The Protective Effects of KAT5 Inhibition on Ocular Inflammation by Mediating the PI3K/AKT Pathway in a Murine Model of Allergic Conjunctivitis.

Purpose: We aimed to explore the effect of lysine acetyltransferase KAT5 on allergic conjunctivitis (AC). Methods: The effect of KAT5 on inflammatory response during AC progression was analyzed in the experimental allergic conjunctivitis (EAC) mouse model. Results: The clinical score, permeability, total IgE, ovalbumin (OVA)-specific IgE, and IgG1/IgG2a were induced in the EAC mice, in which the overexpression of KAT5 could further enhance but KAT5 inhibitor NU9056 reduce the phenotypes. The eosinophilic infiltration was induced in EAC mice, in which the overexpression of KAT5 was able to further promote but NU9056 attenuate the phenotype. The expression of Eotaxin and RANTES and the inflammatory factors were upregulated in EAC mice and KAT5 overexpression increased, but NU9056 decreased the expression in the model. Significantly, the CD11c+ dendritic cells and CD4+ T cells infiltration in the conjunctiva was enhanced in EAC mice, whereas KAT5 overexpression induced but NU9056 suppressed the effect in the model. Mechanically, the phosphorylation of PI3K and Akt and the levels of histone H3 lysine 27 acetylation (H3K27ac) were enhanced in EAC mice, whereas the overexpression of KAT5 promoted and NU9056 repressed the phenotype in the mice. The enrichment of KAT5 and H3K27ac on PI3K promoter was increased in EAC mice, and the overexpression of KAT5 further enhanced the enrichment in the mice. Significantly, we observed similar results in the KAT5 knockout mice as well. Moreover, PI3K/AKT signaling inhibitor LY294002 reversed KAT5 overexpression-mediated phenotypes and inflammatory response after induction AC in vivo. Conclusions: Therefore we concluded that KAT5 inhibition protected against ocular inflammation by mediating the PI3K/AKT pathway in EAC mouse model.

Effect of Yiqihuoxue prescription on myocardial energy metabolism after myocardial infarction via cross talk of liver kinase B1-dependent Notch1 and adenosine 5'-monophosphate-activated protein kinase.

OBJECTIVE: To investigate the effect of Yiqihuoxue prescription (YQHX) from Traditional Chinese Medicine (TCM) on myocardial glucose and lipid metabolism after myocardial infarction via the cross talk between the liver kinase B1 (LKB1)-dependent Notch1 and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK). YQHX was prepared with substances with properties that benefit, to activate blood circulation based on the TCM theory. METHODS: Animal models of myocardial infarction were established by ligating Sprague Dawley rats' left anterior descending coronary arteries. The animals were randomly divided into a myocardial infarction (MI) group, a YQHX group, a perindopril group, a r-secretase inhibitor, Notch signal inhibitor (DAPT) group, a DAPT+YQHX group and a sham group. The related drugs were administered on the second day after operation, and changes in the relevant indexes were examined on weeks 1 and 4. Changes in cardiac structure and function were examined by echocardiography. The glucose and free fatty acids (FFA) were examined by ELISA. The expression of Notch, LKB1 and AMPK mRNA was examined by a real-time fluorescence quantitative method. The expression of glucose transporter 4 (GLUT4), and the expression of total acetyl-CoA carboxylase (ACC) and its phosphorylation were examined by western blotting. RESULTS: Compared with the sham group, the expression of Notch, LKB1 and AMPK mRNA in the MI group was lower. Compared with the MI group, the expression of these mRNAs in the YQHX and perindopril groups was higher, and their expression in the DAPT group was lower. At all time points, the protein expression of GLUT4 and pACC decreased in the MI group. On week 1, the expression of pACC protein was higher. In the DAPT group, the expression of pACC protein decreased. Compared with the YQHX group, the expression of pACC protein in the DAPT + YQHX group was lower. On week 4, compared with the MI group, the expression of GLUT4 protein in the YQHX group and the perindopril group was higher. The expression of GLUT4 protein in the DAPT group decreased. Compared with the YQHX group, the expression of GLUT4 protein in the DAPT+YQHX group was lower. There was no significant difference in the expression of ACC protein between the groups. CONCLUSION: YQHX promoted cross talk between the LKB1-dependent Notch1 and AMPK in myocardial tissue after myocardial infarction. Furthermore, it regulated the glucose and lipid metabolism of cardiomyocytes at different time points, thereby ameliorating the cardiac energy metabolism via different mechanisms and protecting the heart.

Tripartite Motif 8 (TRIM8) Positively Regulates Pro-inflammatory Responses in Pseudomonas aeruginosa-Induced Keratitis Through Promoting K63-Linked Polyubiquitination of TAK1 Protein.

Pseudomonas aeruginosa (PA)-induced keratitis is a rapidly progressive ocular infectious disease that often leads to inflammatory epithelial edema, stromal infiltration, tissue destruction, corneal ulceration, and vision loss. In this study, we investigate the role of tripartite motif 8 (TRIM8) in regulating the inflammatory process of PA-induced keratitis. The expression of TRIM8 was increased in mouse corneas and in vitro-cultured macrophages after PA infection. Knockdown of the expression of TRIM8 significantly inhibited the activation of NF-κB signaling and decreased the production of pro-inflammatory cytokines both in vivo and in vitro after infected with PA. Furthermore, we investigated the potential mechanism and we found after PA infection that TRIM8 could promote K63-linked polyubiquitination of transforming growth factor ß-activated kinase 1 (TAK1), leading to the activation of TAK1 and enhanced inflammatory responses. Taken together, we demonstrated that TRIM8 has pro-inflammatory effect on PA-induced keratitis and suggest TRIM8 as a potential therapeutic target for keratitis.

CT-guided percutaneous transthoracic needle biopsy for paramediastinal and nonparamediastinal lung lesions: Diagnostic yield and complications in 1484 patients.

Computed tomography-guided percutaneous transthoracic needle biopsy (PTNB) is used for identifying paramediastinal lung lesions that cannot be diagnosed by bronchoscopy, but the diagnostic performance and complication rate are unreported.This retrospective study was approved by the institutional review board committee. A total of 1484 patients who underwent PTNB between April 2012 and April 2015 were enrolled. The cohort was divided into a paramediastinal (n = 195) and a nonparamediastinal group (n = 1289) based on lesion location. Diagnostic yield for malignancy and complication rates were analyzed in both groups. Univariate and multivariate logistic regression analysis was used to determine independent risk factors for hemoptysis complication in the paramediastinal group.Percutaneous transthoracic needle biopsy showed 95.6% (109/114) sensitivity and 100% (77/77) specificity for the diagnosis of lesions in the paramediastinal group, with similar accuracy (95.4%, 186/195) to that in the nonparamediastinal group (94.7%, 1221/1289; P = 0.699). Compared with PTNB for nonparamediastinal lesions, PTNB for paramediastinal lesions demonstrated a comparable pneumothorax rate (8.21% vs 8.69%; P = 0.823) and hemothorax rate (2.56% vs 1.47%; P = 0.261), and a higher hemoptysis rate (28.2% vs 19.4%; P = 0.005). Among 6 defined paramediastinal regions, the overall complication rate was the highest in the posterior region (42.4%) and the lowest in the paraventricular region (13.6%). Multivariate analysis revealed that lesion size of 2 to 3 cm (odds ratio [OR] 3.22), intrapulmonary length of needle path >2 cm (OR 8.85), and proximal to pulmonary artery (OR 10.33) were independent risk factors for hemoptysis in the paramediastinal group.Computed tomography-guided PTNB can diagnose paramediastinal lesions with high yield and acceptable complication rates. Given higher rate of hemoptysis in PTNB for paramediastinal lesions, more attention should be paid in cases with high risks.

Construction of doxycycline-mediated BMP-2 transgene combining with APA microcapsules for bone repair.

OBJECTIVES: The repairing of large segmental bone defects is difficult for clinical orthopedists at present. Gene therapy is regarded as a promising method for bone defects repair. The present study aimed to construct an effective and controllable Tet-On expression system for transferring hBMP-2 gene into bone marrow mesenchymal progenitor cells (BMSCs). Meanwhile, with combination of alginate-poly-L-lysine-alginate (APA) microencapsulation technology, we attempted to reduce the influence of immunologic rejection and examine the effect of the Tet-On expression system on osteogenesis of BMSCs. METHODS: The adenovirus encoding hBMP-2 (ADV-hBMP2) was constructed using the means of molecular cloning. The ADV-hBMP2 and Adeno-X Tet-On virus was respectively transfected to the HEK293 for amplification and afterward BMSCs were co-infected with the virus of ADV-hBMP2 and the Adeno-X Tet-On. The expression of hBMP-2 was measured with induction by doxycycline (DOX) at different concentration by means of RT-PCR and ELISA. Combining Tet-On expression system and APA microcapsules with the use of the pulsed high-voltage electrostatic microcapsule instrument, we examined the expression level of hBMP-2 in APA microcapsules by ELISA as well as the osteogenesis by alizarin red S staining. KEY FINDINGS: An effective Tet-On expression system for transferring hBMP-2 gene into BMSCs was constructed successfully. Also, the expression of hBMP-2 could be regulated by concentration of DOX. The data exhibited that BMSCs in APA microcapsules maintained the capability of proliferation and differentiation. The combination of Tet-On expression system and APA microcapsules could promote the osteogenesis of BMSCs. CONCLUSIONS: According to the results, microencapsulated Tet-On expression system showed the effective characteristics of secreting hBMP-2 and enhancing osteogenesis, which would provide a promising way for bone repair.

Distributed flow estimation and closed-loop control of an underwater vehicle with a multi-modal artificial lateral line.

Bio-inspired sensing modalities enhance the ability of autonomous vehicles to characterize and respond to their environment. This paper concerns the lateral line of cartilaginous and bony fish, which is sensitive to fluid motion and allows fish to sense oncoming flow and the presence of walls or obstacles. The lateral line consists of two types of sensing modalities: canal neuromasts measure approximate pressure gradients, whereas superficial neuromasts measure local flow velocities. By employing an artificial lateral line, the performance of underwater sensing and navigation strategies is improved in dark, cluttered, or murky environments where traditional sensing modalities may be hindered. This paper presents estimation and control strategies enabling an airfoil-shaped unmanned underwater vehicle to assimilate measurements from a bio-inspired, multi-modal artificial lateral line and estimate flow properties for feedback control. We utilize potential flow theory to model the fluid flow past a foil in a uniform flow and in the presence of an upstream obstacle. We derive theoretically justified nonlinear estimation strategies to estimate the free stream flowspeed, angle of attack, and the relative position of an upstream obstacle. The feedback control strategy uses the estimated flow properties to execute bio-inspired behaviors including rheotaxis (the tendency of fish to orient upstream) and station-holding (the tendency of fish to position behind an upstream obstacle). A robotic prototype outfitted with a multi-modal artificial lateral line composed of ionic polymer metal composite and embedded pressure sensors experimentally demonstrates the distributed flow sensing and closed-loop control strategies.

Subconjunctival injection of in vitro transforming growth factor-ß-induced regulatory T cells prolongs allogeneic corneal graft survival in mice.

This study is to investigate the effect of subconjunctival injection of in vitro induced regulatory T cells (iTregs) on the survival of corneal allografts. iTregs were expanded by culturing CD4(+)T cells with TGF-ß in vitro. Foxp3, LAP and GARP were analyzed and the suppression ability of iTregs was assayed by co-culturing with effective T cells. Allogeneic transplantations in mice were modeled and randomly classified into PBS control, iTregs and TA groups. The allografts were observed for 60 days. CD25, Foxp3, LAP and GARP in CD4(+)T cells were analyzed on day 21 after the surgery. Inflammatory cells infiltrated in allografts were detected by flow cytometry and histopathological examination. Expressions of Foxp3, GARP and LAP in iTregs were high. iTregs suppressed the proliferation of effective T cells in vitro. The corneal allograft survival time for PBS, iTregs and TA groups was (18 ± 1.73) days, (38.6 ± 1.14) days and (60 ± 0) days, respectively. The corneal allograft survival time in iTregs group was significantly prolonged compared with PBS group (P < 0.05), but shorter than that in TA group (P < 0.05). No significant difference was observed in expressions of CD25, Foxp3, LAP or GARP in CD4(+)T cells (P > 0.05). Finally, CD3(+)CD4(+)T cell infiltration and fewer inflammatory cells were reduced in allografts in iTregs and TA groups compared with PBS group. The survival time of allografts were prolonged in mice after subconjunctival injection of iTregs. Local immune modulation might be involved in the mechanism.

A new nonhuman primate model of severe dry eye.

PURPOSE: The aim of this study was to establish a new rhesus monkey model of severe dry eye. METHODS: A total of 8 rhesus monkeys were used for the study. Four monkeys had their main lacrimal gland and nictitating membrane surgically removed (group 1). Another 4 monkeys had a similar surgery with further application of 50% trichloroacetic acid on the bulbar conjunctiva (group 2). The ocular surface was evaluated before and after the surgery (1, 4, 8, 12, and 24 weeks) using Schirmer-1 test, corneal fluorescein staining, and the lissamine green test. Conjunctival impression cytology was also performed before and 24 weeks after the surgery. Finally, the cornea and the conjunctiva were evaluated using light microscopy. RESULTS: A significant decrease in tear secretion was observed in all operated eyes. Schirmer test data measured were ≤4 mm in all the operated eyes. Slit-lamp examination also revealed abnormal staining in all the operated eyes that remained stable until the end of the experiment. In group 2, corneal fluorescein staining and lissamine green test values were always ≥5 (max 12) and ≥4 (max 9), respectively. Impression cytology specimens of both the treated groups showed conjunctival squamous metaplasia and a decreased number of goblet cells. Under light microscopy, the corneal epithelium appeared irregular with edematous basal epithelial cells. The conjunctiva showed a decreased goblet cell density with infiltration of inflammatory cells. CONCLUSIONS: Complete removal of the principal lacrimal gland and nictitating membrane associated with the application of 50% trichloroacetic acid on the conjunctiva could induce severe dry eye in rhesus monkeys.

CD154 blockade modulates the ratio of Treg to Th1 cells and prolongs the survival of allogeneic corneal grafts in mice.

Administration of anti-CD154 monoclonal antibody (mAb) may prolong the survival of an allograft; however, the associated therapeutic mechanisms remain poorly understood. This study aimed to evaluate the effects of anti-CD154 mAb on T-cell responses in a mouse model of corneal allograft transplantation. BALB/c mice were transplanted with corneal grafts from C57BL/6 mice and treated intraperitoneally with 250 µg anti-CD154 mAb or isotype IgG on days 0, 3 and 6 post surgery. The transparency of the corneal grafts was evaluated for potential rejection signs by slit-lamp biomicroscopy and histopathology. The percentages of CD4+ T, Tim-3+CD4+ T helper (Th) 1 and CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the spleen, ipsilateral draining lymph nodes and corneal grafts, and the frequency of splenic IFN-γ+ and IL-10+ expression in CD4+ T cells were determined by flow cytometry. Moreover, the ratio of Tregs to Th1 cells was calculated and the suppressive activity of splenic Tregs was measured. Anti-CD154 neutralization significantly prolonged the survival of the corneal allograft (P=0.0012) and reduced the numbers of inflammatory infiltrates in the corneal graft. In the spleen and lymph nodes, anti-CD154 treatment reduced the frequency of CD4+ T cells, Tregs and particularly Th1 cells. In the corneal allografts, anti-CD154 treatment downregulated graft-infiltrated CD4+ T cells and Th1 cells, but increased graft-infiltrated Tregs. Furthermore, anti-CD154 treatment increased the frequency of splenic IL-10+CD4+ T cells and decreased the concentration of splenic IFN-γ+CD4+ T cells. As a result, the ratio of Tregs to Th1 cells in the anti-CD154-treated recipients increased. Anti-CD154 treatment did not enhance the suppressive activity of Tregs in the recipients. The results indicate that the therapeutic effects of anti-CD154 mAb on prolonging the survival of the corneal allograft may be associated with an increased ratio of Tregs to Th1 cells in mice.

Tim-1 blockade with RMT1-10 increases T regulatory cells and prolongs the survival of high-risk corneal allografts in mice.

Anti-Tim-1 monoclonal antibody (mAb) RMT1-10 is effective in promoting allograft survival through blocking Tim-1. However, its role in corneal transplantation is unclear. This study aims to evaluate the effect of RMT1-10 on high-risk corneal transplantation. BALB/c mice were transplanted with corneal grafts from C57BL/6 mice and intraperitoneally injected with RMT1-10 or isotype IgG. The transparency of corneal graft was evaluated by slit lamp biomicroscopy. Flow cytometry was used to determine the phenotype of CD4(+) T cells, including CD154, Tim-3, CD25 and Foxp3, and to analyze the proliferation capacity of CD4(+) T cells and the suppressive capacity of T regulatory (Treg) cells. The levels of interferon-gamma (IFN-γ), IL-4 and transforming growth factor-beta1 (TGF-ß1) were investigated by intracellular staining and/or ELISA assay. The delayed-type hypersensitivity (DTH) response was evaluated by ear swelling assay. RMT1-10 therapy delayed the onset of rejection and significantly prolonged the survival of corneal allograft. In RMT1-10 treated mice, percentages of CD4(+)CD154(+) cells and CD4(+)Tim-3(+) cells were significantly decreased while the frequency of CD4(+)CD25(+)Foxp3(+) Treg cells was significantly up-regulated, compared with those of isotype IgG treated mice. And, in vitro proliferation of CD4(+) T cells was significantly inhibited by RMT1-10. In addition, percentage of intracellular expression of IFN-γ and IL-4 in CD4(+) T cells isolated from RMT1-10 treated mice was significantly reduced. After co-culturing with RMT1-10 in vitro, CD4(+) T cells produced significantly decreased levels of IFN-γ and IL-4 and significantly increased levels of TGF-ß1. Furthermore, RMT1-10 inhibited DTH response of recipient mice and enhanced the suppressive capacity of Treg cells isolated from RMT1-10 treated mice. Our data indicate that Tim-1 blockade with RMT1-10 could suppress immunological rejection and prolong the survival of corneal allograft through regulating T cell responses.