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.     

Measurement of Outflow Facility Using iPerfusion

Elevated intraocular pressure (IOP) is the predominant risk factor for glaucoma, and reducing IOP is the only successful strategy to prevent further glaucomatous vision loss. IOP is determined by the balance between the rates of aqueous humour secretion and outflow, and a pathological reduction in the hydraulic conductance of outflow, known as outflow facility, is responsible for IOP elevation in glaucoma. Mouse models are often used to investigate the mechanisms controlling outflow facility, but the diminutive size of the mouse eye makes measurement of outflow technically challenging. In this study, we present a new approach to measure and analyse outflow facility using iPerfusion^™, which incorporates an actuated pressure reservoir, thermal flow sensor, differential pressure measurement and an automated computerised interface. In enucleated eyes from C57BL/6J mice, the flow-pressure relationship is highly non-linear and is well represented by an empirical power law model that describes the pressure dependence of outflow facility. At zero pressure, the measured flow is indistinguishable from zero, confirming the absence of any significant pressure independent flow in enucleated eyes. Comparison with the commonly used 2-parameter linear outflow model reveals that inappropriate application of a linear fit to a non-linear flow-pressure relationship introduces considerable errors in the estimation of outflow facility and leads to the false impression of pressure-independent outflow. Data from a population of enucleated eyes from C57BL/6J mice show that outflow facility is best described by a lognormal distribution, with 6-fold variability between individuals, but with relatively tight correlation of facility between fellow eyes. iPerfusion represents a platform technology to accurately and robustly characterise the flow-pressure relationship in enucleated mouse eyes for the purpose of glaucoma research and with minor modifications, may be applied in vivo to mice, as well as to eyes from other species or different biofluidic systems.     

Aflatoxin — induced alteration in the levels of membrane chemicals of subcellular organelles isolated from excised,incubated Glycine max,cv. ‘Essex’ roots

Isolates of aflatoxin-producing strains of Aspergillus grow on autoclaved and field-grown (lesser extent) Glycine max beans. Both mixed and aflatoxin B₁ inhibit G. max, cv. Essex bean germination and elongation of either attached or excised cultured roots. Because B₁ impairs the latter roots' ability to intracellularize [¹⁴C]-leucine, it may alter plasmalemma structure and/or function. To determine whether incubation of excised roots for 18 hours in toxin-containing medium could affect cellular membrane chemical content, organelles were isolated by differential centrifugation (1 000, 40 000, and 80 000 xg) of homogenates and characterized chemically. Statistically significant differences between treated and untreated roots in acid insoluble protein but not either sterol or lipid phosphorus levels were observed for both 40,000 and 80,000 xg pellets. Protein and sterol recoveries were 81 (treated) and 84 (untreated) % for the former and 77 (treated) and 79 (untreated) % for the latter. Lipid phosphorus recoveries were 87.3 (treated) and 136 (untreated) % with and 96 (treated) and 83 (untreated) without membrane stabilization. Protein:sterol:lipid phosphorus were 35.74.51 (1 000 xg), 18.93.61 (40000 xg), 26.34.61 (80 000 xg) and 1,010291 (80 000 xg supernatant) for untreated and 36.93.31 (1,000 xg), 23.13.81 (40 000 xg), 36.24.81 (80 000 xg) and 1,05321.71 (80 000 xg supernatant) for treated roots. Significant differences in RNA content between treated and untreated roots were found for both 1 000 and 40 000 xg pellets but not for the 80 000 xg pellet and its supernatant. Whereas a significant increase in the 1 000 xg pellet occurred upon treatment, a decrease was noted for the 40 000 xg pellet but not for the 80 000 xg pellet and its supernatant. Similar pH 6 (plasmalemma marker enzyme) and 9 (mitochondrial marker enzyme) K⁺-stimulated ATPase activities were demonstrated for 40 000 and 80 000 xg pellets. The 1 000 xg pellet contained greater than 50% of the NADH-cytochrome c-reductase activity (endoplasmic reticulum marker enzyme) recovered from fractions examined for this activity which was absent from the 40 000 xg pellet. Both the 80 000 xg pellet and its supernatant possessed equivalent reductase activities. Inosine diphosphatase activity (dictyosome marker enzyme) was not present in 1 000 xg pellets obtained from either treated or untreated roots but was in both 40 000 and 80 000 xg pellets. Based on these results, a tentative assignment of organelles to each fraction (xg force) is reported.Abbreviations used AFB₁ aflatoxin B₁ - AFB₂ aflatoxin, B₂ - AFG₁ aflatoxin G₁ - AFG₂ aflatoxin G₂ - ATPase adenosine triphosphatase - IDPase inosine diphosphatase - NADH reduced nicotinamide-adenine dinucleotide - PCA perchloric acid - TCA trichloroacetic acid - 2, 4-D 2,4-dichlorophenoxyacetic acid Aided by grant IN-127 from the American Cancer Society to WVD and funds from the Departments of Biology, West Virginia University and Virginia Commonwealth University as well as a Sigma Xi award to JMD.     

用rAAV8-1.3HBV制备两种品系小鼠乙型肝炎病毒感染模型的比较研究

我们先前用rAAV8-1.3HBV静脉注射C57BL/6小鼠成功地制备了慢性乙型肝炎病毒(Hepatitis B virus,HBV)感染模型。为了探讨不同品系的小鼠对rAAV8-1.3HBV静脉注射是否具有不同反应,本研究比较了C57BL/6和BALB/c小鼠静脉注射重组病毒后外周血中HBV抗原和抗体水平、病毒载量和肝脏组织HBcAg表达情况,以及不同剂量重组病毒注射与这些指标的关系。将低(4×109 Viral genome,vg)、中(4×1010vg)和高(4×1011vg)三种剂量的rAAV8-1.3HBV通过尾静脉注射至C57BL/6和BALB/c小鼠,分别利用ELISA和荧光定量PCR方法检测血清中的HBV抗原、抗体水平以及HBV DNA,利用免疫组化检测肝脏组织HBcAg的表达。结果发现,对于C57BL/6小鼠,三种不同剂量rAAV8-1.3HBV注射均可造成100%小鼠出现HBV持续感染;血清HBsAg、HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,其表达水平随重组病毒注射剂量的增加而升高,高剂量注射时可造成超过40%的肝细胞感染HBV,血清中HBV DNA可达105 IU/mL以上;未检测到针对HBV的抗体。对于BALB/c小鼠,三种不同剂量rAAV8-1.3HBV注射也可造成100%小鼠出现HBV持续感染;血清HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,但是血清HBsAg在重组病毒注射2周之后显著下降甚至消失;在中剂量注射组的BALB/c小鼠血清中检测到低水平的Anti-HBs;血清HBeAg和肝组织HBcAg的表达水平随重组病毒注射剂量的增加而增高,并且各剂量组表达水平均高于C57BL/6小鼠,高剂量注射时可造成超过50%的肝细胞感染HBV。本研究表明,低至4×109 vg剂量的rAAV8-1.3HBV注射即可造成C57BL/6和BALB/c两种品系小鼠出现HBV持续感染,并且HBV复制水平随重组病毒注射剂量增加而增高;BALB/c小鼠对HBV的免疫反应强于C57BL/6小鼠,可以产生针对HBsAg的体液免疫反应而使血清HBsAg转阴,但无法清除携带HBV的肝细胞。     

Quantitative Addition of Dissolved Oxygen to In Situ Benthic Chamber Systems by Use of Catalase and Hydrogen Peroxide

A methodology for reoxygenation of in situ benthic chamber systems by enzymatic catalysis of hydrogen peroxide with catalase was developed. For a 10-liter benthic chamber, the injection of 1 ml of catalase suspension (26,000 U ml⁻¹) followed by 10 ml of 0.5 M hydrogen peroxide solution resulted in complete reoxygenation within 2.5 min at 25°C.     

Mechanism of action of angiostatic steroids: Suppression of plasminogen activator activity via stimulation of plasminogen activator inhibitor synthesis

Recently, a novel class of angiostatic steroids which block angiogenesis in several systems has been described. Since the elaboration of proteases is believed to be an important component of angiogenesis, we tested whether these steroids blocked the fibrinolytic response of endothelial cells to the angiogenic protein, basic fibroblast growth factor [bFGF]). Cultured bovine aortic endothelial (BAE) cells were incubated with bFGF and/or medroxyprogesterone acetate (MPA), an angio-static steroid which has been shown to inhibit vascularization, collagenolysis, and tumor growth. When bFGF (3 ng/ml) was added to confluent monolayers of BAE cells, plasminogen activator (PA) activity in the medium was increased threefold. In contrast, MPA at 10^?6 M, 10^?7 M, 10^?8 M, and 10^?9 M decreased PA levels in the medium by 83%, 83%, 75%, and 39%, respectively. The stimulation of PA levels in BAE cells by bFGF (3 ng/ml) was abrogated by the presence of 10^?6 M MPA. This decrease in PA activity was found to be mediated by a significant increase in plasminogen activator inhibitor type-1 (PAI-1) production. MPA, therefore, negated one of the important enzymatic activities associated with the angiogenic process. In contrast to the decreased levels of secreted PA in cultures exposed simultaneously to MPA and bFGF, cell-associated PA levels remained high, consistent with earlier observations indicating that PAI-1 does not inhibit cell-associated PA. Thus, angiostatic steroids may exert their inhibitory effects on angiogenesis by increasing the synthesis of PAI-1. This, in turn, inhibits PA activity and, therefore, plasmin generation, which is essential for the invasive aspect of angiogenesis. © 1993 Wiley-Liss, Inc.     

Intrasanguine host-mediated assay with Salmonella typhimurium.

A host-mediated assay in the mouse was tested, in which strains of S. typhimurium (TA 98, TA 1535) were used as indicator organisms and administered intrasanguinally. The bacterial suspension was injected intravenously at a cell density of 10¹¹/ml in a volume of 0.2 ml. The test substances were administered three times at intervals of one hour, orally, intraperitoneally or subcutaneously, the last dose being given immediately before the injection of the indicator organisms. The bacteria were re-isolated one hour later from the liver, and the total bacterial counts and mutation rates were determined. The mutagenic activity of the substances was assessed by reference to the quotients of the mutation rates in the various dosage groups over the control rate. The compounds tested were diethylnitrosamine, cyclophosphamide, dimethylaminoazobenzene, thiotepa and EMS.The bacterial recovery rates in the controls and treated groups ranged from 2.72 to 23.5%, which proved entirely adequate. All the known mutagens tested caused a measurable mutagenic effect in this assay.Comparison of the results with already published data reveals that the intrasanguine host-mediated assay is more sensitive than the intraperitoneal assay system, and that the chosen strains of S. typhimurium are well suited for this method.     

Immunological and molecular characterization of plasminogen activator inhibitors 1 and 2 in baboon (Papio anubis) placental tissues

Two major plasminogen activator inhibitors (PAI-1 and PAI-2) increase in the peripheral circulation during pregnancy in humans. PAI-1 is of vascular endothelial origin whereas PAI-2 is produced primarily by human placental tissues. This study was undertaken to determine a) if PAI-1 and PAI-2 are also present in the baboon and b) their association with pregnancy. Citrated plasma was obtained from pregnant baboons sequentially at 15 +/- 3-day intervals between Days 30 and 140 of pregnancy. PAI activity increased significantly (p less than 0.05) at Day 120 (15.3 IU/ml) and 140 (21.8 IU/ml) of gestation and returned to baseline (2.6 IU/ml) 48 h post cesarean section. Placental tissues obtained at cesarean section during the third trimester were either placed in explant culture, fixed for immunocytochemistry, or frozen for RNA extraction. Western blot analysis of tissue culture media (TCM) indicated that the polyclonal antibody to PAI-1 reacted with a major band (Mr 47 000) in TCM from placental tissues while the PAI-2 antibody reacted primarily with a doublet (Mr 67 000 and 69 000) in these same media. PAI-1 was immunocytochemically localized primarily in the chorioamniotic tissue (CAM-D) and PAI-2 was found predominantly in placental villi. Slot blot hybridization with cDNAs to PAI-1 and PAI-2 indicated that the mRNA for PAI-2 was found primarily in placental villi, whereas the mRNA for PAI-1 was present in all three tissue compartments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rho GTPase-mediated cytoskeletal organization in Schlemm's canal cells play a critical role in the regulation of aqueous humor outflow facility

The increased intraocular pressure (IOP) has been considered to be an increased resistance of the aqueous humor outflow through the inner wall of Schlemm's canal (SC) and/or the juxtacanalicular tissue (JCT). The Rho GTPase-regulated actomyosin organization appears to be an important mechanistic determinant of aqueous humor outflow facility. Therefore, in this study, we have evaluated the effects of modulating Rho GTPase activity on actomyosin cytoskeletal organization, monolayer permeability/barrier function of human SC cells, and aqueous humor outflow facility in enucleated porcine eyes ex vivo. Human SC cells, isolated from cadaver eyes, were treated with either Rho GTPase activators such as thrombin and lysophosphatidic acid (LPA), or a specific inhibitor (C3-exoenzyme) of Rho GTPases. Treatment of SC cells with thrombin and LPA led to increased formation of stress fibers, focal adhesion, and increased myosin light chain phosphorylation, whereas treatment with C3-exoenzyme showed the opposite effects like H-7 and ECA, known for increasing the outflow facility in porcine eyes. The findings presented here suggest that LPA and thrombin, presumably through activation of Rho GTPase-mediated actomyosin cytoskeletal reorganization in SC cells, cause a decrease in monolayer permeability of SC cells as well as a decrease in outflow facility of porcine eyes in ex vivo. Our results suggest that decrease in aqueous humor outflow may be correlated better with the changes in cytoskeletal organizations of SC, which could be the prime locus of the outflow resistance.     

Effects of nine weeks of unloading on neuromuscular activities in adult rats

Effects of 9-week hindlimb suspension and 8-week recovery on locomotor performance and electromyogram (EMG) activities of soleus (Sol), plantaris (Pl), lateral gastrocnemius (LG), and tibialis anterior (TA), were studied in adult rats. Hyperextension of knee and ankle joints, noted after nine weeks of suspension, did not recover during 8-week ambulation. Growth of Sol was fully inhibited by suspension and did not recover completely within 8 weeks of ambulation. The EMG levels in Sol, Pl, and LG 1 day after suspension were 52-76% less than the pre-suspension level (resting on the floor). These activities were recovered to or near the pre-suspension level around 1 week, but decreased again to 10-29% of controls from 7 to 9 weeks. The integrated EMG of TA was elevated during the first week of suspension but then gradually declined to control levels within four weeks. At the end of suspension, the Sol and Pl, not the LG, EMGs remained reduced and the TA EMG remained hyperactive. Co-activation of dorsi- and plantar- flexors occurred often during quadripedal walking following suspension. Such a phenomenon was not observed in the control rats. These phenomena were recovered within 1 week. It is suggested that the unloading-related alterations of neuromuscular activities and/or locomotion, but not the hyperextension of knee and ankle joints, in rats are reversible.     

Feasibility of Histological Scoring and Colony Count for Evaluating Infective Severity in Mouse Vaginal Candidiasis

Qualitative measurement of the infective level is relatively difficult in experimental vaginal candidiasis. Female BALB/c mice aged 8 to 10 weeks were randomly divided into E1, E2 and E0 groups, which received subcutaneous injection of 0.05 mg, 0.1 mg of estradiol benzoate or 0.1 ml soybean oil 3 days before vaginal inoculation, respectively, and hormone treatment continued every other day thereafter. Each group was further divided into infected and noninfected subgroups. The infected mice were inoculated intravaginally with 10 µl (5 × 10⁴ conidia) of Candida albicans suspension, while the noninfected mice were inoculated with 10 µl phosphate-buffered saline. Direct microscopic examination, colony count and vaginal histopathology including infection degree and inflammation extent were performed at 3, 7 and 14 days post inoculation. Estrogen treatment increased the vaginal fungal burden and extent of infection and inflammation compared with the control group, and 0.3 mg/week estrogen generally induced more severe infection and inflammation than 0.15 mg/week estrogen did. Colony count peaked on day 3 and decreased remarkably after 7 days. Infection score increased gradually during the first 7 days and decreased on day 14, while inflammation extent exacerbated progressively over the course of 14 days. This study demonstrates that the modified histological scoring system might be more feasible than colony count for evaluation of infectivity and dynamic change in experimental vaginal candidiasis.     

Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 × 10⁵ cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 × 10⁷ cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.     

氧诱导视网膜病模型中曲安奈德抑制新生血管生长的机制

目的观察氧诱导视网膜病模型(OIR)中曲安奈德(TA)对CD14+细胞聚集及VEGF表达的干预作用,初步探讨曲安奈德抑制视网膜新生血管生长的可能机制。方法清洁级C57BL/6哺乳期小鼠36只(共36个眼球),随机将其分为四组:①正常对照组(6个眼球),6只17日龄正常小鼠先行FFA眼底造影,然后每鼠随机摘取一个眼球,行眼球石蜡切片HE染色。②单纯高氧组(6个眼球),6只17日龄OIR模型小鼠处理同单纯对照组。③TA高氧组(12个眼球),12只12日龄OIR模型小鼠随机一眼注射TA 2μL,再于17日龄后行FFA眼底造影后摘除术眼,其中6个眼球行眼球石蜡切片HE染色及视网膜CD14和VEGF免疫组化染色,另6个眼球行视网膜VEGF mRNA的real-time PCR检测。④BSS高氧(12个眼球),12只12日龄OIR模型小鼠随机一眼注射BSS 2μL,余处理同TA高氧组。用t检验两两比较各组视网膜突破内界膜的内皮细胞核数,视网膜CD14与VEGF免疫组化染色的平均吸光度值(IOD/AOI),及VEGF mRNA的相对含量值(2-ΔCt×105)。结果与正常对照组相比,高氧诱导组突破视网膜内界膜的内皮细胞核数...     

Synergism therapeutic and immunoregulatory effects of Albendazole + rAd-mIL-28B against Echinococcosis in experiment-infected mice with protoscoleces

The metacestode stage of Echinococcus granulosus can cause cystic echinococcosis (CE), which still widely occurs around the world. Since the early 1970s, benzimidazoles have been shown to inhibit the growth of cysts and used to treat CE. However, benzimidazoles are still ineffective in 20%-40% of cases. In order to explore the new agents against CE, we have investigated the therapeutic effect of the recombinant adenoviral vector expressing mouse IL-28B (rAd-mIL-28B) on protoscoleces-infected mice. In our study, we successfully established the model mice which infected with protoscoleces intraperitoneally. At 18 weeks post-infection, the mice received rAd-mIL-28B (1×10⁷ PFU) weekly by intramuscular injection for 6 weeks. Compared with the untreated control (13.1 ± 2.2 g), there was a significant reduction in cysts wet weight in rAd-mIL-28B group (8.3 ± 3.5 g) (P < 0.05), especially in Albendazole (ABZ) + rAd-mIL-28B group (5.8 ± 1.4 g) (P < 0.01). We also observed the severe damage of the germinal layer and the laminated layer of cysts after treatment. rAd-mIL-28B group showed a prominent increase in the level of Th1 type cytokines (such as IFN-γ, IL-2 and TNF-α). Meanwhile, the frequency of Foxp3⁺ T cells was decreased in the rAd-mIL-28B group (4.83 ± 0.81%) and ABZ + rAd-mIL-28B group (4.60 ± 0.51%), comparing with the untreated group (8.13 ± 2.60%) (P < 0.05). In addition, compared with the untreated control (122.14 ± 81.09 pg/ml), the level of IFN-γ significantly increased in peritoneal fluid in the rAd-mIL-28B group (628.87 ± 467.16 pg/ml) (P < 0.05) and ABZ + rAd-mIL-28B group (999.76 ± 587.60 pg/ml) (P < 0.001). Taken together, it suggested that ABZ + IL-28B may be a potential therapeutic agent against CE.     

Ocular Localization and Transduction by Adenoviral Vectors Are Serotype-Dependent and Can Be Modified by Inclusion of RGD Fiber Modifications

Purpose

To evaluate localization and transgene expression from adenoviral vector of serotypes 5, 35, and 28, ± an RGD motif in the fiber following intravitreal or subretinal administration.

Methods

Ocular transduction by adenoviral vector serotypes ± RGD was studied in the eyes of mice receiving an intravitreous or subretinal injection. Each serotype expressed a CMV-GFP expression cassette and histological sections of eyes were examined. Transgene expression levels were examined using luciferase (Luc) regulated by the CMV promoter.

Results

GFP localization studies revealed that serotypes 5 and 28 given intravitreously transduced corneal endothelial, trabecular, and iris cells. Intravitreous delivery of the unmodified Ad35 serotype transduced only trabecular meshwork cells, but, the modification of the RGD motif into the fiber of the Ad35 viral vector base expanded transduction to corneal endothelial and iris cells. Incorporation of the RGD motif into the fiber knob with deletion of RGD from the penton base did not affect the transduction ability of the Ad5 vector base. Subretinal studies showed that RGD in the Ad5 knob shifted transduction from RPE cells to photoreceptor cells. Using a CMV-Luc expression cassette, intravitreous delivery of all the tested vectors, such as Ad5-, Ad35- and Ad28- resulted in an initial rapid induction of luciferase activity that thereafter declined. Subretinal administration of vectors showed a marked difference in transgene activity. Ad35-Luc gene expression peaked at 7 days and remained elevated for 6 months. Ad28-Luc expression was high after 1 day and remained sustained for one month.

Conclusions

Different adenoviral vector serotypes ± modifications transduce different cells within the eye. Transgene expression can be brief or extended and is serotype and delivery route dependent. Thus, adenoviral vectors provide a versatile platform for the delivery of therapeutic agents for ocular diseases.     

Triamcinolone acetonide protects the rat retina from STZ-induced acute inflammation and early vascular leakage

Streptozotocin (STZ) has been commonly used to induce in vivo and in vitro hyperglycemic diabetes and its toxicity leads to inflammation and vascular injury. Triamcinolone acetonide (TA), as an anti-angiogenic/anti-inflammatory drug, is clinically used to improve the visual acuity in neovascular and edematous ocular diseases. The aim of this study was to investigate the effect of TA on early inflammation and vascular leakage in the retina of STZ-induced hyperglycemic rats. Hyperglycemia was induced in 8-week-old male Sprague-Dawley (SD) rats by a single intraperitoneal injection of STZ (65 mg/kg); only rats with blood glucose levels >13.9 mmol/l 1 day after STZ injection were included in STZ-hyperglycemic group. Sex- and age-matched SD rats injected with buffer were used as the control group. One day before STZ and buffer injection, 2 microl TA (4 mg/ml in saline) and 2 microl saline were intravitreal-injected into the right and the left eyes of rats, respectively. Retinal vascular leakage was measured using the Evans-blue method. Changes in pro-inflammatory target genes, such as tumor necrotic factor (TNF)-alpha, intracellular adhesion molecule (ICAM)-1, and vascular endothelial growth factor (VEGF) were assessed by immunoblottings, immunostaining, and ELISA analyses. Vascular hyperleakage and up-regulation of most pro-inflammatory genes peaked within a few days after STZ injection and had recovered. However, these changes were blocked by TA pretreatment. Our data suggest that TA controls STZ-induced early vascular leakage and temporary pro-inflammatory signals in the rat retina.     

Evaluation of diallate and triallate herbicides for genotoxic effects in a battery of in vitro and short-term in vivo tests

Commercial-grade preparations of two thiocarbamate herbicides, diallate and triallate, were evaluated for their mutagenic potential in a battery of short-term bioassays. All in vitro bioassays were performed with and without mammalian metabolic activation, and all such tests were repeated after an interval of at least 1 week. Diallate and triallate were tested in the Salmonella/microsome assay over dose ranges of 0.59 to 118.0 micrograms/plate and 6.37 to 1273 micrograms/plate, respectively. Both diallate and triallate gave positive results in S. typhimurium strains TA1535, TA98, and TA100 only in the presence of a rat-liver metabolic activation system. In Saccharomyces cerevisiae strain D7, diallate was tested at concentrations from 1.18 to 29.50 micrograms/ml, and triallate was tested at 0.955 to 9.548 micrograms/ml. Both diallate and triallate gave negative results for mitotic gene conversion, mitotic crossing-over, and reverse mutation. In the mouse lymphoma L5178Y TK+/- assay, diallate was tested at concentrations ranging from 1 to 72 micrograms/ml, and triallate was tested at 0.5 to 60 micrograms/ml. Both herbicides produced mutagenic responses in the mouse lymphoma assay in the presence of metabolic activation. In the Drosophila sex-linked recessive lethal test, flies were exposed to 0.0004% diallate and 0.001% triallate. In this assay, diallate was considered mutagenic, whereas triallate did not produce a detectable mutagenic response.     

S1P₂ receptor regulation of sphingosine-1-phosphate effects on conventional outflow physiology

Elevated intraocular pressure is the main risk factor in primary open-angle glaucoma, involving an increased resistance to aqueous humor outflow in the juxtacanalicular region of the conventional outflow pathway which includes the trabecular meshwork (TM) and the inner wall of Schlemm's canal (SC). Previously, sphingosine-1-phosphate (S1P) was shown to decrease outflow facility in porcine and human eyes, thus increasing outflow resistance and intraocular pressure. Owing to S1P's known effect of increasing barrier function in endothelial cells and the robust expression of the S1P? receptor on the inner wall of SC, we hypothesized that S1P? receptor activation promotes junction formation and decreases outflow facility. The effects of subtype-specific S1P receptor compounds were tested in human and porcine whole-eye perfusions and human primary cultures of SC and TM cells to determine the receptor responsible for S1P effects on outflow resistance. The S1P?-specific agonist SEW2871 failed to both mimic S1P effects in paired human eye perfusions, as well as increase myosin light chain (MLC) phosphorylation in cell culture, a prominent outcome in S1P-treated SC and TM cells. In contrast, the S1P? antagonist JTE-013, but not the S1P? or S1P?,? antagonists, blocked the S1P-promoted increase in MLC phosphorylation. Moreover, JTE-013 prevented S1P-induced decrease in outflow facility in perfused human eyes (P < 0.05, n = 6 pairs). Similarly, porcine eyes perfused with JTE-013 + S1P did not differ from eyes with JTE-013 alone (P = 0.53, n = 3). These results demonstrate that S1P? , and not S1P? or S1P?, receptor activation increases conventional outflow resistance and is a potential target to regulate intraocular pressure.     

比较不同血清型AAV携带HBV基因组建立乙肝小鼠模型效果

近年来,用8型腺相关病毒携带1.3拷贝HBV(Hepatitis B virus)基因组建立的HBV持续感染小鼠模型受到越来越多的关注。本研究比较了除AAV8之外的其他4种血清型重组腺相关病毒(Recombinant adeno-associated virus,rAAV)建立乙肝小鼠模型效果。首先,将携带1.3拷贝ayw亚型HBV基因组的1型、2型、5型、8型、9型腺相关病毒分别以1×10~(11) vg/只(Viral genome,vg)的剂量尾静脉注射C57BL/6J小鼠;利用ELISA方法监测小鼠血清中HBeAg和HBsAg表达水平;用定量PCR方法检测小鼠血清和肝脏中HBV DNA拷贝数;用免疫组化方法检测小鼠肝脏中HBc Ag的表达;用HE染色检测小鼠肝脏病理变化。结果显示,在持续8周中,5组小鼠血清中都检测到HBeAg和HBsAg的表达,血清和肝脏中均检测到HBV DNA的存在。HBeAg、HBsAg、HBV DNA表达水平高低依次为AAV8AAV9AAV1AAV5AAV2。5组小鼠用免疫组化方法都检测到肝脏中HBcAg表达,HE染色病理检测均观察到不同程度的肝损伤。本研究扩大了能用于建立乙肝小鼠持续感染模型可选择的AAV载体种类,发现虽然AAV1、2、5、9的建模效果不如AAV8,但它们都可以介导建立持续感染的乙肝小鼠模型,建模效果依次为AAV8AAV9AAV1AAV5AAV2。其中AAV9介导的建模效果与AAV8载体最为接近,可以替代AAV8载体用于有效地建立HBV持续感染的小鼠模型。