Using salivary glands as a tissue target for gene therapeutics

Gene transfer offers a potential way to correct local and systemic protein deficiency disorders by using genes as drugs, so called gene therapeutics. Salivary glands present an interesting target site for gene therapeutic applications. Herein, we review proofs of concept achieved for salivary glands with in vivo animal models. In that context we discuss problems (general and salivary tissue-specific) that limit immediate clinical use for this application of gene transfer. Ongoing efforts, however, suggest that salivary glands may be suitable as gene therapeutic target sites for drug delivery in the near future.     

Prospects for gene-based immunopharmacology in salivary glands

The clinical potential of gene transfer is increasing. One likely major application of this emerging biotechnology will be for gene therapeutics, the use of a gene as a drug. Salivary glands provide an unusual but increasingly valuable target site for gene transfer. Studies in animal salivary glands from several laboratories, including our own, have provided proof of this concept. In this review, we provide a background and perspective on possible strategies for gene-based immunopharmacology in salivary glands. We use as a target disease model the autoimmune exocrinopathy Sj?gren's syndrome.     

Imidacloprid-mediated alterations on the salivary glands of the Neotropical brown stink bug,Euschistus heros

The management of the Neotropical brown stinkbug Euschistus heros (Hemiptera: Pentatomidae) in soybean fields has been heavily dependent on the application of neonicotinoid insecticides. Neonicotinoids act primarily by impairing the function of the nicotinic acetylcholine receptors of the nervous system. These compounds also target specific organs (e.g., salivary glands), which may potentiate their insecticidal efficacy. Here, we evaluated whether the exposure to the neonicotinoid imidacloprid would cause cytomorphological changes in the salivary glands of E. heros. First, we determined the lethal concentrations (LCs) of imidacloprid through contact and ingestion. Subsequently, the cytomorphology of the salivary glands were evaluated in insect groups that survived exposure to the LC₅ (3.75 mg a.i./L), LC₅₀ (112.5 mg a.i./L), or LC₇₅ (375.0 mg a.i./L, equivalent to the recommended field rate) doses. Imidacloprid induced apoptosis and necrosis in the salivary gland cells according to the insecticide concentration and salivary gland region. All concentrations increased apoptosis and injured cells (e.g., vacuolization, chromatin condensation, swelling of organelles, and plasma membrane rupture) in the principal and accessory salivary glands. Individuals that survived exposure to the highest concentrations (i.e., LC₅ and LC₅₀) were more affected, and exhibited several necrotic cells on their main principal salivary glands. Collectively, our results indicate that imidacloprid exerts toxic effects on the non-target organs, such as the salivary glands, which increases the efficacy of this compound in the management of stink bug infestations.

     

Improved stomach selectivity of gene expression following microinstillation of plasmid DNA onto the gastric serosal surface in mice

Stomach-selective gene transfer is a promising approach as a therapeutic strategy for refractory gastric diseases. In this study, we improved the stomach selectivity of gene expression following microinstillation of naked plasmid DNA (pDNA) onto the gastric serosal surface in mice. pDNA encoding firefly luciferase was used as a reporter gene. It was confirmed that the gene expression level in the stomach 6h after gastric serosal surface microinstillation of pDNA was significantly higher than after intragastric, intraperitoneal and intravenous administration. Regarding selectivity of gene expression, the gene expression level in the stomach after gastric serosal surface microinstillation of 1 microg/1 microL (dose/volume) pDNA was 5.7 times higher than that in the spleen. In our previous study (30 microg/30 microL), the expression level in the stomach was 2.7 times higher than that in the spleen; therefore, the selectivity was 2.1 times higher in this study. When we investigated gene expression at various pDNA solution concentrations, the ratio of the gene expression level in the stomach to that in the spleen was the highest as 1 microg/1 microL of pDNA, which was considered the optimal concentration. Information in this study is useful for further development of target organ-selective gene delivery systems.     

CP‐25 Alleviates Experimental Sjögren's Syndrome Features in NOD/Ltj Mice and Modulates T Lymphocyte Subsets

Primary Sjögren's syndrome (pSS) is a chronic inflammatory autoimmune illness of the moisture‐producing glands such as salivary glands that is characterized by various immune abnormalities. The aetiology of pSS remains unclear and there is no curative agent. In this study, we investigated the putative therapeutic effects on a NOD/Ltj mouse model of Sjögren's syndrome‐like disorders of an ester derivative of paeoniflorin, paeoniflorin‐6′O‐benzene (termed CP‐25). Our study showed that CP‐25 alleviated effectively clinical manifestations in NOD/Ltj mice resulting, for example, in increased salivary flow and reduced histopathological scores. Furthermore, CP‐25 decreased lymphocyte viability in NOD/Ltj mice and attenuated the infiltration of Th1 cells and Th2 cells into the salivary glands of NOD/Ltj mice. In the spleen on NOD/Ltj mice, CP‐25 skewed the ratio of Th17 and regulatory T cells towards regulatory T cells. After treatment, concentrations of anti‐La/SSB and IgG antibodies were reduced and the titre of the inflammatory cytokines IFN‐γ, IL‐4, IL‐6 and IL‐17A in the serum on NOD/Ltj mice was alleviated. Thus, we define CP‐25 as a novel compound that is a potent therapeutic agent for pSS by modulating T lymphocyte subsets. Future studies will validate the use of CP‐25 as a therapeutic strategy for the treatment of pSS.     

Mercuric dichloride induces DNA damage in human salivary gland tissue cells and lymphocytes

Amalgam is still one of the most frequently used dental filling materials. However, the possible adverse effects especially that of the mercuric component have led to continued controversy. Considering that mercury may be released from amalgam fillings into the oral cavity and also reach the circulating blood after absorption and resorption, it eventually may contribute to tumorigenesis in a variety of target cells. The present investigation focuses on genotoxic effects below a cytotoxic dose level of mercuric dichloride (HgCl₂) in human samples of salivary glands and lymphocytes to elucidate a possible role in tumor initiation. DNA migration due to single strand breaks, alkali labile sites and incomplete excision repair was quantified with the aid of the single cell microgel electrophoresis (Comet) assay. The concepts of Olive Tail Moment, percentage of DNA in the Tail and Tail Length were used as measures of DNA damage. To control for cytotoxic effects, the trypan blue exclusion test was applied. Human samples of the parotid salivary gland and lymphocytes of ten donors were exposed to HgCl₂ concentrations from 1 to 50 μM. N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and dimethyl sulfoxide (DMSO) served as controls. Increasing dose-dependent DNA migration could be demonstrated after exposure to HgCl₂ in cells of the salivary glands and lymphocytes. In both cell types a significant increase in DNA migration could be shown starting from HgCl₂ concentrations of 5 μM in comparison to the negative control. The viability of the cell systems was not affected except at the highest concentration (50 μM) tested. These data indicate genotoxic effects of mercuric dichloride in human salivary glands and lymphocytes at concentrations not leading to cytotoxic effects or cell death. Consequently, a contributory role in oral salivary gland tumor initiation warrants further investigation. The authors declare that there is no conflict of interest.     

Human artificial chromosome-based gene delivery vectors for biomedicine and biotechnology

Introduction: Human artificial chromosomes (HACs) have several advantages over viruses as gene delivery vectors, including stable episomal maintenance in a single copy and the ability to carry large gene inserts.

Areas covered: In this review, we summarise recent work on gene transfer into mammalian cells using the HACs. HACs allow therapeutic transgenes to be expressed in target cells under conditions that recapitulate the physiological regulation of endogenous loci.

Expert opinion: Based on the published data, the HAC vectors have a great potential for gene therapy, regenerative medicine, screening of anticancer drugs and biotechnology.     

Alleviating effect of paeoniflorin-6′-O-benzene sulfonate in antigen-induced experimental Sjögren’s syndrome by modulating B lymphocyte migration via CXCR5-GRK2-ERK/p38 signaling pathway

Primary Sjögren's syndrome (pSS) is an autoimmune disease of unresolved aetiology that affects the exocrine glands. Clinical symptoms frequently also involve skin, liver, kidney and neurovascular components. The pathogenesis of pSS is still unclear but B cell hyperactivity has been identified as a hallmark of pSS. Currently, a curative therapeutic agent is lacking. In this study, we explored whether paeoniflorin-6′-O-benzene (CP-25) exerted therapeutic effects through regulating B lymphocyte migration via CXCR5-GRK2-MAPK mediated signaling pathways in a mouse model of antigen-induced, experimental Sjögren's syndrome (ESS). We found that CP-25 increased the salivary flow and alleviated the histopathology of ESS. Furthermore, CP-25 reduced the viability of B lymphocyte and limited the target organs index. In the peripheral blood and salivary gland of ESS mice, CP-25 down-regulated the proportion of total B cells, CXCR5+ B cells and PDCA1 + CD19- and limited the presence of phosphorylated (p-) p38 and ERK (p-ERK). Besides, CP-25 increased the percentage of memory B cells in the peripheral blood and reduced it in salivary gland. Furthermore, in vitro, CP-25 down-regulated p-p38, p-ERK, CXCR5 and membrane GRK2, and increased cytoplasm GRK2 in Maver-1 cells, a mantle cell lymphoma cell line, causing a lower migration ability of Maver-1 cells. Thus, we define CP-25 as a novel compound that is a potent therapeutic agent for pSS which modulates B lymphocyte subsets and impacts the migration of B lymphocytes through regulating the CXCR5-GRK2-ERK/p38 signaling pathway.     

Carbon nanotubes as vectors for gene therapy: Past achievements,present challenges and future goals

Promising therapeutic and prophylactic effects have been achieved following advances in the gene therapy research arena, giving birth to the new generation of disease-modifying therapeutics. The greatest challenge that gene therapy vectors still face is the ability to deliver sufficient genetic payloads in order to enable efficient gene transfer into target cells. A wide variety of viral and non-viral gene therapy vectors have been developed and explored over the past 10 years, including carbon nanotubes. In this review we will address the application of carbon nanotubes as non-viral vectors in gene therapy with the aim to give a perspective on the past achievements, present challenges and future goals. A series of important topics concerning carbon nanotubes as gene therapy vectors will be addressed, including the benefits that carbon nanotubes offer over other non-viral delivery systems. Furthermore, a perspective is given on what the ideal genetic cargo to deliver using carbon nanotubes is and finally the geno-pharmacological impact of carbon nanotube-mediated gene therapy is discussed.     

The potential of adeno-associated viral vectors for gene delivery to muscle tissue

Introduction: Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8⁺ T-cell response against AAV capsid in humans.

Areas covered: In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application.

Expert opinion: Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application.     

Cardiac adenovirus-associated viral Presenilin 1 gene delivery protects the left ventricular function of the heart via regulating RyR2 function in post-ischaemic heart failure

Post-ischaemic heart failure is a major cause of death worldwide. Reperfusion of infarcted heart tissue after myocardial infarction has been an important medical intervention to improve outcomes. However, disturbances in Ca²⁺ and redox homeostasis at the cellular level caused by ischaemia/reperfusion remain major clinical challenges. In this study, we investigated the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of a Type-2 ryanodine receptor (RyR2) degradation-associated gene, Presenilin 1 (PSEN1), to combat post-ischaemic heart failure. Adeno-associated viral PSEN1 gene delivery elevated PSEN1 protein expression in a post-infarction rat heart failure model, and this administration normalised the contractile dysfunction of the failing myocardium in vivo and in vitro by reversing myocardial Ca²⁺ handling and function. Moreover, PSEN1 gene transfer to failing cardiomyocytes reduced sarcoplasmic reticulum (SR) Ca²⁺ leak, thereby restoring the diminished intracellular Ca²⁺ transients and SR Ca²⁺ load. Moreover, PSEN1 gene transfer reversed the phosphorylation of RyR2 in failing cardiomyocytes. However, selective autophagy inhibition did not prevent the PSEN1-induced blockade of RyR2 degradation, making the participation of autophagy in PSEN1-associated RyR2 degradation unlikely. Our results established a role of the cardiac expression of PSEN1 with AAV9 vectors as a promising therapeutic approach for post-ischaemic heart failure.     

104例涎腺肿瘤的临床分析

通过分析104例涎腺肿瘤的临床表现、诊断及治疗,发现男女患者的比值为1:1.21;良性肿瘤80例,占76.9％;恶性肿瘤24例,占23.0％;良性肿瘤多见于21～50岁组(63.25％),主要为混合瘤、腺淋巴瘤;恶性肿瘤多见于51～60岁组(58.33％),主要为粘液表皮样癌、恶性混合瘤、腺样囊性癌;肿瘤的好发部位依次为:腮腺部、颌下腺部、小涎腺部及舌下腺部。其中腮腺及颌下腺部良性肿瘤高于恶性肿瘤;小涎腺中的恶性肿瘤比重大大增加。诊断中强调了细针吸细胞学检查的价值,并对肿瘤的手术原则及预后作了讨论。     

截短的丙型肝炎病毒核心区基因的克隆和表达

目的  克隆和表达截短的丙型肝炎病毒（hepatitis C virus,HCV）核心区基因,为制备HCV核心区抗体准备抗原。方法  根据软件分析选取带有优势抗原表位的HCV核心区多肽,找出对应DNA序列,逆转录PCR扩增目的基因,并将其克隆到原核表达载体中进行诱导表达。表达的目的蛋白纯化后用间接ELISA法检测免疫活性。结果  获得了序列正确的HCV目的基因片段。表达的目的蛋白为可溶性蛋白,能被很好地纯化。使用该纯化蛋白检测各种样本,结果HCV阴性样本的平均吸光度（A）值为0.081,HCV阳性样本与阴性对照的A值之比均>2.1,乙型肝炎和梅毒阳性样本的A值都在0.101以下。结论 成功地克隆和表达了HCV核心区小片段基因,获得了具有良好抗原性的截短HCV核心区多肽。     

利用图像分析技术对基因转导至颌下腺后腺体组织的研究

目的通过图像分析仪研究腺病毒介导荧光素酶基因转导至大白鼠颌下腺后腺体组织结构变化。方法将40只Wistar大鼠随机分为5组,经颌下腺导管转导腺病毒荧光素酶基因重组体即AdCMVLuc,3d,1、2、4、8周后观察其基因表达及病理变化。结果颌下腺转基因表达3d最高,以后逐渐下降,至4、8周时仍可测到表达。组织结构:3d~1周可见腺细胞核面积和周长增大,2周腺细胞核面积和周长开始减小,8周腺细胞核面积和周长基本恢复正常。结论腺病毒介导的大鼠颌下腺基因转导后腺体有明显炎症反应,腺细胞核面积和周长有明显的变化,提示腺体蛋白合成体系功能活跃。     

Potential of microneedle-assisted micro-particle delivery by gene guns: a review

Abstact

Context: Gene guns have been used to deliver deoxyribonucleic acid (DNA) loaded micro-particle and breach the muscle tissue to target cells of interest to achieve gene transfection.

Objective: This article aims to discuss the potential of microneedle (MN) assisted micro-particle delivery from gene guns, with a view to reducing tissue damage.

Methods: Using a range of sources, the main gene guns for micro-particle delivery are reviewed along with the primary features of their technology, e.g. their design configurations, the material selection of the micro-particle, the driving gas type and pressure. Depending on the gene gun system, the achieved penetration depths in the skin are discussed as a function of the gas pressure, the type of the gene gun system and particle size, velocity and density. The concept of MN-assisted micro-particles delivery which consists of three stages (namely, acceleration, separation and decoration stage) is discussed. In this method, solid MNs are inserted into the skin to penetrate the epidermis/dermis layer and create holes for particle injection. Several designs of MN array are discussed and the insertion mechanism is explored, as it determines the feasibility of the MN-based system for particle transfer.

Results: This review suggests that one of the problems of gene guns is that they need high operating pressures, which may result in direct or indirect tissue/cells damage. MNs seem to be a promising method which if combined with the gene guns may reduce the operating pressures for these devices and reduce tissue/cell damages.

Conclusions: There is sufficient potential for MN-assisted particle develivery systems.     

Site directed vascular gene delivery in vivo by ultrasonic destruction of magnetic nanoparticle coated microbubbles

Site specific vascular gene delivery for therapeutic implications is favorable because of reduction of possible side effects. Yet this technology faces numerous hurdles that result in low transfection rates because of suboptimal delivery. Combining ultrasonic microbubble technology with magnetic nanoparticle enhanced gene transfer could make it possible to use the systemic vasculature as the route of application and to magnetically trap these compounds at the target of interest. In this study we show that magnetic nanoparticle-coated microbubbles bind plasmid DNA and successfully deliver it to endothelial cells in vitro and more importantly transport their cargo through the vascular system and specifically deliver it to the vascular wall in vivo at sites where microbubbles are retained by magnetic force and burst by local ultrasound application. This resulted in a significant enhancement in site specific gene delivery compared with the conventional microbubble technique. Thus, this technology may have promising therapeutic potential.From the Clinical EditorThis work focuses on combining ultrasonic microbubble technology with magnetic nanoparticle enhanced gene transfer to enable targeted gene delivery via the systemic vasculature and magnetic trapping of these compounds at the target of interest.