Delivery of antisense oligonucleotide to the cornea by iontophoresis

We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the design of specific antiangiogenic strategy in diseases of the cornea.     

Effects of intravitreous sodium hydrosulfide on intraocular pressure and retinopathy in ocular hypertensive rats

We investigated the possible neuroprotectant and intraocular pressure (IOP) lowering effects of intravitreous injection of sodium hydrosulfide (NaSH) in a rodent model of experimental glaucoma. Glaucoma currently is treated by controlling IOP using medications and/or surgery. These methods are not entirely adequate for all patients. We divided 24 rats into three groups. For the control group, the right eye was treated with intravitreous saline. For the glaucoma group, ocular hypertension was induced by photocoagulating three episcleral veins and the limbal plexus of the right eye using an argon laser, then saline was injected into the vitreous of these eyes during the third week. For the NaSH group, rats were treated with intravenous NaSH 3 weeks after photocoagulation. IOP was measured each week during the 6 week experimental period. Coagulating the episcleral veins rapidly increased the IOP of rat eyes. Intravitreous injection of NaSH significantly reduced IOP. Intravitreous NaSH prevented degeneration of the retina and decreased the number of apoptotic cells. Intravitreous NaSH appeared to reduce IOP and to prevent IOP induced retinopathy in rats.     

汉防己甲素滴眼液对高眼压模型大鼠的降眼压作用

目的比较观察汉防己甲素滴眼液与0.5%噻吗心安滴眼液对高眼压模型大鼠及正常大鼠降眼压的作用。方法正常SD大鼠共分4组：不同浓度的汉防己甲素滴眼液组（0.1%、0.2%、0.3%）及阳性对照组0.5%噻吗心安,药物滴右眼各一滴,阴性对照组生理盐水滴左眼、测量滴药前24h和滴药后1、3、6、24、48、72h的眼压。应用倍频532激光对SD大鼠右眼上巩膜静脉以及小梁网所在区域实施光凝术建立高眼压大鼠模型。高眼压模型鼠共分5组：不同浓度的汉防己甲素滴眼液0.05%、0.1%、0.2%、0.3%及阳性对照组0.5%噻吗心安,右眼即模型眼滴用药物,左眼作为空白对照。测量术前后的眼压。结果汉防己甲素滴眼液对大鼠正常眼压无降压作用（P〉0.05）。对高眼压大鼠用药后24h、72h、1周后,0.3%汉防己甲素滴眼液组降低眼压的幅度与0.5%噻吗心安滴眼液降低眼压的幅度相似（P〉0.05）;0.05%、0.1%、0.2%汉防己甲素滴眼液组也有明显的降压作用,但与0.5%噻吗心安滴眼液相比,降压幅度低于后者（P〈0.05）。结论0.05%、0.1%、0.2%、0.3%汉防己甲素滴眼液均有降低大鼠高眼压的作用,其中0.3%浓度的汉防己甲素滴眼液降眼压效果与0.5%的噻吗心安类似。汉防已甲素滴眼液作为一种治疗青光眼的药物有着良好应用前景。     

Knockdown of caveolin-1 by antisense oligonucleotides impairs angiogenesis in vitro and in vivo

Knock-out of the gene coding for caveolin-1, the main organizer of caveolae, has not yet been performed. We devised a strategy to knock-down caveolin-1 gene expression using antisense oligodeoxynucleotides (ODNs). Seven ODNs, covering different regions of caveolin-1 mRNA, were screened by Western blot analysis of caveolin-1 levels. The most active and specific was found to reduce caveolin-1 protein levels by 70% at 1 microM concentration and its action, as demonstrated by a marked reduction (about 50%) in caveolin-1 mRNA levels, was due to a true antisense mechanism. In HUVEC treated with the active ODN, caveolae were undetectable by confocal and electron microscopy, while their number was not affected when cells were treated with a scrambled ODN. Using the fibrin gel 3 D angiogenesis test we established that the active (but not the scrambled) ODN strongly suppressed capillary-like tube formation. Moreover, an antisense tailored against chicken caveolin-1 mRNA, when tested using the chorio-allantoic membrane technique, dramatically reduced vessel formation at doses (10-20 microg) under which control ODNs were ineffective and devoid of toxicity. Thus, it is likely that caveolin-1 down regulation, followed by caveolae disruption, impairs angiogenesis in vitro and in vivo.     

Antisense oligonucleotide inhibition of angiotensinogen in the brains of rats and sheep

Phosphorothioated antisense oligodeoxynucleotides (ODNs) that were complementary to various parts of the rat or sheep mRNA encoding angiotensinogen were synthesized by conventional techniques. Their effectiveness as blockers of angiotensinogen synthesis in the brain was tested by bioassay. This involved measuring the effect of centrally administered antisense ODNs on water drinking that occurred in response to intracerebroventricular injection of hog renin. Renin-induced drinking requires brain angiotensinogen for the generation of angiotensin I and then angiotensin II to stimulate thirst. Intracerebroventricular injection of an 18-mer antisense ODN (0.5 microg twice in 24 h) complementary to the 5'-end start codon for rat angiotensinogen mRNA caused a pronounced inhibition of renin-induced drinking. This effect appeared to be specific for this region of the codon because antisense ODNs directed against other regions of rat angiotensinogen mRNA were ineffective, and renin-induced drinking was not inhibited by intracerebroventricular injection of scrambled or mismatched sequences of the effective ODN or by intraperitoneal injection of it. Intracerebroventricular injection of antisense ODN (0.5 microg twice in 24 h) did not inhibit appetite or affect water drinking in response to some other dipsogenic stimuli, thus demonstrating the specificity of its action against renin-induced drinking. By contrast, intracerebroventricular administration of 625 microg of an antisense ODN directed against the corresponding 5'-end start codon region of sheep angiotensinogen mRNA did not inhibit intracerebroventricular renin-induced drinking in sheep. These data show that while intracerebroventricularly administered antisense may be used effectively in rodents, the method is not necessarily applicable in larger mammals.     

珍珠明目液消炎作用观察

目的观察珍珠明目液对眼结膜刺激性及其对实验性兔眼部炎症的影响.方法给兔眼眶内滴入珍珠明目液(每次0.1ml/眼,1天3次,连续7天),观察给药7天后角膜、虹膜及结膜的反应;用30%巴豆油造成兔眼结膜炎,致炎前及致炎后局部应用珍珠明目液滴眼(0.1ml/眼),并设醋酸可的松眼药水治疗组和生理盐水对照组.于致炎后2、4、12、24、36、48及72h,根据结膜炎充血、水肿及分泌物轻重程度进行评分.结果珍珠明目液对兔眼角膜、虹膜及结膜未见明显异常反应.珍珠明目液治疗30%巴豆油所致兔眼炎症评分值显著低于相应生理盐水组(P＜0.01),与醋酸可的松治疗组比较无显著性差异.结论珍珠明目液无眼刺激性,对巴豆油致兔眼结膜炎具有较好的消炎作用.     

p70s6 kinase is a functional target of insulin activated Akt cell-survival signaling

Insulin administration attenuates cardiac ischemia-reperfusion apoptosis via activation of Akt-mediated cell-survival signaling. As p70s6 kinase is a cognate Akt-mediated phosphorylation target we evaluated whether p70s6 kinase activation is a functional requirement in insulin-mediated cell survival program during post-ischemic reoxygenation. Human cardiac-derived girardi cells were subjected to 6h of simulated ischemia and 2h of reoxygenation+/-insulin treatment [0.3mU/ml]. Concurrently, cells were pre-treated with anti-sense oligodeoxynucleotides (ODNs) corresponding to the initiation start-site of human p70s6 kinase mRNA. Sense ODN and scrambled ODN were used as controls. Cell viability was measured using lactate dehydrogenase (LDH) release and propidium iodide (PI) exclusion. Insulin at reoxygenation enhanced cell viability with attenuated LDH release (>or=50% , p<0.001 vs. ischemic controls) and reduced PI uptake by >or=30% vs. ischemic controls. The protection afforded by insulin was abolished by anti-sense ODN targeting p70s6 kinase, but not by the sense or scrambled ODNs. In parallel, insulin administration at reoxygenation significantly increased p70s6 kinase levels and activity compared with controls. P70s6 kinase activity was abolished by pre-treatment with anti-sense ODNs. Collectively, these data demonstrate that p70s6 kinase activation is a functional target of Akt following insulin-activated cytoprotection during ischemia-reoxygenation-induced injury.     

Suppression of complement regulatory proteins (CRPs) exacerbates experimental autoimmune anterior uveitis (EAAU)

This study was undertaken to explore the role of complement regulatory proteins (CRPs) in experimental autoimmune anterior uveitis (EAAU). We observed that the levels of CRPs, Crry and CD59, in the eyes of Lewis rats increased during EAAU and remained elevated when the disease resolved. The in vivo role of these CRPs in EAAU was explored using neutralizing mAbs, antisense oligodeoxynucleotides (AS-ODNs), and small interfering RNAs against rat Crry and CD59. Suppression of Crry in vivo at days 9, 14, or 19 by neutralizing mAb or AS-ODNs resulted in the early onset of disease, the exacerbation of intraocular inflammation, and delayed resolution. Suppression of CD59 was only effective when the Abs and ODNs were given before the onset of disease. The most profound effect on the disease was observed when a mixture of Crry and CD59 mAbs or AS-ODNs was administered. A similar effect was observed with a combination of Crry and CD59 small interfering RNA. There was no permanent histologic damage to ocular tissue after the inflammation cleared in these animals. Increased complement activation as determined by increased deposition of C3, C3 activation fragments, and membrane attack complex was observed in the eyes of Lewis rats when the function and/or expression of Crry and CD59 was suppressed. Thus, our results suggest that various ocular tissues up-regulate the expression of Crry and CD59 to avoid self-injury during autoimmune uveitis and that these CRPs play an active role in the resolution of EAAU by down-regulating complement activation in vivo.     

Sweet delivery - sugar translocators as ports of entry for antisense oligodeoxynucleotides in plant cells

Antisense oligodeoxynucleotides (ODNs) are short (12-25 nt long) stretches of single-stranded DNA that may be delivered to a cell, where they hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. Here we used confocal microscopy to monitor the uptake and trafficking of ODNs in barley tissues. We conclude that uptake of ODNs across the plant plasma membrane is mediated by active transport of mono- or disaccharides through sugar translocators. We demonstrate that sugar transport can deliver ODNs to barley seeds, and that this strategy may be employed to suppress gene activity in endosperm cells by antisense ODN inhibition. We further found that sucrose compared favorably with oligofectamine as a vehicle for ODN delivery to human cells in a low-serum environment.     

Effects of antisense oligodeoxynucleotides against opioid receptors on the receptor mRNA contents

It was demonstrated in the previous study that the microinjection of antisense oligodeoxynucleotide (AS ODN) against mu-opioid receptor (MOR) into periaqueductal gray (PAG) of rat brain selectively decreased the MOR mRNA content in PAG, and the decrease in MOR mRNA content was enhanced by pretreatment of the PAG with MOR AS ODN. In the present investigation, effects of the pretreatment of PAG with AS ODN against kappa- or delta-opioid receptor (KOR or DOR) on the decrease in the MOR mRNA content induced by MOR AS ODN were examined. Both KOR and DOR AS ODNs significantly decreased the target mRNA contents, while they did not significantly change MOR mRNA content. The decrease in MOR mRNA content induced by MOR AS ODN, however, was significantly enhanced by the pretreatment of PAG with either KOR or DOR AS ODNs. Results show that the AS ODN has both the specific target mRNA decreasing action and the nonspecific enhancing action on the AS-induced decrease in the mRNA content.     

The effect of two CpG oligodeoxynucleotides with different sequences on haemocytic immune responses of giant freshwater prawn,Macrobrachium rosenbergii

A previous study has demonstrated that the intrahaemocytic phenoloxidase (PO) activity of the prawn Macrobrachium rosenbergii was enhanced by CpG oligodeoxynucleotide (ODN) 2006, but not by os-ODN13. The study described herein determined the binding characteristics of the two ODNs to haemocytes. Treating haemocytes with FAM-ODN or FAM-ODN plus different concentrations of the same ODN revealed that both ODNs specifically bound to haemocytes. Results from haemocytes treated with FAM-ODN2006 plus ROX-os-ODN13 in a competitive assay indicated that about 91% of haemocytes were simultaneously bound by the two ODNs and that the remaining haemocytes were only bound by ODN2006; moreover, ODN2006 binding to haemocytes was stronger than that of os-ODN13. To clarify the interactive effect of the two ODNs on haemocytic function, mRNA levels of haemocytic genes from single or double ODN-injected prawns were determined by semi-quantitative RT-PCR. The expression of either the prophenoloxidase (proPO) or peroxinectin (pon) genes was elevated by ODN2006 but reduced by os-ODN13; furthermore, ODN2006-increased proPO expression was abated following treatment with os-ODN13. In comparison with ODN-injected prawns alone, the NF-κB inhibitor PDTC reduced the proPO mRNA levels induced by ODN2006, but it elevated proPO expression inhibited by os-ODN13. These results support the notion that os-ODN13 may be able to neutralise or negate the enhancing effect of ODN2006 on proPO expression via the NF-κB signalling pathway as well as an unknown signalling pathway.     

Anti-inflammatory effect of the alpha-melanocyte stimulating hormone in animal eyes undergoing extracapsular lens extraction

In this study anti-inflammatory effects of the alpha-melanocyte stimulating hormone (alpha-MSH) on ocular inflammation caused by extracapsular lens extraction (ECLE) have been investigated and the potential mechanism of an anti-inflammatory effect is discussed. Pigmented rabbit eyes after ECLE were treated locally with alpha-MSH, dexamethasone, diclofenac, or saline 4 times a day (q.i.d.) for 4 weeks. The inhibitory effect of alpha-MSH on infiltrating cells in the aqueous humor (AqH) was almost twice as good as that of dexamethasone or diclofenac for 3 days, 1 week, and 2 weeks after the operation. The eyes of Sprague-Dawley rats were treated with an intravenous injection of alpha-MSH or saline immediately after ECLE. Six hours postoperatively, the iris/ciliary body exhibited increased expression of TNF-alpha and IL-6 mRNAs, which were significantly decreased after alpha-MSH treatment. The number of activated NF-kappa B (NFkappaB)-positive cells in the iris/ciliary body was also significantly reduced by the alpha-MSH treatment. These results suggested that alpha-MSH could effectively reduce ocular inflammation after ECLE, and the potential mechanism for this is by down-regulating the expression of proinflammatory cytokines and inhibiting the NFkappaB-dependent signaling pathway.     

Differential distribution of oligodeoxynucleotides in developing organs with epithelial-mesenchymal interactions.

The use of antisense oligodeoxynucleotides (ODNs) to inhibit gene expression is a usefull method for determining protein function and has potential therapeutic applications. However, there is still great variability in the successfull application of antisense technology to individual systems. In order to assess the ability of different cell types to take up ODNs, developing embryonic tissues were cultured in vitro in the presence of fluoresceine labelled, phosphorothioate substituted ODNs. The distribution of ODNs in individual cell populations was assayed by fluorescent microscopy and the tissue sections were counterstained for epithelial basement membrane formation. High intracellular levels of ODNs were observed in all mesenchymal cells of the lung, salivary gland, kidney, ovary and testis. However, a significant decrease in ODN levels was observed with the formation of new epithelium in kidney and gonads, whereas mature epithelial cells in all tissues had no detecable levels of ODNs. The ability to inhibit gene expression in mesenchymal cells, but not in epithelial cells, was consistent with the distribution pattern of labeled ODNs. These results may indicate a general resistance of epithelial cells to take up ODNs in culture and bear directly on the ability of ODNs to affect gene expression in complex organs with epithelial components.     

Intraocular gene transfer of pigment epithelium-derived factor rescues photoreceptors from light-induced cell death

In this study, we investigated whether intraocular gene transfer of pigment epithelium-derived factor (PEDF) ameliorates the extent of light-induced photoreceptor cell death. Lewis rats received intravitreous injection of 3 x 10(9) particles of adenoviral vector expressing PEDF (AdPEDF.11) in one eye and 3 x 10(9) particles of empty adenoviral vector (AdNull.11) in the contralateral eye. The rats were then dark-adapted for 3 days after which they were continuously exposed to fluorescent light (2,500 lux) for 0, 6, 24, 96, and 168 h. Both eyes were then enucleated and processed for morphometric analysis. Cell death in the retina was examined using TUNEL staining with a propidium iodide counterstain. The photoreceptor cell counts in each of the three groups were significantly different (P < 0.001). Eyes that received intravitreous injection of AdNull.11 or no injection showed a greater number of pyknotic photoreceptor cells and a reduced photoreceptor cell density as compared to eyes treated with intravitreous AdPEDF.11 injection. AdNull.11 treated eyes showed a lesser but still significant protection of photoreceptor cells when compared to untreated eyes. Fewer TUNEL-positive photoreceptor cells were present in AdPEDF.11 treated eyes than in AdNull.11 treated or untreated eyes (P = 0.004). The amplitudes of the ERG a-wave, b-wave, and oscillatory potentials (OPs) were increased significantly by treatment (P < 0.05). These data suggest that adenovirus vector-mediated intraocular expression of PEDF significantly increases photoreceptor cell survival following excessive light exposure. Neuroprotection may result from inhibition of light-induced apoptotic processes. This study provides proof of concept for a gene transfer approach to modulating retinal cell death resulting from photo-oxidative damage and supports the hypothesis that gene transfer of PEDF is broadly applicable to modulating apoptosis in the retina.     

CpG oligodeoxynucleotides discriminately enhance binding capacity of human naïve B cells to Hepatitis B virus epitopes

CpG oligodeoxynucleotides (CpG ODN) have the potential to enhance the antigen-presenting cells function of human na?ve B cells. In this study, we aim to define the effect of CpG ODNs on the binding capacity of human na?ve B cells for different Hepatitis B virus (HBV) epitopes. Three HLA-A2 restricted epitopes were selected to incubate with CpG ODN-primed human na?ve B cells. Binding capacity for each epitope and expression of CD80, CD86, class I major histocompatibility complex (MHC), and class II MHC of na?ve B cells was tested, respectively, by flow cytometry. CpG ODNs, especially ODN 2216, enhanced the binding capacity of human na?ve B cells for HBV epitopes (p < 0.01), and induced markedly higher expression of CD80, CD86, class I MHC, and class II MHC. The binding capacity of CpG-treated naive B cells for each epitope was significantly different. In all the 3 subjects, CpG ODN 2216-primed na?ve B cells showed the highest binding ability for Env172-180 compared with the other epitopes with a high expression of co-stimulatory and MHC molecules. CpG ODN showed the potential to selectively enhance the binding capacity of human na?ve B cells for HBV epitopes. These results suggest new strategies for development of vaccine design.     

Efficacy of the antioxidant ebselen in experimental uveitis.

Inflammation results in the production of free radicals. In a model of experimental uveitis upon subcutaneous injection of endotoxin to Lewis rats, i.e., endotoxin-induced experimental uveitis (EIU), we have evaluated the status of the antioxidant capacity of ocular tissues. EIU results in a decrease of glutathione (GSH) content and glutathione peroxidase (GPx) activity in whole eye homogenates 24-h after endotoxin administration. Furthermore, an increase in malondialdehyde (MDA) content was observed in these same samples, thus confirming the involvement of oxidative stress in the pathophysiology of the process. In view of the ability of the antioxidant ebselen as GPx enzyme mimic, we tested the effect of the oral treatment with two doses of 100 mg/kg body weight of ebselen (first dose administered at the same time of endotoxin, and the second after 12 h). Ebselen administration normalized the GSH and MDA contents and protected the GPx activity of the EIU rat eyes. The GPx activity in the eye homogenate of the treated rats could be completely acounted for by the ebselen-dependent GPx-like activity, i.e., GPx activity measured in the acidic supernatant of the homogenate after neutralization. Unmodified ebselen was detected in whole eye homogenates, thus it shows for the first time the penetration of ebselen through the blood-aqueous and blood-retina barrier. The results herein may allow the proposal of ebselen as a suitable antiinflammatory agent in ocular tissues.