral injections of naked DNA plasmids. Bioluminescent imaging (BLI) of injected animals at the 11th postinjection week revealed significantly higher transgene activity in animals receiving compacted DNA plasmids when compared to animals receiving naked DNA. There was minimal evidence of brain inflammation. Intrastriatal injections of a compacted plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) resulted in a significant overexpression of GDNF protein in the striatum 1-3 weeks after injection.
Increased airway smooth muscle (ASM) mass, a characteristic finding in asthma, may be caused by hyperplasia or hypertrophy. Cell growth requires increased translation of contractile apparatus mRNA, which is controlled, in part, by glycogen synthase kinase (GSK)-3beta, a constitutively active kinase that inhibits eukaryotic initiation factor-2 activity and binding of methionyl tRNA to the ribosome. Phosphorylation of GSK-3beta inactivates it, enhancing translation. We sought to quantify the contributions of hyperplasia and hypertrophy to increased ASM mass in ovalbumin (OVA)-sensitized and -challenged BALB/c mice and the role of GSK-3beta in this process. Immunofluorescent probes, confocal microscopy, and stereological methods were used to analyze the number and volume of cells expressing alpha-smooth muscle actin and phospho-Ser(9) GSK-3beta (pGSK). OVA treatment caused a 3-fold increase in ASM fractional unit volume or volume density (Vv) (PBS, 0.006 +/- 0.0003; OVA, 0.014 +/- 0.001), a 1.5-fold increase in ASM number per unit volume (Nv), and a 59% increase in volume per cell (Vv/Nv) (PBS, 824 +/- 76 microm(3); OVA, 1,310 +/- 183 mum(3)). In OVA-treated mice, there was a 12-fold increase in the Vv of pGSK (+) ASM, a 5-fold increase in the Nv of pGSK (+) ASM, and a 1.6-fold increase in Vv/Nv. Lung homogenates from OVA-treated mice showed increased GSK-3beta phosphorylation and lower GSK-3beta activity. Both hyperplasia and hypertrophy are responsible for increased ASM mass in OVA-treated mice. Phosphorylation and inactivation of GSK-3beta are associated with ASM hypertrophy, suggesting that this kinase may play a role in asthmatic airway remodeling.
Unlabelled:
This study was undertaken to test whether there is structural remodeling of lung parenchyma that could lead to tissue mechanical changes at an early phase of varying degrees of acute lung injury (ALI). Tissue resistance (R), dynamic elastance (E), and hysteresivity (eta) were analyzed during sinusoidal oscillations of rat lung parenchymal strips 24 h after intraperitoneal injection of saline (C) or paraquat (P [10, 15, 25, and 30 mg/kg]). These strips were also stained in order to quantify the amount of collagen and of three types of elastic fibers (elaunin, oxytalan, and fully developed elastic fibers) in the alveolar septa. E augmented progressively from C to P25, but the data from the P25 and P30 groups were not different (p < 0.0001). R and eta increased from C to P10 and from P15 to P25 (p < 0.001). Collagen fiber content increased exponentially with the severity of the injury. Elaunin and fully developed elastic fibers remained unchanged in the five groups, while oxytalan fibers increased only in the P25 and P30 groups. In conclusion, the pronounced mechanical changes at the tissue level and fibroelastogenesis happened at an early phase of the disease and even in mildly abnormal lung parenchyma.
Keywords:
elastance; collagen fibers; elastin; paraquat
Treatment of chronic obstructive pulmonary disease (COPD) with inhaled corticosteroids does not appear to be as effective as similar treatment of asthma. It seems that only certain subgroups of patients with COPD benefit from steroid treatment. A study was undertaken to examine whether inhaled fluticasone propionate (FP) had an effect on lung function and on indices of inflammation in a subgroup of COPD patients with bronchial hyperresponsiveness (BHR).
Twenty three patients with COPD were studied. Patients had to be persistent current smokers between 40 and 70 years of age. Non-specific BHR was defined as a PC(20) for histamine of <or=8 mg/ml. Patients received either 2 x 500 microg FP or placebo for 6 months. Expiratory volumes were measured at monthly visits, BHR was determined at the start of the study and after 3 and 6 months, and bronchial biopsy specimens were taken at the start and after 6 months of treatment. Biopsy specimens from asymptomatic smokers served as controls.
In contrast to asthma, indices of BHR were not significantly influenced by treatment with FP. Forced expiratory volume in 1 second (FEV(1)) showed a steep decline in the placebo group but remained stable in patients treated with FP. FEV(1)/FVC, and maximal expiratory flows at 50% and 25% FVC (MEF(50), MEF(25)) were significantly increased in the FP treated patients compared with the placebo group. Biopsy specimens were analysed for the presence of CD3+, CD4+, CD8+, MBP+, CD15+, CD68+, CD1a, and tryptase cells. FP treatment resulted in marginal reductions in these indices of inflammation.
In patients with COPD and BHR, FP has a positive effect on indices of lung function compared with placebo. Bronchial inflammation analysed in bronchial biopsy specimens is only marginally reduced.
Nanoparticles containing DNA compacted with poly-l-lysine modified on an N-terminal cysteine with polyethylene glycol can effectively transfect cells of the airway epithelium when applied by the luminal route. To evaluate the toxicity of these nanoparticles, we administered 10 and 100 microg DNA compacted into nanoparticles suspended in normal saline by the intranasal route to mice and determined the pulmonary and systemic responses to this challenge, compared to administration of saline alone, and in some experiments, compared to administration of naked DNA, Escherichia coli genomic DNA, or lipofectin-complexed naked DNA. There was no systemic response to either dose of nanoparticles in serum chemistries, hematologic parameters, serum complement, IL-6, or MIP-2 levels or in the activity, growth, and grooming of the mice. Nanoparticles containing 10 microg DNA induced responses comparable to saline in all measures, including BAL cell counts and differentials and cytokine levels and histology. However, mice dosed with 100 microg DNA in nanoparticles had modest increases in BAL neutrophils 48 and 72 h after dosing, modest increases in BAL IL-6 and KC beginning 24 and 48 h, respectively, after dosing, and, on histology of the lung, a trace to 1+ mononuclear cell infiltrates about the pulmonary veins at 48 h, which were markedly reduced by 10 days and gone by 28 days after dosing. BAL neutrophil and cytokine responses were no greater than those entrained by naked DNA for up to 24 h. However, compared to administration of only 10 microg E. coli genomic DNA, the response to compacted DNA was much less. A low dose of lipofectin-complexed DNA (5 microg DNA) induced the same response as 20-fold higher doses of DNA nanoparticles. These data indicate that DNA nanoparticles have no measurable toxic effect at a dose of 10 microg and a very modest effect, which is not limiting, at a dose of 100 microg, which gives maximal gene expression. This favorable toxicity profile encourages development of stabilized compacted DNA for airway administration.
DNA can be compacted using polyethylene glycol-substituted poly-L-lysine into discrete unimolecular (with respect to DNA) nanoparticles with minor diameter < 20 nm that are stable in normal saline for at least 23 months at 4 degrees C. We compared the activity of firefly luciferase in lungs of C57BL/6 mice that received 100 microg compacted plasmid in 25 microl saline (shown to be the optimal dose) via intratracheal or intranasal instillation with levels in animals given 100 microg naked plasmid or in untreated mice. Mice dosed with compacted DNA nanoparticles had peak activity of luciferase in lung at 2 days postinstillation, which declined in log-linear fashion with a half-life of 1.4 days. Luciferase activity in animals dosed with naked DNA was 200-fold less. Addition of polyethylene glycol to the complex was necessary for efficient gene transfer and animals that received DNA compacted with unmodified poly-L-lysine did not exhibit luciferase activity above background. Immunohistochemical staining for bacterial beta-galactosidase 2 days after administration of a compacted lacZ expression plasmid (n = 8) revealed expression predominantly in the dependent portions of the right lungs of mice, in alveolar and airway epithelial cells, though macrophages and sometimes endothelial cells also were transfected. No staining for beta-galactosidase was observed in uninjected animals (n = 4) or those dosed with naked lacZ plasmid (n = 7). Tissue survey for transgene expression shows expression only in lung and trachea following intranasal administration. Stable compacted DNA nanoparticles transfer exogenous genes to airway epithelium and show promise for lung gene therapy.
This study tested the hypotheses that chronic allergic inflammation induces not only bronchial but also lung parenchyma remodeling, and that these histologic changes are associated with concurrent changes in respiratory mechanics. For this purpose, airway and lung parenchyma remodeling were evaluated by quantitative analysis of collagen and elastin, immunohistochemistry (smooth-muscle actin expression, eosinophil, and dendritic cell densities), and electron microscopy. In vivo (airway resistance, viscoelastic pressure, and static elastance) and in vitro (tissue elastance, resistance, and hysteresivity) respiratory mechanics were also analyzed. BALB/c mice were sensitized with ovalbumin and exposed to repeated ovalbumin challenges. A marked eosinophilic infiltration was seen in lung parenchyma and in large and distal airways. Neutrophils, lymphocytes, and dendritic cells also infiltrated the lungs. There was subepithelial fibrosis, myocyte hypertrophy and hyperplasia, elastic fiber fragmentation, and increased numbers of myofibroblasts in airways and lung parenchyma. Collagen fiber content was increased in the alveolar walls. The volume proportion of smooth muscle-specific actin was augmented in distal airways and alveolar duct walls. Airway resistance, viscoelastic pressure, static elastance, and tissue elastance and resistance were significantly increased. In conclusion, prolonged allergen exposure induced remodeling not only of the airway wall but also of the lung parenchyma, leading to in vivo and in vitro mechanical changes.
A double-blind, dose escalation gene transfer trial was conducted in subjects with cystic fibrosis (CF), among whom placebo (saline) or compacted DNA was superfused onto the inferior turbinate of the right or left nostril. The vector consisted of single molecules of plasmid DNA carrying the cystic fibrosis transmembrane regulator- encoding gene compacted into DNA nanoparticles, using polyethylene glycol-substituted 30-mer lysine peptides. Entry criteria included age greater than 18 years, FEV1 exceeding 50% predicted, and basal nasal potential difference (NPD) isoproterenol responses (> or = -5 mV) that are typical for subjects with classic CF. Twelve subjects were enrolled: 2 in dose level I (DLI) (0.8 mg DNA), 4 in DLII (2.67 mg), and 6 in DLIII (8.0 mg). The primary trial end points were safety and tolerability, and secondary gene transfer end points were assessed. In addition to routine clinical assessments and laboratory tests, subjects were serially evaluated for serum IL-6, complement, and C-reactive protein; nasal washings were taken for cell counts, protein, IL-6, and IL-8; and pulmonary function and hearing tests were performed. No serious adverse events occurred, and no events were attributed to compacted DNA. There was no association of serum or nasal washing inflammatory mediators with administration of compacted DNA. Day 14 vector polymerase chain reaction analysis showed a mean value in DLIII nasal scraping samples of 0.58 copy per cell. Partial to complete NPD isoproterenol responses were observed in eight subjects: one of two in DLI, three of four in DLII, and four of six in DLIII. Corrections persisted for as long as 6 days (1 subject to day 28) after gene transfer. In conclusion, compacted DNA nanoparticles can be safely administered to the nares of CF subjects, with evidence of vector gene transfer and partial NPD correction.
Nanoparticles consisting of single molecules of DNA condensed with polyethylene glycol-substituted lysine 30-mers efficiently transfect lung epithelium following intrapulmonary administration. Nanoparticles formulated with lysine polymers having different counterions at the time of DNA mixing have distinct geometric shapes: trifluoroacetate or acetate counterions produce ellipsoids or rods, respectively. Based on intracytoplasmic microinjection studies, nanoparticle ellipsoids having a minimum diameter less than the 25 nm nuclear membrane pore efficiently transfect non-dividing cells. This 25 nm size restriction corresponds to a 5.8 kbp plasmid when compacted into spheroids, whereas the 8-11 nm diameter of rod-like particles is smaller than the nuclear pore diameter. In mice, up to 50% of lung cells are transfected after dosing with a rod-like compacted 6.9 kbp lacZ expression plasmid, and correction of the CFTR chloride channel was observed in humans following intranasal administration of a rod-like compacted 8.3 kbp plasmid. To further investigate the potential size and shape limitations of DNA nanoparticles for in vivo lung delivery, reporter gene activity of ellipsoidal and rod-like compacted luciferase plasmids ranging in size between 5.3 and 20.2 kbp was investigated. Equivalent molar reporter gene activities were observed for each formulation, indicating that microinjection size limitations do not apply to the in vivo gene transfer setting.
Thymulin is a thymic peptide possessing hypophysiotropic activity and antiinflammatory effects in the brain. We constructed a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin, and subsequently cloned it into different expression vectors. A sequence optimized for expression of met-FTS in rodents, 5'-ATGCAGGCCAAGTCGCAGGGGGGGTCGAACTAGTAG-3', was cloned in the mammalian expression vectors pCDNA3.1(+) and phMGFP (which expresses the Monster Green Fluorescent Protein), thus obtaining pcDNA3.1-metFTS and p-metFTS-hMGFP, which express met-FTS and the fluorescent fusion protein metFTS-hMGFP, respectively. The synthetic sequence was also used to construct the adenoviral vector RAd-metFTS, which expresses met-FTS. Transfection of HEK293 and BHK cells with pcDNA3.1-metFTS (experimental groups) or pcDNA3.1 (control), led to high levels of thymulin bioactivity (>600 versus <0.1 pg/ml in experimental and control supernatants, respectively). Transfection of HEK293 and BHK cells with pmetFTS-hMGFP revealed a cytoplasmic and nuclear distribution of the fluorescent fusion protein. A single intramuscular (i.m.) injection (10(7) plaque forming units (PFU)/mouse or 10(8) PFU/rat) of RAd-metFTS in thymectomized animals (nondetectable serum thymulin) restored serum thymulin levels for at least 110 and 130 days post-injection in mice and rats, respectively. We conclude that RAd-metFTS constitutes a suitable biotechnological tool for the implementation of thymulin gene therapy in animal models of chronic brain inflammation.
Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.
Until recently, nonviral vectors were outside the mainstream of gene transfer technology. Recent problems in clinical trials using viral vectors renewed interest in these methods. The clinical usefulness of nonviral methods is still hindered by their relatively low gene delivery/transgene expression efficiencies. Vectors must navigate a series of obstacles before the therapeutic gene can be expressed. This review considers these barriers and the properties of components of nonviral vectors that are essential for nucleic acid transfer. Although developments of new physical methods (hydrodynamic delivery, ultrasound, electroporation) have made a significant impact on gene transfer efficiency, various chemical carriers (lipids and polymers) have been shown to achieve high-level gene delivery and functional expression. Success of nonviral gene targeting will depend not only on the efficacy, but also safety of this methodology, and this aspect is also discussed. Understanding problems associated with nonviral targeting can also help in designing better viral vectors. In fact, interplay between viral and nonviral technologies should lead to a continued refinement of both methodologies.
The eye is an excellent candidate for gene therapy as it is immune privileged and much of the disease-causing genetics are well understood. Towards this goal, we evaluated the efficiency of compacted DNA nanoparticles as a system for non-viral gene transfer to ocular tissues. The compacted DNA nanoparticles examined here have been shown to be safe and effective in a human clinical trial, have no theoretical limitation on plasmid size, do not provoke immune responses, and can be highly concentrated.
Here we show that these nanoparticles can be targeted to different tissues within the eye by varying the site of injection. Almost all cell types of the eye were capable of transfection by the nanoparticle and produced robust levels of gene expression that were dose-dependent. Most impressively, subretinal delivery of these nanoparticles transfected nearly all of the photoreceptor population and produced expression levels almost equal to that of rod opsin, the highest expressed gene in the retina.
As no deleterious effects on retinal function were observed, this treatment strategy appears to be clinically viable and provides a highly efficient non-viral technology to safely deliver and express nucleic acids in the retina and other ocular tissues.
The pathogenesis of pulmonary hypertension (PH) includes an inflammatory response. Thymulin, a zinc-dependent thymic hormone, has important immunobiological effects by inhibiting various proinflammatory cytokines and chemokines. We investigated morphological and hemodynamic effects of thymulin administration in a rat model of monocrotaline (MCT)-induced PH, as well as the pattern of proinflammatory cytokine gene expression and the intracellular pathways involved. Adult Wistar rats received an injection of MCT (60 mg/kg, sc) or an equal volume of saline. One day after, the animals randomly received during 3 wk an injection of saline, vehicle (zinc plus carboxymethyl cellulose), or thymulin (100 ng/kg, sc, daily). At d 23-25, the animals were anesthetized for hemodynamic recordings, whereas heart and lungs were collected for morphometric and molecular analysis. Thymulin prevented morphological, hemodynamic, and inflammatory cardiopulmonary profile characteristic of MCT-induced PH, whereas part of these effects were also observed in MCT-treated animals injected with the thymulin's vehicle containing zinc. The pulmonary thymulin effect was likely mediated through suppression of p38 pathway.
Inhaled corticosteroids are used extensively in the treatment of asthma and chronic obstructive lung disease due to their demonstrated efficacy in reducing exacerbations and improving overall control. Their beneficial effect is based on their broad anti-inflammatory and immunosuppressive effects. The available inhaled corticosteroids vary in their therapeutic index based on individual drug potency, pharmacokinetics and specific delivery system. Although ICS are used in all age groups including children, younger and smaller children may be at a greater risk for adverse systemic effects as they can receive higher mg/kg doses of ICSs compared to older children. Most of the benefit from ICSs occurs in the low-medium dose range with minimal additional improvement with higher doses, although some patients may benefit from these higher doses. While ICS are the preferred agents for managing persistent asthma in all ages, their benefit in COPD is less convincing. When used appropriately, ICSs have few adverse events at low-medium doses but risk increases with high dose ICS. Although several new drugs are being developed and evaluated, it is unlikely that any of these new medications will replace ICS as the preferred initial long-term controller therapy for asthma but more effective initial controller therapy could be developed for COPD.
Asthma is a disease characterised by persistent inflammation and structural changes in the airways, referred to as airway remodelling. The mechanisms underlying these processes may be interdependent or they may be separate processes that are driven by common factors. The levels of a variety of growth factors (including transforming growth factor β, granulocyte macrophage colony stimulating factor, and vascular endothelial growth factor) are known to be changed in the asthmatic airway. These and other growth factors can contribute to the development and persistence of inflammation and remodelling. One of the prominent features of the structural changes of the airways is the increased deposition and alterations in the composition of the extracellular matrix proteins. These proteins include fibronectin, many different collagen types and hyaluronan. There is a dynamic relationship between the extracellular matrix proteins and the airway mesenchymal cells such that the changes in the extracellular matrix proteins can also contribute to the persistence of inflammation and the airway remodelling. This review aims to summarise the role growth factors and extracellular matrix proteins play in the regulation of inflammation and airway remodelling in the asthmatic airway.
Multiple extra- and intracellular obstacles, including low stability in blood, poor cellular uptake, and inefficient endosomal escape and disassembly in the cytoplasm, have to be overcome in order to deliver nucleic acids for gene therapy. This review introduces the recent advances in tackling the key challenges in achieving efficient, targeted, and safe nonviral gene delivery using various nucleic acid-containing nanomaterials that are designed to respond to various extra- and intracellular biological stimuli (e.g., pH, redox potential, and enzyme) as well as external artificial triggers (e.g., light and ultrasound). Gene delivery in combination with molecular imaging and targeting enables diagnostic assessment, treatment monitoring and quantification of efficiency, and confirmation of cure, thus fulfilling the great promise of efficient and personalized medicine. Nanomaterials platform for combined imaging and gene therapy, nanotheragnostics, using stimuli-responsive materials is also highlighted in this review. It is clear that developing novel multifunctional nonviral vectors, which transform their physico-chemical properties in response to various stimuli in a timely and spatially controlled manner, is highly desired to translate the promise of gene therapy for the clinical success.
Highly compacted DNA nanoparticles (DNPs) composed of polyethylene glycol linked to a 30-mer of poly-l-lysine via a single cysteine residue (CK(30)PEG) have previously been shown to provide efficient gene delivery to the brain, eyes and lungs. In this study, we used a combination of flow cytometry, high-resolution live-cell confocal microscopy, and multiple particle tracking (MPT) to investigate the intracellular trafficking of highly compacted CK(30)PEG DNPs made using two different molecular weights of PEG, CK(30)PEG(10k) and CK(30)PEG(5k). We found that PEG MW did not have a major effect on particle morphology nor nanoparticle intracellular transport. CK(30)PEG(10k) and CK(30)PEG(5k) DNPs both entered human bronchial epithelial (BEAS-2B) cells via a caveolae-mediated pathway, bypassing degradative endolysosomal trafficking. Both nanoparticle formulations were found to rapidly accumulate in the perinuclear region of cells within 2h, 37±19% and 47±8% for CK(30)PEG(10k) and CK(30)PEG(5k), respectively. CK(30)PEG(10k) and CK(30)PEG(5k) DNPs moved within live cells at average velocities of 0.09±0.04μm/s and 0.11±0.04μm/s, respectively, in good agreement with reported values for caveolae. These findings show that highly compacted DNPs employ highly regulated trafficking mechanisms similar to biological pathogens to target specific intracellular compartments.
Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of poly-l-lysine and 10kDa polyethylene glycol (CK(30)PEG(10k)), mediate effective gene delivery to the brain, eyes and lungs in vivo. Nevertheless, we found that CK(30)PEG(10k) DNA nanoparticles are immobilized by mucoadhesive interactions in sputum that lines the lung airways of patients with cystic fibrosis (CF), which would presumably preclude the efficient delivery of cargo DNA to the underlying epithelium. We previously found that nanoparticles can rapidly penetrate human mucus secretions if they are densely coated with low MW PEG (2-5kDa), whereas nanoparticles with 10kDa PEG coatings were immobilized. We thus sought to reduce mucoadhesion of DNA nanoparticles by producing CK(30)PEG DNA nanoparticles with low MW PEG coatings. We examined the morphology, colloidal stability, nuclease resistance, diffusion in human sputum and in vivo gene transfer of CK(30)PEG DNA nanoparticles prepared using various PEG MWs. CK(30)PEG(10k) and CK(30)PEG(5k) formulations did not aggregate in saline, provided partial protection against DNase I digestion and exhibited the highest gene transfer to lung airways following inhalation in BALB/c mice. However, all DNA nanoparticle formulations were immobilized in freshly expectorated human CF sputum, likely due to inadequate PEG surface coverage.
Airway remodeling, or structural changes of the airway wall arising from injury and repair, plays an important role in the pathophysiology of asthma. Remodeling is characterized as structural changes involving the composition, content, and organization of many of the cellular and molecular constituents of the bronchial wall. These structural changes can include epithelial injury, subepithelial thickening/fibrosis, airway smooth muscle hyperplasia, goblet cell hypertrophy and hyperplasia, and angiogenesis. Historically, these changes are considered a consequence of long-standing airway inflammation. Recent infant and child studies, however, suggest that remodeling occurs in parallel with inflammation in asthmatic subjects. Despite advancements in the recognition of key cellular and molecular mechanisms involved in remodeling, there remains a paucity of information about which treatments or interactions are most likely to regulate these processes. Furthermore, it is unclear as to when is the best time to initiate treatments to modify remodeling, which components to target, and how best to monitor interventions on remodeling. Indeed, inhaled corticosteroids, which are generally considered to have limited influence on remodeling, have been shown to be beneficial in studies in which the dose and duration of treatment were increased and prolonged, respectively. Moreover, several studies have identified the need to identify novel asthma indices and phenotypes that correlate with remodeling and, as a consequence, might specifically respond to new therapies, such as anti-IgE, anti-IL-5, and anti-TNF-α mAbs. Our review will evaluate the development of remodeling in asthmatic subjects and the effects of treatment on these processes.
Allergic diseases are complex genetic diseases resulting from the effect of multiple genetic and interacting environmental factors on their pathophysiology. Recent years have seen considerable progress in unraveling the contribution of these factors to an individual subject's susceptibility to, subsequent development of, and severity of disease. This has resulted in increasing insight into novel areas of allergic disease pathophysiology, for example the significant role played by locally acting tissue susceptibility factors like epithelial/epidermal barrier function and remodeling, such as filaggrin, ADAM33, and GSDML/ORMDL3, in patients with atopic dermatitis and asthma. Furthermore, studies of gene-environment interactions and Mendelian randomization approaches have led to increased insight into the importance of environmental triggers for allergic disease. Studies of the timing of action of genetic variants in determining disease susceptibility have highlighted the importance of in utero development and early life in determining susceptibility to allergic disease. In the future, genetic discoveries in allergic disease will potentially lead to better endophenotyping, prognostication, prediction of treatment response, and insights into molecular pathways to develop more targeted therapy for these conditions.
In this review we will overview novel nanotechnological nanocarrier systems for cancer therapy focusing on recent development in polyelectrolyte capsules for targeted delivery of antineoplastic drugs against cancer cells. Biodegradable polyelectrolyte microcapsules (PMCs) are supramolecular assemblies of particular interest for therapeutic purposes, as they can be enzymatically degraded into viable cells, under physiological conditions. Incorporation of small bioactive molecules into nano-to-microscale delivery systems may increase drug's bioavailability and therapeutic efficacy at single cell level giving desirable targeted therapy. Layer-by-layer (LbL) self-assembled PMCs are efficient microcarriers that maximize drug's exposure enhancing antitumor activity of neoplastic drug in cancer cells. They can be envisaged as novel multifunctional carriers for resistant or relapsed patients or for reducing dose escalation in clinical settings.
Inhaled combination therapy composing of long-acting β2-agonist and corticosteroid has been widely applied in the management of asthma, but observed treatment responses vary. The aim of this study was to evaluate the pharmacogenetic effect of the adenylyl cyclase type 9 (ADCY9) gene polymorphism on combination therapy.
Eighty-six mild to moderate Korean asthmatics were enrolled in this clinical trial. After the 2-week 'run-in' period, patients received budesonide (an inhaled corticosteroid) and formoterol (long-acting β2-agonist) during the following 12-week active treatment period. Forced expiratory volume in 1 s (FEV(1) ) and maximum mid-expiratory flow (MMEF) levels were measured at all visits as primary outcome. ADCY9 (Ile772Met, 150127 C/T, 150130 C/T, 150397 C/T, 150479 C/T, TTTA (5/4) ) and β2-adrenergic receptor (ADRB2, Arg16Gly) gene polymorphisms were genotyped.
Significant associations were observed between the ADCY9 single nucleotide polymorphisms and percent changes in FEV(1) (Ile772Met T/C, P = 0·030) and MMEF (150397 C/T, P = 0·016) after 8 weeks of combination therapy. Haplotype associations were also observed with respect to percent changes in FEV(1) after 8 weeks of therapy (Ht3[TTCC], P = 0·017). Additive therapeutic effect was observed in those with the ADCY9 Ile772Met and ADRB2 Arg16Gly gene polymorphisms in terms of percent change in FEV(1) after 8 and 12 weeks of therapy (P = 0·002 and P = 0·027 respectively).
Our results suggest that ADCY9 gene polymorphisms may alone, and in combination with ADRB2 gene polymorphisms, contribute to individual response to combination therapy in mild to moderate asthmatics.
The Global Initiative for Asthma (GINA) is an initiative started in the early 1990s to develop guidelines for asthma diagnosis and management that were applicable to both developed and developing countries. GINA now recommends that achieving overall asthma control is the goal of therapy. Overall asthma control consists of 2 domains: to achieve day-to-day (or current) asthma control and to minimize future risk measured by the absence of asthma exacerbations, the prevention of accelerated decline in lung function over time, and no side effects from medications. The GINA treatment paradigm consists of 5 treatment steps. At each step a preferred option and other alternatives are identified. Step 1 is as needed (prn) rapid-acting inhaled β2-agonist. The other 4 treatment steps include a controller option, ranging from low-dose inhaled corticosteroids (ICSs) as the preferred treatment option at Step 2, to high-dose ICSs plus long-acting inhaled β2-agonist combinations together with oral corticosteroids at Step 5. Once the level of asthma control has been established, consideration should be given to reducing the amount of treatment. By contrast, if asthma is uncontrolled, treatment needs to be increased to the next step. In an effort to remain current, a yearly update, based on an extensive review of the previous year's peer-reviewed literature on asthma management, is available on the web version of the GINA guidelines.
We hypothesized that bone marrow-derived mononuclear cells (BMDMC) would attenuate the remodeling process in a chronic allergic inflammation model. C57BL/6 mice were assigned to two groups. In OVA, mice were sensitized and repeatedly challenged with ovalbumin. Control mice (C) received saline under the same protocol. C and OVA were further randomized to receive BMDMC (2 × 10⁶) or saline intravenously 24 h before the first challenge. BMDMC therapy reduced eosinophil infiltration, smooth muscle-specific actin expression, subepithelial fibrosis, and myocyte hypertrophy and hyperplasia, thus causing a decrease in airway hyperresponsiveness and lung mechanical parameters. BMDMC from green fluorescent protein (GFP)-transgenic mice transplanted into GFP-negative mice yielded lower engraftment in OVA. BMDMC increased insulin-like growth factor expression, but reduced interleukin-5, transforming growth factor-β, platelet-derived growth factor, and vascular endothelial growth factor mRNA expression. In conclusion, in the present chronic allergic inflammation model, BMDMC therapy was an effective pre-treatment protocol that potentiated airway epithelial cell repair and prevented inflammatory and remodeling processes.
Gene and nucleic acid therapy are expected to play a major role in the next generation of medicine. We recently developed a multifunctional envelope-type nano device (MEND) for use as a novel non-viral gene delivery system. Poly(ethylene glycol) (PEG)ylation is a useful method for achieving a longer circulation time for delivery of the MEND to a tumour via the enhanced permeability and retention (EPR) effect. However, PEGylation strongly inhibits cellular uptake and endosomal escape, which results in significant loss of activity for the delivery system. For successful gene delivery for cancer treatment, the crucial issue associated with the use of PEG, the 'PEG dilemma' must be addressed. In this review, we describe the development and applications of MEND, and discuss strategies for overcoming the PEG dilemma, based on the manipulation of intracellular trafficking of cellular uptake and endosomal release using functional devices such as specific ligands, cleavable PEG systems and endosomal fusogenic/disruptic peptides.
DNA nanoparticles (DNPs) formed by compacting DNA with polyethyleneglycolylated poly-L-lysine are a nonviral vector shown to be safe and efficacious in animals and humans. To extend our capabilities of assessing the efficacy and duration of expression achieved by DNPs, we tested the utility of bioluminescent imaging (BLI) of transgene expression in wildtype and cystic fibrosis (CF) mouse models. We tested the effect of route of administration, mouse coat color, anesthesia, dose, and promoter sequence on the level and duration of expression. Furthermore, we investigated the correlation between imaging and direct analysis of luciferase expression in lung homogenates. We found that intratracheal instillation, and the use of deep and prolonged anesthesia with avertin produced significantly higher expression compared with intranasal administration, and the use of lighter anesthesia with isoflurane. Although similar expression was observed for both dark and light coat animals, imaging signal intensity was attenuated in mice with dark fur. Furthermore, good correlation between imaging and direct homogenate analysis was observed for single dose (r = 0.96), and dose response studies in wildtype (r = 0.82) and CF mice (r = 0.87). Finally, we used imaging to track gene expression over a 56-day time course. We found that the human ubiquitin B promoter gives stable transgene expression up to 49 days following nanoparticle administration, while expression with the cytomegalovirus promoter diminished after 2 days and returned to background levels by day 14. Taken together, our results demonstrate that BLI is an effective and useful modality for measuring gene expression conferred by DNPs in the lung.
Glucocorticoids are the most effective anti-inflammatory therapy for many chronic inflammatory and immune diseases, such as asthma, but are relatively ineffective in other diseases such as chronic obstructive pulmonary disease (COPD). Glucocorticoids suppress inflammation by several mechanisms. Glucocorticoids suppress the multiple inflammatory genes that are activated in chronic inflammatory diseases, such as asthma, by reversing histone acetylation of activated inflammatory genes through binding of liganded glucocorticoid receptors (GR) to coactivator molecules and recruitment of histone deacetylase-2 (HDAC2) to the activated transcription complex. At higher concentrations of glucocorticoids GR homodimers interact with DNA recognition sites to activate transcription through increased histone acetylation of anti-inflammatory genes and transcription of several genes linked to glucocorticoid side effects. Decreased glucocorticoid responsiveness is found in patients with severe asthma and asthmatics who smoke, as well as in all patients with COPD and cystic fibrosis. Several molecular mechanisms of glucocorticoid resistance have now been identified. HDAC2 is markedly reduced in activity and expression as a result of oxidative/nitrative stress so that inflammation becomes resistant to the anti-inflammatory actions of glucocorticoids. Dissociated glucocorticoids have been developed to reduce side effects but so far it has been difficult to dissociate anti-inflammatory effects from adverse effects. In patients with glucocorticoid resistance alternative anti-inflammatory treatments are being investigated as well as drugs that may reverse the molecular mechanism of glucocorticoid resistance.
The thymus gland is a central lymphoid organ in which bone marrow-derived T cell precursors undergo differentiation, eventually leading to migration of positively selected thymocytes to the peripheral lymphoid organs. This differentiation occurs along with cell migration in the context of the thymic microenvironment, formed of epithelial cells, macrophages, dendritic cells, fibroblasts, and extracellular matrix components. Various interactions occurring between microenvironmental cells and differentiating thymocytes are under neuroendocrine control. In this review, we summarize data showing that thymus physiology is pleiotropically influenced by hormones and neuropeptides. These molecules modulate the expression of major histocompatibility complex gene products by microenvironmental cells and the extracellular matrix-mediated interactions, leading to enhanced thymocyte adhesion to thymic epithelial cells. Cytokine production and thymic endocrine function (herein exemplified by thymulin production) are also hormonally controlled, and, interestingly in this latter case, a bidirectional circuitry seems to exist since thymic-derived peptides also modulate hormonal production. In addition to their role in thymic cell proliferation and apoptosis, hormones and neuropeptides also modulate intrathymic T cell differentiation, influencing the generation of the T cell repertoire. Finally, neuroendocrine control of the thymus appears extremely complex, with possible influence of biological circuitry involving the intrathymic production of a variety of hormones and neuropeptides and the expression of their respective receptors by thymic cells.
Bleomycin (BLM), an antitumour drug, is known to cause interstitial pneumonia followed by pulmonary fibrosis, and has often been used to produce an animal model of pulmonary fibrosis. In the present study, we examined the effect of a nonapeptide thymic hormone, facteur thymique serique (FTS), on the murine lung fibrosis induced by intratracheal instillation of BLM. Treatment with FTS ameliorated BLM-induced fibrotic changes in a dose-dependent manner, as indicated by the reduced accumulation of hydroxyproline (HP). In addition, FTS suppressed BLM-induced cellular inflammatory response in the lungs, as evidenced by inhibition of increased lung weight, reduced accumulation of inflammatory leucocytes, including lymphocytes and neutrophils, but not macrophages, and less pronounced histopathological changes. Finally, BLM challenge increased the local synthesis of proinflammatory cytokines, TNF-alpha and IL-1beta and chemokines, MCP-1, MIP-1alpha RANTES, MIP-2 and KC, while administration of FTS suppressed the production of these cytokines, except for MCP-1. These effects of FTS were observed only when mice received intratracheal instillation with BLM. Considered collectively, our results indicated that FTS treatment ameliorated the cellular inflammatory responses and fibrotic changes in the lungs caused by BLM and such inhibition was well correlated with reduced synthesis of several fibrosis-related cytokines, and suggested that FTS may be potentially useful for the treatment of pulmonary fibrosis.
Airway inflammation, airflow obstruction, and bronchial hyperresponsiveness are characteristic phenotypic features of asthma. Clinically, airflow obstruction in asthma often is not fully reversible, and many asthmatic subjects experience an accelerated and progressive loss of lung function over time. Histopathologic studies of the asthmatic airway have demonstrated stereotypic changes that might explain the loss of lung function that many patients with asthma experience. The notion of airway remodeling in asthma postulates that the alteration of the structure and function of key airway constituents, including airway smooth muscle, epithelium, blood vessels, and mucus glands, might explain, at least in part, the progressive loss of lung function that is observed clinically. Inflammation driven by CD4(+) lymphocytes and mediated by effector cells, particularly the eosinophil, appears to modulate the function of mesenchymal cells, including fibroblasts and myofibroblasts, changing the composition of the airway wall matrix. Changes in the airway epithelium might alter the function of the underlying smooth muscle and the composition of the matrix and could drive inflammation. Alterations in the structure and function of airway smooth muscle change the mechanical properties of the airway wall and might also affect the function of other airway constituents. A variety of experimental models have identified candidate mechanisms and mediators for these observed changes, which are thus potential therapeutic targets. However, clinical studies to date have been disappointing, and it remains to be seen whether targeted therapies will prevent the progressive loss of lung function seen in asthma.
Mercury (Hg2+) affects cell-mediated immunity, including thymulin production. Thymulin, a zinc-dependent thymic hormone synthesized by thymic epithelial cells (TECs), is involved in NK cell cytotoxicity and Th1 cytokine production (IL-2 and IFN-gamma), which in turn affect both NKT and classic NK spleen cell cytotoxicity. High doses of Hg2+ induce an inflammatory status, increased production of IL-6 and consequent Th1/Th2 imbalance as well as cell-mediated immune depression. The mechanisms by which Hg+ affects the cell-mediated immune response are still unclear. The nitric oxide (NO) pathway may be implicated. The aim of this work was to further explore its noxious role in innate and adaptive immunity and to study the possible role played by the NO pathway. Young Balb/c mice treated in vivo for 1 month with 1.0 mg HgCl2/kg b.w. showed low thymulin activity, depressed NO production (as measured by nitrite and nitrate plasma levels), impaired classic NK spleen cell cytotoxicity, decreased Th1 (IL-2 and IFN-gamma) cytokine profiles, and increased IL-6 production. In vitro, 10(-6) M of HgCl2 inhibited active thymulin kinetics, TEC proliferation, NKT cell cytotoxicity and Th1 cytokine production, whereas IL-6 increased. L-arginine restored thymulin activity, TEC proliferation, NKT cytotoxicity, cytokine profiles and nitrite and nitrate plasma levels both in vivo and in vitro. Since L-arginine is the substrate for NO production, it may compensate for the cell-mediated immune defect induced by HgCl2, via the arginine-NO-pathway. L-arginine is also able to reduce glomerular kidney IgG antibodies deposits induced by higher dose of HgCl2 administration.
Airway remodeling in asthma is defined by several structural changes including epithelial cell mucus metaplasia, an increase in peribronchial smooth muscle mass, subepithelial fibrosis, and angiogenesis. Cytokines, chemokines, and growth factors released from inflammatory and structural cells in the airway are considered to play a pivotal role in the development of remodeling. Studies of allergen induced airway remodeling in transgenic mice suggest an important role for TGF-beta, VEGF, Th2 cytokines (IL-5, IL-9, IL-13), and epithelial derived NF-kappaB regulated chemokines in airway remodeling. Although studies of bronchial biopsies from human asthmatics also demonstrate expression of TGF-beta, VEGF, IL-5, IL-9, IL-13, and NF-kappaB regulated chemokines, further human intervention studies are required in which individual cytokines or chemokines are neutralized to define their role in airway remodeling.
Models of Lung Disease-Microscopy and Structural Methods
- E R Weibel
Weibel, ER. Models of Lung Disease-Microscopy and Structural Methods. New York, NY, USA:
1990. Morphometry: stereological theory and practical methods; p. 48