Prognostic risk factors in patients with refractory heart failure treated with continuous veno-venous hemofiltration.

INTRODUCTION: Investigation of the prognostic factors in patients with refractory heart failure (HF) undergoing continuous veno-venous hemofiltration (CVVH). METHODS: Clinical data of 146 patients with refractory HF between May 2018 and December 2020 were retrospectively analyzed and divided into survival and death groups according to the prognosis. Vital signs, inflammatory markers, and renal function parameters were compared before and after treatment. RESULTS: Central venous pressure levels were lower, whereas serum levels of brain natriuretic peptide, oxygen saturation, and cardiac output were higher after treatment (p < 0.05). Heart rate, systolic and diastolic blood pressures, serum levels of C-reactive protein, interleukin-6, tumor necrosis factor-α, interleukin-8, blood urea nitrogen, creatinine, and 24-h urinary protein were lower after treatment (p < 0.05). CONCLUSION: CVVH improved renal function and regulated blood pressure and vital signs in patients with refractory HF. Age, APACHE II score, disease duration, and hypotension were risk factors affecting the prognosis.

Non-coding RNAs and related molecules associated with form-deprivation myopia in mice.

The role of miRNAs and its regulatory mechanism in myopia are indeterminate. Our study aimed to investigate potential myopia-associated non-coding RNAs and related molecules by performing a comprehensive bioinformatic analysis of miRNA expression profile of mice with form-deprivation myopia (FDM). Differentially expressed miRNAs in two raw microarray data sets (GSE58124 and GSE84220) from Gene Expression Omnibus (GEO) database were comprehensively analysed using GEO2R. Target genes were predicted using miRDB and enriched with Metascape online tool. Protein-protein interaction (PPI) networks were constructed utilizing STRING and Cytoscape. Significant differentially expressed miRNAs were validated by real-time polymerase chain reaction (qRT-PCR) using RNA extracted from monocular FDM ocular tissues. As result, we identified three upregulated miRNAs (mmu-miR-1936, mmu-miR-338-5p, and mmu-miR-673-3p) significantly associated with myopia in the two microarray data sets (p < 0.05 and |Log (Fold Change) |>1). GO functional analysis suggested these three miRNAs were targeted in genes mostly enriched in morphogenesis and developmental growth of retinal tissues. Enrichment analysis revealed top eight transcription factors, including PAX6 and Smad3, related to myopia. Ten hub genes, including Rbx1, Fbxl3, Fbxo27, Fbxl7, Fbxo4, Cul3, Cul2, Klhl5, Fbxl16 and Klhl42, associated with ubiquitin conjugation were identified. qRT-PCR confirmed the increased expression of mmu-miR-1936 and mmu-miR-338-5p (p < 0.05), but no statistical difference was observed in mmu-miR-673-3p expression in myopic retinas. Our findings indicated mmu-miR-1936, mmu-miR-338-5p and mmu-miR-673-3p upregulation may be associated with myopia development via post-transcriptional gene regulation, and identified potential molecules that could be further explored in future studies of the mechanism in myopia.

Exosomes from Human Umbilical Vein Endothelial Cells Ameliorate Ischemic Injuries by Suppressing the RNA Component of Mitochondrial RNA-processing Endoribonuclease via the Induction of miR-206/miR-1-3p Levels.

Exosomes might mediate the effects of remote ischemic post-conditioning (RIPostC) treatment on vital organs. The present study aimed to explore the role of RNA component of mitochondrial RNA-processing endoribonuclease (RMRP) in the effects of human umbilical vein endothelial cell (HUVEC)-derived exosomes on ischemic injuries in vitro and in vivo. HUVECs were subjected to oxygen-glucose deprivation (OGD) treatment and exosomes were collected OGD-treated human neural cells were incubated with HUVEC-derived exosomes. Changes in cell viability, apoptosis, and RMRP-mediated PI3K/Akt/mTOR pathway activity were detected. The role of RMRP inhibition in the anti-OGD effects of exosomes was further determined by upregulating RMRP expression in human neural cells. The potential RMRP inhibitory factors in exosomes were explored using microarray detection. The effects of exosomes were validated with MCAO mouse models. In OGD neurons incubated with the exosomes, cell viability was improved and cell apoptosis was suppressed. At molecular level, exosomes on downregulated RMRP, p-PI3K, p-Akt, and p-mTOR, while induced eNOS. After the overexpression of RMRP, the cell protective effects of exosomes were counteracted, which was associated with the re-activation of PI3K/Akt/mTOR pathway. Based on the detection of microarray, the induced levels of miR-206 and miR-1-3p by OGD in HVUECs contributed to the RMPR inhibition. Additionally, injection of exosomes restricted infarction area and suppressed RMRP in MCAO mice. Collectively, exosomes from OGD HUVECs could protect neurons against ischemia-induced injuries, and the effects were associated with the suppression of RMRP in neurons via distance transfer of miR-206 and miR-1-3p.

Circ_0132817 facilitates cell proliferation, migration, invasion and glycolysis by regulating the miR-432-5p/NOL4L axis in neuroblastoma.

Neuroblastoma (NB) is one of the most common extracranial solid tumors in children. Circular RNAs (circRNAs) have been shown to be involved in the development of NB. However, the function of circ_0132817 in NB is currently unclear. In this paper, the levels of circ_0132817 and NOL4L were induced in NB tissues and cells, and miR-432-5p expression was on the contrary. MiR-432-5p was verified as a target of circ_0132817 and miR-432-5p could bind to NOL4L. The inhibitory effects of miR-432-5p overexpression on cell proliferation, migration, invasion and glycolysis could be reversed by circ_0132817 facilitation. The suppression of NOL4L knockdown on NB cells progression could be rescued by miR-432-5p inhibition. Besides, knockdown of circ_0132817 repressed tumor growth in vivo. Thus, we came to a conclusion that circ_0132817 promoted the tumorigenesis of NB cells by up-regulating NOL4L and acting as a sponge for miR-432-5p.

Rheumatoid arthritis coexisting with ankylosing spondylitis: A report of 22 cases with delayed diagnosis.

ABSTRACT: Coexisting rheumatoid arthritis (RA) and ankylosing spondylitis (AS) in the same patient is often thought to be rare, and thus misdiagnosis is common. The aim of our study was to describe the main characteristics of RA coexisting with AS in patients with delayed diagnoses and improve awareness of the disease association.Between 2012 and 2018, data from 22 patients who had RA and AS (RA/AS) were retrospectively reviewed. All patients had a history of delayed diagnosis for RA or AS. The clinical features and radiographic changes of RA and AS patients were obtained at baseline and after 2 years. Disease activity score 28 (DAS28) or bath ankylosing spondylitis disease activity index (BASDAI) were used as outcome measures. The mean age at the time of diagnosis of RA/AS was 51.8 years, while the mean duration of diagnostic delay was 5.5 years. Middle-aged women were the most common subgroup among the RA/AS cohort. The common clinical manifestations were systemic, symmetric, peripheral, and axial arthritis. The erythrocyte sedimentation rate and C-reactive protein levels in RA/AS patients were elevated at the time diagnosis of RA/AS. The typical radiologic changes for the 2 diseases coexisted in RA/AS patients. The DAS28 and BASDAI scores at the 2-year follow-up evaluation were lower than the initial assessment.Coexisting RA and AS is often misdiagnosed for many years; a lack of recognition of RA and AS together is one of the most common reasons. Systemic, symmetric, peripheral, and axial arthritis in middle-aged women were the most frequent presentations at onset.

Risk factors related to the severity of COVID-19 in Wuhan.

Objective: To evaluate the characteristics at admission of patients with moderate COVID-19 in Wuhan and to explore risk factors associated with the severe prognosis of the disease for prognostic prediction. Methods: In this retrospective study, moderate and severe disease was defined according to the report of the WHO-China Joint Mission on COVID-19. Clinical characteristics and laboratory findings of 172 patients with laboratory-confirmed moderate COVID-19 were collected when they were admitted to the Cancer Center of Wuhan Union Hospital between February 13, 2020 and February 25, 2020. This cohort was followed to March 14, 2020. The outcomes, being discharged as mild cases or developing into severe cases, were categorized into two groups. The data were compared and analyzed with univariate logistic regression to identify the features that differed significantly between the two groups. Based on machine learning algorithms, a further feature selection procedure was performed to identify the features that can contribute the most to the prediction of disease severity. Results: Of the 172 patients, 112 were discharged as mild cases, and 60 developed into severe cases. Four clinical characteristics and 18 laboratory findings showed significant differences between the two groups in the statistical test (P<0.01) and univariate logistic regression analysis (P<0.01). In the further feature selection procedure, six features were chosen to obtain the best performance in discriminating the two groups with a linear kernel support vector machine. The mean accuracy was 91.38%, with a sensitivity of 0.90 and a specificity of 0.94. The six features included interleukin-6, high-sensitivity cardiac troponin I, procalcitonin, high-sensitivity C-reactive protein, chest distress and calcium level. Conclusions: With the data collected at admission, the combination of one clinical characteristic and five laboratory findings contributed the most to the discrimination between the two groups with a linear kernel support vector machine classifier. These factors may be risk factors that can be used to perform a prognostic prediction regarding the severity of the disease for patients with moderate COVID-19 in the early stage of the disease.

Job Satisfaction and Its Associated Factors Among General Practitioners in China.

OBJECTIVES: Investigation is scare on job satisfaction among general practitioners (GPs) in China. This study aimed to investigate job satisfaction of GPs in China and explore its determinants. METHODS: A multistage-stratified random sampling method was used to collect data with a structured self-administered questionnaire from 3236 GPs (response rate, 99.8%) working in community health institutions in China between October 2017 and February 2018. Multiple linear stepwise regression analysis was used to analyze the associated factors with job satisfaction among GPs. RESULTS: Among these respondents, 1215 (37.5%), 352 (10.9%), and 1669 (51.6%) GPs were dissatisfied, moderate, and satisfied for their current job, respectively. Male GPs, a higher education level, at a higher professional title, at a lower level of income, and those with heavy work stress had a lower job satisfaction. In addition, GPs who often worked overtime, who were at a higher level of emotional exhaustion, at a higher level of depersonalization, at a lower level of personal accomplishment, and who had less occupational development opportunities reported a lower level of job satisfaction. CONCLUSION: These findings suggest that job satisfaction among Chinese GPs is at a moderate level. Region, sex, professional title, education level, working overtime, income level, emotional exhaustion, depersonalization, personal accomplishment, work stress, and occupational development opportunities were significant predictors of job satisfaction.

Treat 2019 novel coronavirus (COVID-19) with IL-6 inhibitor: Are we already that far?

The world is in the midst of the coronavirus disease 2019 (COVID-19) pandemic. Interleukin 6 (IL-6) inhibitor (tocilizumab) had been suggested for the treatment of acute respiratory distress syndrome (ARDS) patients based on the concept of "cytokine storm" in COVID-19. However, we still lack reliable studies to verify "cytokine storm" in COVID-19 pneumonia. Furthermore, IL-6 inhibitor has potential hazards of inducing infectious diseases. The efficacy of IL-6 monoclonal antibody-directed therapy remains to be fully evaluated.

Clinical Characteristics of Children with Coronavirus Disease 2019 in Hubei, China.

Since December 2019, COVID-19 has occurred unexpectedly and emerged as a health problem worldwide. Despite the rapidly increasing number of cases in subsequent weeks, the clinical characteristics of pediatric cases are rarely described. A cross-sectional multicenter study was carried out in 10 hospitals across Hubei province. A total of 25 confirmed pediatric cases of COVID-19 were collected. The demographic data, epidemiological history, underlying diseases, clinical manifestations, laboratory and radiological data, treatments, and outcomes were analyzed. Of 25 hospitalized patients with COVID-19, the boy to girl ratio was 1.27:1. The median age was 3 years. COVID-19 cases in children aged <3 years, 3.6 years, and ≥6-years patients were 10 (40%), 6 (24%), and 9 (36%), respectively. The most common symptoms at onset of illness were fever (13 [52%]), and dry cough (11 [44%]). Chest CT images showed essential normal in 8 cases (33.3%), unilateral involvement of lungs in 5 cases (20.8%), and bilateral involvement in 11 cases (45.8%). Clinical diagnoses included upper respiratory tract infection (n=8), mild pneumonia (n=15), and critical cases (n=2). Two critical cases (8%) were given invasive mechanical ventilation, corticosteroids, and immunoglobulin. The symptoms in 24 (96%) of 25 patients were alleviated and one patient had been discharged. It was concluded that children were susceptible to COVID-19 like adults, while the clinical presentations and outcomes were more favorable in children. However, children less than 3 years old accounted for majority cases and critical cases lied in this age group, which demanded extra attentions during home caring and hospitalization treatment.

Evaluation of Tear Protein Markers in Dry Eye Disease with Different Lymphotoxin-Alpha Expression Levels.

PURPOSE: To compare tear protein markers between normal subjects and patients with dry eye (DE) and high and low lymphotoxin-alpha (LT-α) levels. DESIGN: Prospective cross-sectional study. METHODS: Patients with DE were divided into low (≤700 pg/mL) and high (>700 pg/mL) LT-α groups. Twelve protein markers were measured by microsphere-based immunoassay and ocular surface parameters were determined in right eyes (33 high LT-α DE, 27 low LT-α DE, and 20 control eyes) and left eyes (21 high LT-α DE, 39 low LT-α DE, and 20 control eyes). RESULTS: In both eyes, tumor necrosis factor-α (TNF-α), interleukin (IL)-10, IL-1ß, IL-1 receptor antagonist (IL-1Ra), IL-17A, and IL-12/23 p40 levels in high LT-α DE were significantly higher (P < .01) than in low LT-α DE. Significant correlations identified in high LT-α DE were: Standard Patient Evaluation Eye Dryness with IL-10 (R = 0.43, P = .013), IL-1ß (R = 0.48, P = .005), and IL-12/23 p40 (R = 0.50, P = .003), IL-12/23 p40 with ocular surface disease index (R = 0.35, P = .049), and epidermal growth factor with corneal fluorescein staining score (R = -0.36, P = .038). Significant correlations in low LT-α DE were: Standard Patient Evaluation Eye Dryness with IL-10 (R = -0.39, P = .046), TNF-α (R = -0.39, P = .047), and IL-17A (R = -0.48, P = .013), ocular surface disease index with TNF-α (R = -0.47, P = .017) and IL-17A (R = -0.46, P = .018), and IL-6 with tear breakup time (R = -0.40, P = .044). Lastly, IL-1Ra levels significantly increased in DE patients, positively correlated with temporal conjunctival hyperemia index, and negatively correlated with Schirmer I test (P < .05). CONCLUSIONS: Our study identified tear IL-1Ra level as a potential biomarker to replace the Schirmer I test. Multiple tear protein marker levels increased in high LT-α DE, indicating that high LT-α DE might have a different pathogenesis.

A national survey of turnover intention among general practitioners in China.

Studies on turnover intention among Chinese general practitioners (GPs) at the national level are limited. This study aimed to assess intention to leave and its associated factors among a nationally representative sample of GPs. The participants were selected using a multistage stratified random sampling method. A self-administered structured questionnaire was used to collect data from 3236 GPs in China between October 2017 and February 2018. A multiple linear stepwise regression analysis was used to identify factors associated with turnover intention. Over 70.0% GPs had a moderate or high turnover intention. GPs who were male, were younger, had a higher education level, had a lower professional title, had a lower income level, and had a temporal work contract had higher turnover intention. In addition, GPs who worked night shifts, had low job satisfaction, and had few opportunities for professional development reported higher turnover intention. Substantial gender and regional differences in predictors of turnover intention among GPs were observed. The study showed that turnover intention in Chinese GPs is high, and the factors influencing turnover intention were low professional title and income level, high education level, having a temporary work contract, working night shifts, and limited opportunities for professional development.

Early Intratracheal Administration of Corticosteroid and Pulmonary Surfactant for Preventing Bronchopulmonary Dysplasia in Preterm Infants with Neonatal Respiratory Distress Syndrome: A Meta-analysis.

There is uncertain result with regard to the use of inhalation or instillation steroids to prevent bronchopulmonary dysplasia in preterm infants. This meta-analysis was designed to evaluate the efficacy and safety of early airway administration (within 2 days after birth) of corticosteroids and pulmonary surfactant (PS) for preventing bronchopulmonary dysplasia (BPD) in premature infants with neonatal respiratory distress syndrome (NRDS). The related studies were retrieved in PubMed, EMBASE, the Cochrane Library, Clinical Trial, CNKI, Wanfang and VIP Database from inception to August 2018. Two reviewers independently screened the studies to ensure that all patients with diagnosis of NRDS were enrolled to studies within 1 day after birth, assessed the quality of included studies by GRADEpro system and extracted the data for review. The meta-analysis was performed by RevMan 5.2 software. A subgroup analysis about inhaled corticosteroid (ICS) delivery method was made between ICS inhalation subgroup [inhalation of ICS by nebulizer or metered dose inhaler (MDI)] and ICS intratracheal instillation subgroup (PS used as a vehicle). Eight randomized controlled trials were enrolled in the meta-analysis, 5 trials of which stated the randomized method, grouping and blinded method, and the follow-up procedures were reported. GRADEpro system showed high quality of 4 trials (5 articles), and the rest 4 trials had moderate quality. Meta-analysis showed that the incidence of BPD was decreased in ICS group, the relative risk (RR) was 0.56 (95% CI: 0.42-0.76), and similar trends were found in ICS inhalation subgroup and ICS intratracheal instillation subgroup, with the corresponding RR being 0.58 (95% CI: 0.41-0.82) and 0.47 (95% CI: 0.24-0.95) respectively. ICS could also significantly reduce the mortality risk as compared with placebo control group (RR: 0.67; 95% CI: 0.45-0.99), with RR of ICS inhalation subgroup and ICS intratracheal instillation subgroup being 0.81 (95% CI: 0.34-1.94) and 0.64 (95% CI: 0.41-0.99) respectively. Moreover, the percentage of infants using PS more than one time was lower in ICS group than in the placebo control group, with the RR and 95% CI being 0.55 (95% CI: 0.45-0.67), and that in ICS intratracheal instillation subgroup lower than in ICS inhalation subgroup (RR: 0.56; 95% CI: 0.45-0.69, and RR: 0.35; 95% CI: 0.08-1.52 respectively). There was no significant difference in the incidence of infection or retinopathy of prematurity and neuro-motor system impairment between ICS group and placebo control group, with the corresponding RR being 0.95 (95% CI: 0.59-1.52), 0.92 (95% CI: 0.62-1.38) and 1.13 (95% CI: 0.92-1.39), respectively. It was concluded that early administration of ICS and PS is an effective and safe option for preterm infants with NRDS in preventing BPD and reducing mortality, decreasing the additional PS usage, especially for the ICS intratracheal instillation subgroup. Furthermore, the appropriate dose and duration of ICS, combined use of inhalation or instillation of ICS with PS and the long-term safety of airway administration of corticosteroids need to be assessed in large trials.

Extracellular ATP activates P2X7R-NF-κB (p65) pathway to promote the maturation of bone marrow-derived dendritic cells of mice.

The maturation state of dendritic cell (DC) plays an important role in immune activities. Previously we had found that NF-κB (p65) pathway could promote DC maturation and subsequent immune effects. But the upstream mechanism of this pathway was still unclear. Extracellular adenosine triphosphate (ATP) activating its receptor P2X7R has recently been considered as the fourth signal to activate T lymphocytes. Here we aimed to find out the connection between P2X7R and NF-κB (p65) pathway in DC maturation. Results showed that the expression of P2X7R and the intracellular ATP levels were increased along with the maturation of DC. P2X7R agonist stimulated the morphological changes of DCs into the appearance of mature DCs, and promoted the expression of NF-κB (p65), as well as the release of IFN-γ and IL-12. Whereas, P2X7R inhibitor had the opposite influences. Co-immunoprecipitation assay confirmed the binding of P2X7R and NF-κB (p65). Our study suggested that extracellular ATP could promote DC maturation and release of inflammatory cytokines through the binding of P2X7R and NF-κB (p65). This is the first study to show the P2X7R-NF-κB (p65) pathway in DC. Interference with this pathway may be able to regulate immune responses in areas like infectious diseases, inflammation, transplantation, tumor and autoimmune diseases. In addition, intracellular ATP level could be a new indicator of the maturation state of DC.

Association of smoking with risk of stroke in middle-aged and older Chinese: Evidence from the China National Stroke Prevention Project.

Although the impacts of smoking on health are well established, it is unclear on how they affect the Chinese population aged ≥40 years. This study aimed to investigate the association between smoking and risk of stroke in middle-aged and older Chinese adults, based on the data from the China National Stroke Prevention Project.A community-based cross-sectional study with 12,704 (5681 men, 7023 women) Chinese adults aged ≥40 years was conducted to examine the association of smoking with stroke. Multivariable logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs).Among the study population, a total of 524 stroke survivors were identified. The age-adjusted prevalence of stroke was 4.06% for both sexes, 2.95% for women, and 5.38% for men. The multivariate-adjusted ORs (95% CI) of stroke associated with current cigarette smoking and former cigarette smoking were 1.67 (1.24-2.25) and 1.93 (1.29-2.87), respectively. Compared with those who were never-smokers, the multivariate-adjusted OR of stroke (95% CI) were 1.48 (0.96 to -2.29), 1.75 (1.20-2.56), and 2.37 (1.20 to -4.68) for those who smoked 1 to 10, 11 to 20, and ≥21 cigarettes per day; and 0.51 (0.19 to -1.42), 1.90 (1.36 to -2.67), and 2.01 (1.17 to -3.46) for those who smoked 1 to 19, 20 to 39, and ≥40 years, respectively (both P < .001 for linear trends). Among former smokers, the multivariable-adjusted ORs of stroke by duration of smoking cessation (compared with never smokers) for <5, 5 to 19, and ≥20 years were 3.47 (1.42-8.49), 3.37 (1.95-5.80), and 0.95 (0.49-1.84), respectively (P = .009 for linear trend). The increased odds of stroke with smoking were more evident among participants who were men, >60 years old, or without family history of stroke than their counterparts.This study suggests the increased odds of stroke in current cigarette smokers with a graded increase in prevalent risk that depended on how many cigarettes and how many years were smoked. Moreover, quitting smoking appears to decrease this excess risk substantially.

PCAF/GCN5-Mediated Acetylation of RPA1 Promotes Nucleotide Excision Repair.

The RPA complex can integrate multiple stress signals into diverse responses by activating distinct DNA repair pathways. However, it remains unclear how RPA1 elects to activate a specific repair pathway during different types of DNA damage. Here, we report that PCAF/GCN5-mediated K163 acetylation of RPA1 is crucial for nucleotide excision repair (NER) but is dispensable for other DNA repair pathways. Mechanistically, we demonstrate that the acetylation of RPA1 is critical for the steady accumulation of XPA at damaged DNA sites and preferentially activates the NER pathway. DNA-PK phosphorylates and activates PCAF upon UV damage and consequently promotes the acetylation of RPA1. Moreover, the acetylation of RPA1 is tightly regulated by HDAC6 and SIRT1. Together, our results demonstrate that the K163 acetylation of RPA1 plays a key role in the repair of UV-induced DNA damage and reveal how the specific RPA1 modification modulates the choice of distinct DNA repair pathways.

Blockade of toll-like receptor 2 expression and membrane translocation in rat corneal epithelial cells by glucocorticoid (TobraDex) after penetrating keratoplasty.

PURPOSE: To evaluate the role of Toll-like receptor (TLR) 2 and the effect of glucocorticoid on immune rejection of penetrating keratoplasty. METHODS: Allograft corneal transplantation was performed between host Sprague Dawley (SD) and Wistar donor rats. The expression of TLR2 messenger RNA (mRNA) and protein in corneas was determined by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and immunofluorescence on days 5, 7, and 9 after operation. Three groups were included: allograft, allograft treated with TobraDex (Alcon, Rijksweg, Belgium), and isograft. Normal rat corneas were included as an additional control. RESULTS: Various degrees of congregation of inflammatory cells and neovascularization of grafts were confirmed by histopathology. Immunohistochemistry revealed that TLR2 was expressed in epithelial, stromal, and endothelial cells of normal tissue, and in all of the grafts. Immunofluorescence analysis of TLR2 showed membrane staining of epithelial cells in the allografts on days 7 and 9. This was absent in the isografts and the allografts treated with TobraDex. TLR2 mRNA was detected in normal corneas, and levels were increased in all of the grafts, as determined by quantitative reverse transcription-polymerase chain reaction. By day 9 after transplantation, a 3.6-fold increase in TLR2 mRNA was observed in the allografts compared with the isografts or the allografts treated with TobraDex, which was statistically significant, at P < 0.005. CONCLUSIONS: Expression of TLR2 in the rat cornea was significantly increased and concurred with the allograft rejection, but was effectively blocked by treatment with TobraDex.

Topical dihydroartemisinin inhibits suture-induced neovascularization in rat corneas through ERK1/2 and p38 pathways.

AIM: To determine if topical instillation of dihydroartemisinin (DHA) inhibits corneal neovascularization (NV) in rats and to investigate the role of the extracellular regulated kinases (ERK) 1/2 and p38 pathways in this process. METHODS: Suture-induced corneal NV was produced in rats and the eyes were topically treated with different concentrations of DHA (20mg/L, 10mg/L or 5mg/L) or normal saline 4 times a day for 7 days. The corneal NV was quantified as the proportion of NV area to the whole cornea. Western blot was used to determine the expressions of vascular endothelial growth factor (VEGF) and the phosphorylation status of VEGF receptor-2, ERK1/2 and p38 in the corneas. Immunofluorescent staining was used to determine the expressions of phospho-ERK1/2 and phospho-p38 in the corneal tissues from the eyes treated with 20 mg/L DHA (DHA group) or normal saline (control group). RESULTS: The proportion of corneal NV area in the eyes treated with normal saline or DHA at dosages of 20mg/L, 10mg/L or 5mg/L was (23.74±3.00)%, (15.73±2.88)%, (19.53±2.42)%, and (23.38±2.79)%, respectively. In the eyes treated with 20mg/L or 10mg/L DHA, the corneal NV area was significantly reduced when compared to that in eyes with normal saline (P<0.05). Western blot analyses revealed that 20mg/L DHA significantly inhibited the expressions of VEGF and phospho-VEGFR-2. Both 20mg/L and 10mg/L DHA inhibited the expressions of phospho-ERK1/2 and phospho-p38. Immunofluorescent staining further demonstrated that 20mg/L DHA lowered the expression levels of phospho-ERK1/2 and phospho-p38 in the corneas with suture-induced NV. CONCLUSION: Suture-induced NV in rat corneas was significantly inhibited by topical treatment with 20mg/L and 10mg/L DHA. The results suggest that the effects could be partially dependent on the DHA-mediated inhibitions of the ERK1/2 and p38 pathways.

[Pathological changes of the cornea in rabbits with hyphema and concurrent ocular hypertension].

OBJECTIVE: To evaluate the impact of hyphema secondary to high intraocular pressure on corneal pathology in rabbits. METHODS: Thirty adult New Zealand rabbit were randomized into 3 equal groups, and in each rabbit, one eye served as the experimental eye with the other as the control eye. In the experimental eye, autoblood was injected into the anterior chamber to induce high intraocular pressure maintained for 3, 5, or 8 days. Only saline was injected into the control eye. After the injections, the cornea was observed with slit-lamp microscopy, and at 3, 5, or 8 days, the experimental and control eyes were taken from the 3 groups for microscopic examination of the corneas to detect the occurrence of cornea bloodstain with prolonged high intraocular pressure. Corneal edema, elastic fibers changes, growth of new blood vessels, changes of eosinophils, fibroblasts, lymphocytes and plasma cells, as well as the pathological changes of the corneal layers were observed and compared between the experimental and control eyes. RESULTS: Maintenance of high intraocular pressure for 8 days resulted in the most severe corneal edema and thickening, and histopathologically, the corneal stroma showed widened space between the elastic fibers and obvious fiber distortion. Neovascularization was seen in the marginal cornea where eosinophil infiltration occurred with a small number of lymphocytes, plasma cells and fiber cells. All the three groups showed more obvious edema in the posterior than in the anterior cornea. CONCLUSION: Prolonged hyphema with ocular hypertension results in aggravation of corneal edema, and corneal blood staining does not occur until 8 days of high intraocular pressure but corneal elastic fiber disruption can be seen, suggesting the impending irreversible pathological changes of cornea.

[Role of Toll-like receptor 2 in corneal graft rejection following penetrating keratoplasty].

OBJECTIVE: To gain insight into the role of Toll-like receptor 2 (TLR2) in graft rejection following penetrating keratoplasty, and investigate the expression of TLR2 mRNA in the corneal graft. METHODS: Penetrating keratoplasty was performed in 3 groups of rats for orthotopic autologous corneal transplantation (group A), allograft corneal transplantation (group B), or allograft corneal transplantation with hormone treatment (C). The transparency and neovascularization of the cornea were observed using a slit-lamp microscope and scored according to the rejection index, with normal cornea serving as the control. The corneal tissues were sampled at 5, 7, and 9 days after the transplantation for histopathological examination and detection of TLR2 mRNA expression using RT-PCR. RESULTS: With the passage of time, edema, opacities and neovascularization of the corneal graft occurred after the operation in all the groups. Seven days after the operation, the rejection index of group B, but not that of groups A and C, met the diagnostic criteria for graft rejection with also support by histopathological evidence. The expression of TLR2 mRNA was detected in normal corneas and augmented in the corneal grafts in the 3 transplantation groups. TLR2 mRNA expression in group B was significantly higher than that of group A, and the expression in group C decreased significantly in comparison with that in group B (P<0.05). CONCLUSION: As the recognition receptors of native immune system, TLR2 in the rejected corneal grafts may recognize the allograft antigen and play a role in acute graft rejection after penetrating keratoplasty.

[Recombinant type 1 adeno-associated virus-mediated transfection of ECV304 cells with enhanced green fluorescent protein gene in vitro].

OBJECTIVE: To assess the feasibility of recombinant type 1 adeno-associated virus (rAAV1) as a vector for gene therapy of corneal neovascularization. METHODS: The rAAV1 vector carrying enhanced green fluorescence protein (EGFP) gene (rAAV1-EGFP) was transfected into ECV304 cells at different multiplicities of infection (MOI=5 x 10(3), 5 x 10(4), 5 x 10(5)). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage determined by flow cytometry. MTT assay was used to assess the proliferation of the transfected cells. RESULTS: The cells with rAAV1-EGFP transfection at MOI of 5 x 10(5) began to exhibit GFP expression 2 days after transfection, and the fluorescence intensity increased with the MOI used for transfection. GFP expression reached the maximum on day 7, at the point of which the transduction efficiency of rAAV1-EGFP in ECV304 cells was 45.90%, 58.56% and 68.31% corresponding to MOIs of 5 x 10(3), 5 x 10(4), and 5 x 10(5), respectively. MTT assay did not reveal significant difference in the absorbance between the transfected cells and the control cells at 72 and 96 h after transfection. CONCLUSION: arAAV1-EGFP gene can be stably and efficiently expressed in ECV304 cells without causing cell growth inhibition, suggesting the potential of rAAV1 as a safe and efficient vector for gene therapy of corneal neovascularization.