Changes of Ocular Surface and the Inflammatory Response in a Rabbit Model of Short-Term Exposure Keratopathy

Purpose

To evaluate the ocular surface change and the inflammatory response in a rabbit model of short-term exposure keratopathy.

Methods

Short term exposure keratopathy by continuous eyelid opening was induced in New Zealand white rabbits for up to 4 hours. Ultrasound pachymetry was used to detect central total corneal thickness. In vivo confocal microscopy and impression cytology were performed to evaluate the morphology of ocular surface epithelium and the infiltration of inflammatory cells. Immunohistochemistry for macrophage,neutrophil, CD4(+) T cells, and CD8(+) T cells were performed to classify the inflammatory cells. Scanning electron microscopy(SEM) was performed to detect ocular surface change.The concentrations of IL-8, IL-17, Line and TNF-αwere analyzed by multiplex immunobead assay. TUNEL staining was performed to detect cellular apoptosis.

Results

Significant decrease ofcentral total cornealthickness were found within the first 5 minutes and remained stable thereafter, while there were no changes of corneal epithelial thickness.No significant change of corneal, limbal and conjunctival epithelial morphology was found by in vivo confocal microscopy except the time dependent increase of superficial cellular defects in the central cornea. Impression cytology also demonstrated time dependent increase of sloughing superficial cells of the central cornea. Aggregations ofinflammatory cells were found at 1 hour in the limbal epithelium, 2 hours in the perilimbal conjunctival epithelium, and 3 hours in the peripheral corneal epithelium.In eyes receiving exposure for 4 hours, the infiltration of the inflammatory cells can still be detected at 8 hours after closing eyes.Immunohistochemical study demonstrated the cells to be macrophages, neutrophils, CD4-T cells and CD-8 T cells.SEM demonstrated time-depending increase of intercellular border and sloughing of superficial epithelial cells in corneal surface. Time dependent increase of IL-8, IL-17 and TNF-α in tear was found.TUNEL staining revealed some apoptotic cells in the corneal epithelium and superficial stroma at 3 hours after exposure.

Conclusions

Short term exposure keratopathy can cause significant changes to the ocular surface and inflammatory response. Decrease of central total corneal thickness, aggregation of inflammatory cells, and cornea epithelial cell and superficial keratocyte apoptosis were found no less than 4 hours following the insult.     

Clinical Features of Reported Ethylene Glycol Exposures in the United States

Background

Ethylene glycol is highly toxic and represents an important cause of poisonings worldwide. Toxicity can result in central nervous system dysfunction, cardiovascular compromise, elevated anion gap metabolic acidosis and acute kidney injury. Many states have passed laws requiring addition of the bittering agent, denatonium benzoate, to ethylene glycol solutions to reduce severity of exposures. The objectives of this study were to identify differences between unintentional and intentional exposures and to evaluate the utility of denatonium benzoate as a deterrent.

Methods and Findings

Using the National Poison Data System, we performed a retrospective analysis of reported cases of ethylene glycol exposures from January 2006 to December 2013. Outcome classification was summed for intentionality and used as a basis for comparison of effect groups. There were 45,097 cases of ethylene glycol exposures resulting in 154 deaths. Individuals more likely to experience major effects or death were older, male, and presented with more severe symptoms requiring higher levels of care. Latitude and season did not correlate with increased exposures; however, there were more exposures in rural areas. Denatonium benzoate use appeared to have no effect on exposure severity or number.

Conclusion

Deaths due to ethylene glycol exposure were uncommon; however, there were major clinical effects and more exposures in rural areas. Addition of denatonium benzoate was not associated with a reduction in exposures. Alternative means to deter ingestion are needed. These findings suggest the need to consider replacing ethylene glycol with alternative and less toxic agents.     

Rho kinase inhibitor Y-27632 and Accutase dramatically increase mouse embryonic stem cell derivation

Although it has been 30 yr since the development of derivation methods for mouse embryonic stem (ES) cells, the biology of derivation of ES cells is poorly understood and the efficiency varies dramatically between cell lines. Recently, the Rho kinase inhibitor Y-27632 and the cell dissociation reagent Accutase were reported to significantly inhibit apoptosis of human ES cells during passaging. Therefore, in the current study, C57BL/6×129/Sv mouse blastocysts were used to evaluate the effect of the combination of the two reagents instead of using the conventional 129 line in mouse ES cell derivation. The data presented in this study suggests that the combination of Y-27632 and Accutase significantly increases the efficiency of mouse ES cell derivation; furthermore, no negative side effects were observed with Y-27632 and Accutase treatment. The newly established ES cell lines retain stable karyotype, surface markers expression, formed teratomas, and contributed to viable chimeras and germline transmission by tetraploid complementation assay. In addition, Y-27632 improved embryoid body formation of ES cells. During ES cell microinjection, Y-27632 prevented the formation of dissociation-induced cell blebs and facilitates the selection and the capture of intact cells. The methods presented in this study clearly demonstrate that inhibition of Rho kinase with Y-27632 and Accutase dissociation improve the derivation efficiently and reproducibility of mouse ES cell generation which is essential for reducing variability in the results obtained from different cell lines.     

Construction of a Full-Length cDNA Library and Analysis of Expressed Sequence Tags from Inflorescence of Apomictic Sabaigrass (Eulaliopsis binata)

Eulaliopsis binata, which is a close relative of cereal crops, was recognized as an important research material owing to its high frequency of apospory and autonomous endosperm formation. However, little information is known about its genomics and regulatory pathway participating in reproductive development. For the first step to understand molecular basis in sabaigrass (E. binata), a SMART complementary DNA library from the inflorescence tissue was constructed and characterized. The titers of original and amplified libraries were 5.53 × 10⁶ and 1.49 × 10¹⁰ pfu/ml, respectively. The percentage of recombinants was 96% in the original library. Analysis of sequencing results of 398 out of 437 randomly picked clones showed that 271 (68.1%) expressed sequence tags (ESTs) exhibited significant similarity with known putative functional nucleotide sequences in the GenBank databases, 25 (6.3%) ESTs have significant matches with hypothetical proteins, putative proteins, and unknown proteins, and the other 25.6% ESTs had no significant similarity to sequences in the public databases. Based on molecular function of GO annotation, the four most abundant terms are nucleotide binding, hydrolase activity, ion binding, and protein binding, and these genes were involved in 61 different pathways using the Kyoto Encyclopedia of Genes and Genomes pathway analysis. Besides, simple sequence repeats detection in 398 ESTs was carried out, and several genes were chosen to perform expression analysis. This report represents a first step in expanding molecular-genetic analyses in E. binata and can be used to optimally mine useful information from a relatively small data set.     

Enantioselective Degradation of (2RS, 3RS)‐Paclobutrazol in Rat Liver Microsomes

Paclobutrazol, with two stereogenic centers, but gives only (2R, 3R) and (2S, 3S)‐enantiomers because of steric‐hindrance effects, is an important plant growth regulator in agriculture and horticulture. Enantioselective degradation of paclobutrazol was investigated in rat liver microsomes in vitro. The degradation kinetics and the enantiomer fraction were determined using a Lux Cellulose‐1 chiral column on a reverse‐phase liquid chromatography–tandem mass spectrometry system. The t_1/2 of (2R, 3R)‐paclobutrazol is 18.60 min, while the t_1/2 of (2S, 3S)‐paclobutrazol is 10.93 min. Such consequences clearly indicated that the degradation of paclobutrazol in rat liver microsomes was stereoselective and the degradation rate of (2S, 3S)‐paclobutrazol was much faster than (2R, 3R)‐paclobutrazol. In addition, significant differences between the two enantiomers were also observed in enzyme kinetic parameters. The V_max of (2S, 3S)‐paclobutrazol was more than 2‐fold of (2R, 3R)‐paclobutrazol and the Cl_int of (2S, 3S)‐paclobutrazol was higher than that of (2R, 3R)‐paclobutrazol after incubation in rat liver microsomes. These results may have potential implications for better environmental and ecological risk assessment for paclobutrazol. Chirality 27:344–348, 2015. © 2015 Wiley Periodicals, Inc.     

Different antigen presentation tendencies of granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived macrophages and peritoneal macrophages

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced bone marrow-derived cells (BMCs) and primary peritoneal exudate cells (PECs) are usually used for antigen presentation in in vitro experiments. In order to expound their tendency for uptake and antigen presentation, we compared differences in the degree of phagocytosis, the expression of co-stimulatory molecules, and the activation of T lymphocytes between these two cell types. These assays used the F4/80 marker expression, as it is the general marker for macrophages. The BMC population was found to contain both F4/80(bright) and F4/80(dim) subtypes, while PECs were mainly composed of the F4/80(bright) subtype. Expression levels of cell surface co-stimulatory molecules, CD80, CD86, CD54, and CD40, were significantly higher for F4/80(+)BMCs than F4/80(+)PECs. Their expressions were further upregulated for F4/80(+)BMCs than for F4/80(+)PECs after stimulation with flagellin. F4/80(+)BMCs had a weaker ability to phagocytize microbeads than F4/80(+)PECs (P?相似文献   

Modelling and optimal control of immune response of renal transplant recipients

We consider the increasingly important and highly complex immunological control problem: control of the dynamics of immunosuppression for organ transplant recipients. The goal in this problem is to maintain the delicate balance between over-suppression (where opportunistic latent viruses threaten the patient) and under-suppression (where rejection of the transplanted organ is probable). First, a mathematical model is formulated to describe the immune response to both viral infection and introduction of a donor kidney in a renal transplant recipient. Some numerical results are given to qualitatively validate and demonstrate that this initial model exhibits appropriate characteristics of primary infection and reactivation for immunosuppressed transplant recipients. In addition, we develop a computational framework for designing adaptive optimal treatment regimes with partial observations and low-frequency sampling, where the state estimates are obtained by solving a second deterministic optimal tracking problem. Numerical results are given to illustrate the feasibility of this method in obtaining optimal treatment regimes with a balance between under-suppression and over-suppression of the immune system.     

Development and characterization of 70 novel microsatellite markers for the sea cucumber (Apostichopus japonicus)

The sea cucumber (Apostichopus japonicus) is an important item in Asian cuisine. It is currently produced through aquaculture, especially in China, after being overexploited in the wild in the 1990s. We isolated 70 novel polymorphic microsatellite loci using an enrichment-colony hybridization protocol. All loci were characterized in 48 individuals from a natural population in Rongcheng (Shandong, China) using genomic DNA isolated from muscle tissue. The number of alleles ranged from 2 to 17 (mean 7.0), and the observed and expected heterozygosities varied from 0.0010 to 1.0000 and from 0.2125 to 0.9477, respectively. Thirty-one of the 70 loci exhibited departure from Hardy-Weinberg equilibrium. These microsatellite markers should be useful resources for population genetic studies and for molecular marker-assisted breeding of A. japonicus.     

不同强度及时间窗骨骼肌缺血后处理对兔心肌的保护作用

目的:通过比较不同强度及时间窗骨骼肌缺血后处理对兔缺血/再灌注心肌的保护效能,试图寻找最佳强度和时间窗的骨骼肌缺血后处理方案。方法:健康新西兰大白兔42只(雄性)随机分为7组(n=6):①假手术组(Sham)、②缺血对照组(CON)、③标准骨骼肌后处理组(SP)、④延迟6min骨骼肌后处理组(6M-DSP)、⑤延迟1 min骨骼肌后处理组(1M-DSP)、⑥骨骼肌后处理加强组(SSP)、⑦骨骼肌后处理减弱组(WSP)。以开胸结扎冠状动脉左室支固定部位方法制作缺血/再灌注模型,以游离并夹闭双侧腹股沟髂外动脉固定位置方法造成骨骼肌缺血,再灌注末以TTC法确定心肌梗死范围,并分别于心肌缺血前、后及再灌注1 h、2 h,以生化法测定血清肌酸激酶(CK)及乳酸脱氢酶(LDH)水平。结果:和CON组相比,1M-DSP组心肌梗死重量比及面积比分别下降了42.32%及42.68%、SP组分别下降了49.97%及43.78%、SSP组分别下降了48.36%及48.86%,(P均<0.05),但三组之间相比,心梗范围未见明显差异;而6M-DSP、WSP组与CON组相比未见心肌保护作用;肌酸激酶(CK)的水平和梗死范围变化趋势一致。结论:兔在心肌缺血/再灌注之前完成骨骼肌5 min缺血/1 min再灌注1次循环的缺血后处理,可以起到明显的心肌保护作用。