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AdvIL-10 infects lung A549 epithelial cells to produce detectable vIL-10 protein in the culture media and addition of protease inhibitor cocktail preserves detectable vIL-10. A549 cells at were infected with 1000:1 MOI of either Adlac-Z or AdvIL-10. Supernatants collected +/- protease inhibitor cocktail and assayed for vIL-10 by ELISA. Values represent average of two experiments.
Source publication
Adenovirus and cationic liposome mediated transfer of Interleukin-10 (IL-10), a potent anti-inflammatory cytokine, has been shown to decrease pro-inflammatory cytokine levels and overall lung inflammation in models of lung transplantation and injury. Limitations to current approaches of IL-10 gene therapy include poor vector delivery methods and pr...
Citations
... Use of PFC liquid prior to vector administration improved AdV and AAV vector mediated gene transfer to the airways of rodents [84,86,88] and non-human primates [89]. Inhalation of nebulised PFC vapour has also been explored as a simpler and more clinically appealing technique compared to liquid-based delivery. ...
Gene-based therapeutics are actively being pursued for the treatment of lung diseases. While promising advances have been made over the last decades, the absence of clinically available lung-directed genetic therapies highlights the difficulties associated with this effort. Largely, progress has been hindered by the presence of inherent physical and physiological airway barriers that significantly reduce the efficacy of gene transfer. These barriers include surface mucus, mucociliary action, cell-to-cell tight junctions, and the basolateral cell membrane location of viral receptors for many commonly used gene vectors. Accordingly, airway surface preparation methods have been developed to disrupt these barriers, creating a more conducive environment for gene uptake into the target airway cells. The two major approaches have been chemical and physical methods. Both have proven effective for increasing viral-mediated gene transfer pre-clinically, although with variable effect depending on the specific strategy employed. While such methods have been explored extensively in experimental settings, they have not been used clinically. This review covers the airway surface preparation strategies reported in the literature, the advantages and disadvantages of each method, as well as a discussion about applying this concept in the clinic.
... To achieve basolateral access, airway conditioning with a chemical "tight-junction opener" can be employed to increase paracellular permeability. Airway conditioning with compounds including ethylene glycol tetraacetic acid (EGTA) (Chu et al., 2001), perfluorochemicals (Li et al., 2007), sodium caprate (Gregory et al., 2003), and sulphur dioxide inhalation (Johnson et al., 2000) have proven effective for disrupting tight-junctions and increasing viral-vector mediated gene transfer in proof-of-principle animal studies (Castellani and Conese, 2010). Another compound that has been investigated extensively for this purpose is lysophosphatidylcholine (LPC) (Limberis et al., 2002;Koehler et al., 2005;Cmielewski et al., 2010;Cao et al., 2013;Cmielewski et al., 2014). ...
The lungs have evolved complex physical, biological and immunological defences to prevent foreign material from entering the airway epithelial cells. These mechanisms can also affect both viral and non-viral gene transfer agents, and significantly diminish the effectiveness of airway gene-addition therapies. One strategy to overcome the physical barrier properties of the airway is to transiently disturb the integrity of the epithelium prior to delivery of the gene transfer vector. In this study, chemical (lysophosphatidylcholine, LPC) and physical epithelium disruption using wire abrasion were compared for their ability to improve airway-based lentiviral (LV) vector mediated transduction and reporter gene expression in rats. When luciferase expression was assessed at 1-week post LV delivery, LPC airway conditioning significantly enhanced gene expression levels in rat lungs, while a long-term assessment in a separate cohort of rats at 12 months revealed that LPC conditioning did not improve gene expression longevity. In rats receiving physical perturbation to the trachea prior to gene delivery, significantly higher LacZ gene expression levels were found when compared to LPC-conditioned or LV-only control rats when evaluated 1-week post gene transfer. This proof-of-principle study has shown that airway epithelial disruption strategies based on physical perturbation substantially enhanced LV-mediated airway gene transfer in the trachea.
... 7 Over the last three decades, a number of preclinical investigations have been conducted in which perfluorochemical (PFC)-facilitated pulmonary drug was tested. [9][10][11][12][13][14][15][16] Given that the distribution of inhaled aerosolized agents is complicated by the pathology they are targeted to treat, we posited that PFC liquids facilitate the distribution of the PAs and improve outcomes in ISALI. The benefits of PFC-facilitated drug delivery include that these liquids are inert, can be homogenously distributed in the lung because of low surface tension, mitigate barotrauma, support gas exchange, improve lung mechanics, and recruit lung volume in animal models of ALI. ...
... 19,21,22,53,54 PFCs have also been used to formulate drugs for delivery and distribution in the lung during "liquid ventilation," including the recombinant protein superoxide dismutase, antibiotics, vasoactive substances, or surfactant in the formulation. 9,[11][12][13][14][15][16]55 PFCs are also well tolerated and have direct beneficial effects in the compromised lung (decreased edema and inflammation) independent of the drug delivered. 18,20,51 Given these advantages, we inferred that PFCs would improve airway delivery of PAs and improve outcomes in ISALI. ...
Background:
Effective clinical management of airway clot and fibrinous cast formation of severe inhalational smoke-induced acute lung injury (ISALI) is lacking. Aerosolized delivery of tissue plasminogen activator (tPA) is confounded by airway bleeding; single-chain urokinase plasminogen activator (scuPA) moderated this adverse effect and supported transient improvement in gas exchange and lung mechanics. However, neither aerosolized plasminogen activator (PA) yielded durable improvements in physiologic responses or reduction in cast burden. Here, we hypothesized that perfluorochemical (PFC) liquids would facilitate PA distribution and sustain improvements in physiologic outcomes in ISALI.
Methods:
Spontaneously breathing adult sheep (n = 36) received anesthesia and analgesia and were instrumented, exposed to cotton smoke inhalation, and supported by mechanical ventilation for 48 h. Groups (n = 6/group) were studied without supplemental treatment, or, starting 4 h post injury, they received intratracheal low volume (8 mL) PFC liquid alone or a dose range of tPA/PFC or scuPA/PFC suspensions (4 or 8 mg in 8 mL PFC) every 8 h. Outcomes were evaluated by sequential measurements of cardiopulmonary parameters, lung histomorphology, and biochemical analyses of bronchoalveolar lavage fluid.
Results:
Dose-response and PA-type comparisons of outcomes demonstrated sustained superiority with low-volume PFC suspensions of scuPA over tPA or PFC alone, favoring the highest dose of scuPA/PFC suspension over lower doses, without airway bleeding.
Conclusions:
We propose that this improved profile over previously reported aerosolized delivery is likely related to improved dose distribution. Sustained salutary responses to scuPA/PFC suspension delivery in this translational model are encouraging and support the possibility that the observed outcomes could be of clinical importance.
... The distribution of the coxsackie-adenovirus receptor (CAR) on basolateral surfaces of cells has led to the proposed use of reagents that disrupt tight junctions and improve transduction of airway epithelial cells345. As an alternative approach to improve distribution, several studies have used an intratracheal pulsechase method in the lung to deliver recombinant adenovirus (rAd) and adeno-associated virus vectors in a small volume of saline administered followed by a larger volume of perfluorochemical (PFC) liquid [3,67891011. These studies have demonstrated increased total lung gene expression, distribution of gene expression, and enhanced delivery to the distal lung over saline alone. ...
WE COMPARED LUNG DELIVERY METHODS OF RECOMBINANT ADENOVIRUS (RAD): (1) rAd suspended in saline, (2) rAd suspended in saline followed by a pulse-chase of a perfluorochemical (PFC) liquid mixture, and (3) a PFC-rAd suspension. Cell uptake, distribution, and temporal expression of rAd were examined using A549 cells, a murine model using luciferase bioluminescence, and histological analyses. Relative to saline, a 4X increase in transduction efficiency was observed in A549 cells exposed to PFC-rAd for 2-4 h. rAd transgene expression was improved in alveolar epithelial cells, and the level and distribution of luciferase expression when delivered in PFC-rAd suspensions consistently peaked at 24 h. These results demonstrate that PFC-rAd suspensions improve distribution and enhance rAd-mediated gene expression which has important implications in improving lung function by gene therapy.
... Retrovirus vector is commonly used because of its stable expression, relative safety, and efficiency of in vitro transfection in a wide range of host cells. [8][9][10] Our laboratory has successfully constructed a recombinant retrovirus carrying vIL-10. The present report is the first of this kind in China on the ex vivo targeting of vIL-10 with a retroviral vector in an animal model. ...
Interleukin 1beta (IL-1beta) is the principal mediator in the pathogenesis of rheumatoid arthritis. Continuous injection of interleukin 1beta (IL-1beta) into the knee articular cavities of animals can induce models that resemble rheumatoid arthritis. The objective of this study was to evaluate the feasibility of local recombinant retrovirus viral interleukin 10 (rRV-vIL-10) gene transfer treatment of a rabbit model of arthritis induced by IL-1beta.
An hIL-1beta-induced rabbit rheumatoid arthritis model was established using the MFG-hIL-1beta-neo-HIG-82 cell line, which is capable of continuous secretion of hIL-1beta. After transfecting the rabbit synovial fibroblast cell line (MFG-hIL-1beta-neo-HIG-82) with rRV-vIL-10, G418 was then added to identify the positive clone. The rRV-vIL-10 positive clone was injected into the established rabbit rheumatoid arthritis model through intra-articular injection. Successful gene transfer was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The levels of IL-1beta before and after treatment were determined by enzyme- linked immunosorbent assay.
Retrovirus vector was an effective vector both to synoviocytes in vitro and synovium tissue in vivo as confirmed by RT-PCR and immunohistochemistry. The rabbit arthritis model treated with rRV-vIL-10 showed a dramatic remission of arthritis and a decline in the level of cytokines such as IL-1beta.
Retrovirus-mediated transfection of vIL-10 successfully transferred the gene into rabbit synovium ex vivo and was able to suppress intra-articular inflammation response to IL-1beta.
Remarkable advances in three-dimensional (3D) cell cultures and organ-on-a-chip technologies have opened the door to recapitulate complex aspects of human physiology, pathology, and drug responses in vitro. The challenges regarding oxygen delivery, throughput, assay multiplexing, and experimental complexity are addressed to ensure that perfused 3D cell culture organ-on-a-chip models become a routine research tool adopted by academic and industrial stakeholders. To move the field forward, we present a throughput-scalable organ-on-a-chip insert system that requires a single tube to operate 48 statistically independent 3D cell culture organ models. Then, we introduce in-well perfusion to circumvent the loss of cell signaling and drug metabolites in otherwise one-way flow of perfusate. Further, to augment the relevancy of 3D cell culture models in vitro, we tackle the problem of oxygen transport by blood using, for the first time, a breathable hemoglobin analog to improve delivery of respiratory gases to cells, because in vivo approximately 98% of oxygen delivery to cells takes place via reversible binding to hemoglobin. Next, we show that improved oxygenation shifts cellular metabolic pathways toward oxidative phosphorylation that contributes to the maintenance of differentiated liver phenotypes in vitro. Lastly, we demonstrate that the activity of cytochrome P450 family of drug metabolizing enzymes is increased and prolonged in primary human hepatocytes cultured in 3D compared to two-dimensional (2D) cell culture gold standard with important ramifications for drug metabolism, drug-drug interactions and pharmacokinetic studies in vitro.
Background:
Cutaneous wound healing is a complex process involving various regulatory factors at the molecular level. Aloe vera is widely used for cell rejuvenation, wound healing, and skin moisturizing.
Hypothesis/purpose:
This study aimed to investigate the effects of aloesin from Aloe vera on cutaneous wound healing and mechanisms involved therein.
Study design:
This study consisted of both in vitro and in vivo experiments involving skin cell lines and mouse model to demonstrate the wound healing effects of aloesin by taking into account several parameters ranging from cultured cell migration to wound healing in mice.
Methods:
The activities of Smad signaling molecules (Smad2 and Smad3), MAPKs (ERK and JNK), and migration-related proteins (Cdc42, Rac1, and α-Pak) were assessed after aloesin treatment in cultured cells (1, 5 and 10µM) and mouse skin (0.1% and 0.5%). We also monitored macrophage recruitment, secretion of cytokines and growth factors, tissue development, and angiogenesis after aloesin treatment using IHC analysis and ELISAs.
Results:
Aloesin increased cell migration via phosphorylation of Cdc42 and Rac1. Aloesin positively regulated the release of cytokines and growth factors (IL-1β, IL-6, TGF-β1 and TNF-α) from macrophages (RAW264.7) and enhanced angiogenesis in endothelial cells (HUVECs). Aloesin treatment accelerated wound closure rates in hairless mice by inducing angiogenesis, collagen deposition and granulation tissue formation. More importantly, aloesin treatment resulted in the activation of Smad and MAPK signaling proteins that are key players in cell migration, angiogenesis and tissue development.
Conclusion:
Aloesin ameliorates each phase of the wound healing process including inflammation, proliferation and remodeling through MAPK/Rho and Smad signaling pathways. These findings indicate that aloesin has the therapeutic potential for treating cutaneous wounds.
Fopius arisanus (Sonan) is a dominant egg-pupal parasitoid of many fruit fly pests. Bactrocera dorsalis (Hendel) is one of F. arisanus' hosts and is the usual host used for mass rearing F. arisanus. To obtain parasitoids without hosts suitable for the release of F. arisanus in the field—skipping the need to separate parasitoids and unparasitized hosts—radiation (15, 20, 25 and 30 Gy) was applied to B. dorsalis eggs of five different ages (24, 27, 30, 33, and 36 h). Hatch rates, pupation rates, pupae weights, emergence rates, sex ratios, flight abilities, mortality rates and fertilities were analyzed during parasitoid development from irradiated eggs to adults. Irradiation of eggs aged 30 to 36 h at 20 Gy might be the optimal age and dosage when using B. dorsalis as hosts for mass rearing of F. arisanus. Furthermore, we investigated the viability of the parents and offspring of F. arisanus when the parents were raised from irradiated eggs under both laboratory and field cage conditions. The parasitism percentage, emergence rate, sex ratio (female: male) and longevity of parasitoids reared with irradiated hosts were similar to those of parasitoids obtained from non-irradiated host eggs. Significantly, the results revealed that unparasitized B. dorsalis previously irradiated with an appropriate dose at appropriate ages do not emerge: a finding that solves the problem of separating unparasitized emerging flies during mass rearing. This experiment also demonstrates that F. arisanus reared on irradiated hosts can find, identify, and parasitize hosts normally—both indoors and outdoors—and that the resulting parasitoids can be directly applied to control pest populations in the field.
The development of a new physiological multi-parameter remote monitoring system is based on the B/S model. The system consists of a server monitoring center, Internet network and PC-based multi-parameter monitors. Using the B/S model, the clients can browse web pages via the server monitoring center and download and install ActiveX controls. The physiological multi-parameters are collected, displayed and remotely transmitted. The experimental results show that the system is stable, reliable and operates in real time. The system is suitable for use in physiological multi-parameter remote monitoring for family and community healthcare.
Unlabelled:
Acupuncture is widely used clinically to treat acute and chronic pain conditions, but the mechanisms underlying its effect are not fully understood. Although endocannabinoids are involved in modulation of nociception in animal models and in humans, their role in acupuncture analgesia has not been assessed. In this report, we determined the effect of electroacupuncture (EA) on the level of anandamide in the skin tissue and the role of cannabinoid CB1 and CB2 receptors in the analgesic effect of EA in an animal model of inflammatory pain. Inflammatory pain was induced by local injection of complete Freund's adjuvant (CFA) into the hind paw of rats. Thermal hyperalgesia was tested with a radiant heat stimulus, and mechanical allodynia was quantified with von Frey filaments. The anandamide concentration in the skin tissue was measured by using high-performance liquid chromatography. EA, applied to GB30 and GB34, at 2 and 100Hz significantly reduced thermal hyperalgesia and mechanical allodynia induced by CFA injection. Compared with the sham group, EA significantly increased the anandamide level in the inflamed skin tissue. Local pretreatment with a specific CB2 receptor antagonist, AM630, significantly attenuated the antinociceptive effect of EA. However, the effect of EA was not significantly altered by AM251, a selective CB1 receptor antagonist. These findings suggest that EA potentiates the local release of endogenous anandamide from inflamed tissues. Activation of peripheral CB2 receptors contributes to the analgesic effect of EA on inflammatory pain.
Perspective:
This study shows that electroacupuncture increases the anandamide level in inflammatory skin tissues, and CB2 receptors contribute to the analgesic effect of electroacupuncture in a rat model of inflammatory pain. This information improves our understanding of the mechanisms involved in the analgesic effect of acupuncture.
