Recombinant sulfated CCR2 peptide trap reduces retinal degeneration in mice.

Chemokine receptors are generally sulfated at tyrosine residues of the N-terminal region. Tyrosine sulfation of the C-C chemokine receptor type 2 (CCR2) enhances its interaction with the chemokine ligand CCL2. Here, we generated a recombinant sulfated CCR2 peptide trap (mCCR2-S2) and investigated its effects on retinal degeneration in mice. Treatment with mCCR2-S2 reduced choroidal neovascularization (CNV) in a laser-induced CNV mouse model. In NaIO3-injected mice, treatment with mCCR2-S2 increased the outer nuclear layer thickness and rhodopsin expression in the retinas compared to that in mice treated with mCCR2-wild-type or glutathione S-transferase controls. Furthermore, glial fibrillary acidic protein (GFAP) expression and macrophage infiltration were decreased in mCCR2-S2-treated retinas. Recombinant mCCR2-S2 suppressed CNV development and retinal degeneration, possibly by regulating macrophage infiltration. Thus, the sulfated form of the CCR2 peptide trap may be a useful tool for treating patients with retinal degeneration, such as those with age-related macular degeneration and intraocular inflammatory disorders.

Non-invasive tests for liver disease severity and the hepatocellular carcinoma risk in chronic hepatitis B patients with low-level viremia.

BACKGROUND & AIMS: We tested whether non-invasive tests for liver disease severity can stratify hepatocellular carcinoma (HCC) risk in chronic hepatitis B virus (HBV)-infected patients showing low-level viremia (LLV, HBV DNA <2000 IU/mL). METHODS: A retrospective cohort of 1006 chronic hepatitis B patients showing persistently LLV, defined by at least two consecutive assessments in the year before enrolment, was assessed for HCC development. Two non-invasive serum biomarkers, the aspartate aminotransferase to platelet ratio index (APRI) and the Fibrosis-4 (FIB-4), were tested. Cirrhosis was defined with ultrasonography. RESULTS: During a median 5.1 years of follow-up, HCC developed in 36 patients. HCC incidence rate at 5 years was significantly higher for cirrhotic patients (19/139, 13.7%), but was not null for non-cirrhotic patients (17/867, 2.0%, P<.001). APRI at a cut-off of 0.5 was more specific but less sensitive for HCC development, and FIB-4 at a cut-off of 1.45 was more sensitive but less specific. When both APRI and FIB-4 were used to group patients, the 5-year cumulative HCC incidence rate was 13.9%, 1.4% and 1.2% for both high, any high, and both low APRI and FIB-4 score among all patients (n=1006, P<.001), respectively, and was 11.4%, 1.5% and 0.4% in the same respective order among non-cirrhotic patients (n=867, P<.001). CONCLUSIONS: The combined use of two non-invasive serum biomarkers (APRI and FIB-4) could stratify HCC risk for chronic HBV-infected patients with LLV.

Sphingosine-1-phosphate is involved in inflammatory reactions in patients with Graves' orbitopathy.

OBJECTIVE: Graves' orbitopathy (GO) is initiated by excessive amount of various inflammatory mediators produced by orbital fibroblasts. This study aimed to assess the crucial role of sphingosine-1-phosphate (S1P) in the inflammatory process of GO. METHODS: Orbital adipose/connective tissue samples were obtained from 10 GO patients and 10 normal control individuals during surgery. Primary orbital fibroblast culture was done. After the expression of S1P receptors and sphingosine kinase (SphK) was assessed with the treatment of interleukin (IL)-1ß, we evaluated the expression of pro-inflammatory factors [intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2) and IL-6] after treating S1P. S1P receptor antagonists and SphK 1 inhibitor were pretreated and the expression of the pro-inflammatory factors was assessed. RESULTS: IL-1ß exacerbated the inflammatory process by enhancing the expression of S1P receptors and SphK in GO orbital fibroblasts. IL-1ß also induced the expressions of ICAM-1, COX-2, and IL-6 in GO orbital fibroblasts, and these expressions were effectively inhibited by S1P receptor antagonists and SphK1 inhibitor. CONCLUSION: S1P has an important role in the pathological inflammatory process of GO, which is mediated through the SphK1-S1P- S1P receptor pathway. SphK1 inhibitors and S1P receptors or antagonists could be potential approaches for controlling the inflammatory process of GO.

Homeobox A9 directly targeted by miR-196b regulates aggressiveness through nuclear Factor-kappa B activity in non-small cell lung cancer cells.

MicroRNAs (miRNAs) are recognized as crucial posttranscriptional regulators of gene expression, and play critical roles as oncogenes or tumor suppressors in various cancers. Here, we show that miR-196b is upregulated in mesenchymal-like-state non-small cell lung cancer (NSCLC) cells and lung cancer tissues. Moreover, miR-196b upregulation stimulates cell invasion and a change in cell morphology to a spindle shape via loss of cell-to-cell contacts. We identified homeobox A9 (HOXA9) as a target gene of miR-196b by using public databases such as TargetScan, miRDB, and microRNA.org. HOXA9 expression is inversely correlated with miR-196b levels in clinical NSCLC samples as compared to that in corresponding control samples, and with the migration and invasion of NSCLC cells. Ectopic expression of HOXA9 resulted in a suppression of miR-196b-induced cell invasion, and HOXA9 reexpression increased E-cadherin expression. Furthermore, HOXA9 potently attenuated the expression of snail family zinc finger 2 (SNAI2/SLUG) and matrix metallopeptidase 9 (MMP9) by controlling the binding of nuclear factor-kappa B to the promoter of SLUG and MMP9 genes, respectively. Therefore, we suggest that HOXA9 plays a central role in controlling the aggressive behavior of lung cancer cells and that miR-196b can serve as a potential target for developing anticancer agents. © 2015 Wiley Periodicals, Inc.

Effect of Floor Space Allowance on Pig Productivity across Stages of Growth: A Field-scale Analysis.

A total of 152 pig farms were randomly selected from the five provinces in South Korea. During the experiment, the average temperature and relative humidity was 24.7°C and 74% in summer and 2.4°C and 53% in winter, respectively. The correlation between floor space allowance (FSA) and productivity index was analyzed, including non-productive sow days (NPD), number of weaners (NOW), survival rate (SR), appearance rate of A-grade pork (ARA), and days at a slaughter weight of 110 kg (d-SW) at different growth stages. The objectives of the present study were i) to determine the effect of FSA on the pig productivity index and ii) to suggest the minimum FSA for pigs based on scientific baseline data. For the pregnant sow, NPD could be decreased if pregnant sows were raised with a medium level (M) of FSA (3.10 to 3.67 m(2)/head) while also keeping the pig house clean which improves hygiene, and operating the ventilation system properly. For the farrowing sows, the NOW tended to decrease as the FSA increased. Similarly, a high level of FSA (H) is significantly negative with weaner SR of farrowing sows (p-value = 0.017), indicating this FSA tends to depress SR. Therefore, a FSA of 2.30 to 6.40 m(2)/head (very low) could be appropriate for weaners because a limited space can provide a sense of security and protection from external interruptions. The opposite trend was observed that an increase in floor space (>1.12 m(2)/head) leads to increase the SR of growing pigs. For the fattening pigs, H level of FSA was negatively correlated with SR, but M level of FSA was positively correlated with SR, indicating that SR tended to increase with the FSA of 1.10 to 1.27 m(2)/head. In contrast, ARA of male fattening pigs showed opposite results. H level of FSA (1.27 to 1.47 m(2)/head) was suggested to increase productivity because ARA was most affected by H level of space allowance with positive correlation (R(2) = 0.523). The relationship between the FSA and d-SW of fattening pigs was hard to identify because of the low R(2) value. However, the farms that provided a relatively large floor space (1.27 to 1.54 m(2)/head) during the winter period showed d-SW was significantly and negatively affected by FSA.

Lithium chloride inhibits TGF-ß1-induced myofibroblast transdifferentiation via PI3K/Akt pathway in cultured fibroblasts from Tenon's capsule of the human eye.

Excess scarring of the conjunctiva after glaucoma filtration surgery is a major cause of failure. Transforming growth factor (TGF)-ß is critically involved in post-operative scarring. Lithium inhibits TGF-ß-induced gene protein expression in corneal fibroblasts and inhibits TGF-ß-induced epithelial mesenchymal transition. Here, we investigated the effects of LiCl on TGF-ß1-mediated signaling pathways and on myofibroblast transdifferentiation of human Tenon's capsule fibroblasts (HTFs). LiCl treatment reduced expression of TGF-ß1-induced α-SMA expression in HTFs. LiCl also decreased Akt phosphorylation induced by TGF-ß1. TGF-ß1-induced α-SMA expression was significantly decreased by LY294002 and Akt siRNA indicating that these changes are mediated by the PI3K/Akt pathway. Thus, LiCl induces the suppression of transdifferentiation stimulated by TGF-ß1 by the regulation of PI3K/Akt signaling in HTFs.

Sulfated CXCR3 Peptide Trap Use as a Promising Therapeutic Approach for Age-Related Macular Degeneration.

BACKGROUND AND OBJECTIVES: Chemokines have various biological functions and potential roles in the development or progression of neuroinflammatory diseases. However, the specific pathogenic roles of chemokines in the major cause for vision loss among the elderly, the leading cause of blindness in older individuals, remain elusive. Chemokines interact with their receptors expressed in the endothelium and on leukocytes. The sulfation of tyrosine residues in chemokine receptors increases the strength of ligand-receptor interaction and modulates signaling. Therefore, in the present study, we aimed to construct a human recombinant sulfated CXCR3 peptide trap (hCXCR3-S2) and mouse recombinant sulfated CXCR3 peptide trap (mCXCR3-S2) to demonstrate in vivo effects in preventing choroidal neovascularization (CNV) and chemotaxis. MATERIALS AND METHODS: We generated expression vectors for mCXCR3-S2 and hCXCR3-S2 with GST domains and their respective cDNA sequences. Following overexpression in E. coli BL21 (DE3), we purified the fusion proteins from cell lysates using affinity chromatography. First, the impact of hCXCR3-S2 was validated in vitro. Subsequently, the in vivo efficacy of mCXCR3-S2 was investigated using a laser-induced CNV mouse model, a mouse model of neovascular age-related macular degeneration (AMD). RESULTS: hCXCR3-S2 inhibited the migration and invasion of two human cancer cell lines. Intravitreal injection of mCXCR3-S2 attenuated CNV and macrophage recruitment in neovascular lesions of mouse models. These in vitro and in vivo effects were significantly stronger with CXCR3-S2 than with wild-type CXCR3 peptides. CONCLUSION: These findings demonstrate that the sulfated form of the CXCR3 peptide trap is a valuable tool that could be supplemented with antivascular endothelial growth factors in AMD treatment.

Inhibitory effect of recombinant tyrosine­sulfated madanin­1, a thrombin inhibitor, on the behavior of MDA­MB­231 and SKOV3 cells in vitro.

Thrombin, which plays a crucial role in hemostasis, is also implicated in cancer progression. In the present study, the effects of the thrombin­targeting recombinant tyrosine­sulfated madanin­1 on cancer cell behavior and signaling pathways compared with madanin­1 wild­type (WT) were investigated. Recombinant madanin­1 2 sulfation (madanin­1 2S) and madanin­1 WT proteins were generated using Escherichia coli. SKOV3 and MDA­MB­231 cells were treated with purified recombinant proteins with or without thrombin stimulation. Migration and invasion of cells were analyzed by wound healing assay and Transwell assay, respectively. Thrombin markedly increased cell migration and invasion in both SKOV3 and MDA­MB­231 cells, which were significantly suppressed by madanin­1 2S (P<0.05). Madanin­1 2S also significantly suppressed thrombin­induced expression of phosphorylated (p)­Akt and p­extracellular signal­regulated kinase in both cell lines (P<0.05), whereas madanin­1 WT had no effect on the expression levels of these proteins in MDA­MB­231 cells. Furthermore, madanin­1 2S significantly reversed the effects of thrombin on E­cadherin, N­cadherin and vimentin expression in MDA­MB­231 cells (P<0.05), whereas madanin­1 WT did not show any effect. In conclusion, madanin­1 2S suppressed the migration and invasion of cancer cells more effectively than madanin­1 WT. It is hypothesized that inhibiting thrombin via the sulfated form of madanin­1 may be a potential candidate for enhanced cancer therapy; however, further in vivo validation is required.

The evolution of chemokine release supports a bimodal mechanism of spinal cord ischemia and reperfusion injury.

BACKGROUND: Paraplegia remains a devastating complication of thoracic aortic surgery. The mechanism of the antecedent spinal cord ischemia and reperfusion injury (IR) remains poorly described. IR involves 2 injuries, an initial ischemic insult and subsequent inflammatory amplification of the injury. This mechanism is consistent with the clinical phenomenon of delayed onset paraplegia. This study sought to characterize the inflammatory response in the spinal cord after IR and hypothesized that this would support a bimodal mechanism of injury. METHODS AND RESULTS: Male C57Bl/6 mice were subjected to 5 minutes of aortic arch and left subclavian occlusion with subsequent reperfusion to generate spinal cord ischemia. Functional outcomes were scored at 12-hour intervals. Spinal cords were harvested after 0, 6, 12, 18, 24, 36, and 48 hours of reperfusion. Cytokine levels were analyzed using a mouse magnetic bead-based multiplex immunoassay. Inflammatory chemokine concentrations (interleukin [IL]-1ß, IL-6, keratinocyte-derived cytokine, macrophage inflammatory protein-1α, monocyte chemotactic protein-1, RANTES, and tumor necrosis factor-α) peaked at 6 hours and 36 to 48 hours after reperfusion. Functional scores reflected initial gain in function with subsequent decline, inversely proportional to cytokine levels. Immunofluorescent staining demonstrated microglia activation at 12 and 48 hours. CONCLUSIONS: Spinal cord ischemia and reperfusion injury occurs in 2 phases, correlating to increases in inflammatory chemokines release and microglial activation. These observations chronologically parallel the too-common clinical syndrome of delayed-onset paraplegia. Understanding the molecular pathogenesis of this injury may allow future intervention to prevent this devastating complication.

ox-LDL induces PiT-1 expression in human aortic valve interstitial cells.

BACKGROUND: The aortic valve interstitial cell (AVIC) has been implicated in the pathogenesis of calcific aortic stenosis. When appropriately stimulated, AVICs undergo a phenotypic change from that of a myofibroblast to that of a bone-forming-like cell. An elevated blood level of low-density lipoprotein (LDL) cholesterol is a clinical risk factor for aortic stenosis, and oxidized LDL (ox-LDL) cholesterol has been consistently found in calcified aortic valve leaflets. However, whether it plays a role in the pathogenesis of aortic stenosis is unknown. The process of aortic valve leaflet calcification has been associated with the deposition of calcium phosphate, mediated in part by the phosphate inorganic transporter 1 (PiT-1), a sodium-phosphate ion cotransporter. Therefore, we hypothesized that ox-LDL induces an osteogenic change in human AVICs marked by the induction of PiT-1. Using isolated human AVICs, the purpose of the present study was to examine the effect of ox-LDL on the expression of PiT-1 and the osteogenic factor bone morphogenetic protein 2 (BMP-2), which is a protein necessary for bone formation. METHODS: Human AVICs were isolated from nonstenotic aortic valves obtained from the explanted hearts of patients undergoing cardiac transplantation (n = 4) and grown in culture. The cells were treated with serum-free media, serum-free media with dimethyl sulfoxide (vehicle control), 40 µg/mL of ox-LDL, or 40 µg/mL of ox-LDL plus 2.5 mM phosphonoformate hexahydrate acid. Phosphonoformate hexahydrate acid is a competitive inhibitor of PiT-1 by mimicking inorganic phosphate. Cell lysis was performed at 24 h after treatment. Cell lysates were analyzed using immunoblot and densitometry for PiT-1 and BMP-2. Statistical analysis was performed using analysis of variance. P < 0.05 was significant. RESULTS: ox-LDL stimulation of AVICs induced an increase in PiT-1 and BMP-2. ox-LDL induced increased production of the phosphate transporter, PiT-1, and the osteogenic factor, BMP-2. Inhibition of PiT-1 with phosphonoformate hexahydrate acid prevented ox-LDL-induced BMP-2 expression. CONCLUSIONS: These data offer mechanistic insight into the pathogenesis of calcific aortic stenosis.

Paeoniflorin, a monoterpene glycoside, attenuates lipopolysaccharide-induced neuronal injury and brain microglial inflammatory response.

Chronic activation of microglial cells endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. Paeoniflorin (PF), a water-soluble monoterpene glycoside found in the root of Paeonia lactiflora Pall, has a wide range of pharmacological functions, such as anti-oxidant, anti-inflammatory, and anti-cancer effects. Neuroprotective potential of PF has also been demonstrated in animal models of neuropathologies. Here, we have examined the efficacy of PF in the repression of inflammation-induced neurotoxicity and microglial inflammatory response. In organotypic hippocampal slice cultures, PF significantly blocked lipopolysaccharide (LPS)-induced hippocampal cell death and productions of nitric oxide (NO) and interleukin (IL)-1ß. PF also inhibited the LPS-stimulated productions of NO, tumor necrosis factor-α, and IL-1ß from primary microglial cells. These results suggest that PF possesses neuroprotective activity by reducing the production of proinflammatory factors from activated microglial cells.

Activation state of stromal inflammatory cells in murine metastatic pancreatic adenocarcinoma.

The histologic presence of macrophages (tumor-associated macrophages, TAMs) and neutrophils (tumor-associated neutrophils, TANs) has been linked to poor clinical outcomes for solid tumors. The exact mechanism for this association with worsened prognosis is unclear. It has been theorized that TAMs are immunomodulated to an alternatively activated state and promote tumor progression. Similarly, TANs have been shown to promote angiogenesis and tumor detachment. TAMs and TANs were characterized for activation state and production of prometastatic mediators in an immunocompetent murine model of pancreatic adenocarcinoma. Specimens from liver metastases were evaluated by immunofluorescence and immunoblotting. TAMS have upregulated expression of CD206 and CD163 markers of alternative activation, (4.14 ± 0.55-fold and 7.36 ± 1.13-fold over control, respectively, P < 0.001) but do not have increased expression of classically activated macrophage markers CCR2 and CCR5. TAMs also express oncostatin M (OSM). We found that TANs, not TAMs, predominantly produce matrix metalloproteinase-9 (MMP-9) in this metastatic tumor microenvironment, while MMP-2 production is pan-tumoral. Moreover, increased expression of VEGF colocalized with TAMs as opposed to TANs. TAMs and TANs may act as distinct effector cells, with TAMs phenotypically exhibiting alternative activation and releasing OSM and VEGF. TANs are localized at the invasive front of the metastasis, where they colocalize with MMP-9. Improved understanding of these interactions may lead to targeted therapies for pancreas adenocarcinoma.

Effect of fibrin glue as an adjuvant to hang-back surgery.

BACKGROUND: The hang-back surgery is a useful technique in the field of strabismus surgery. The aim of this study is to determine the stabilizing effects of fibrin glue as an adjuvant to hang-back surgery. MATERIALS AND METHODS: Four (4)-mm hang-back recessions of the superior rectus muscle was performed in 32 eyes of 16 rabbits. Only in the left eye of the 16 rabbits, fibrin glue was applied between the recessed muscle bed and the sclera at the end of hang-back surgery (fibrin glue group). After 6 weeks, we compared the stability of the recessed rectus muscle between the fibrin glue group and the control group by evaluating the displacement of the muscle. RESULTS: The frequency of stable insertion of the recessed muscle at the intended site was greater in the fibrin glue group (9 eyes) compared to the control group (3 eyes) (p=0.028). In the control group, 5 eyes showed anterior displacement and 8 eyes showed posterior displacement and in the fibrin glue group, 1 eye showed anterior displacement, and 6 eyes showed posterior displacement. Anterior displacement was more common in the control group (6.3% Vs 31.3%). The control group and the fibrin glue group showed similar histological findings on microscopic examination. CONCLUSIONS: Fibrin glue is effective in stabilizing the new rectus muscle insertion and decreasing the displacement in the hang-back surgery.

TGF-ß-stimulated aberrant expression of class III ß-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells.

The class III ß-tubulin isotype (ß(III)) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III ß-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-ß (TGF-ß) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-ß on the aberrant expression of class III ß-tubulin and the intracellular signaling pathway mediating these changes. TGF-ß-induced aberrant expression and O-linked-ß-N-acetylglucosamine (O-GlcNac) modification of class III ß-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-ß also stimulated phosphorylation of ERK. TGF-ß-induced aberrant expression of class III ß-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-ß stimulated aberrant expression of class III ß-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-ß stimulation and provide useful information towards understanding the pathogenesis of proliferative vitreoretinal diseases.

The change of the quantitative HBsAg level during the natural course of chronic hepatitis B.

BACKGROUND: There is insufficient information about HBsAg levels and their correlation with serum hepatitis B virus (HBV) DNA in chronic hepatitis B (CHB). AIMS: We aimed to describe HBsAg levels during various phases of CHB and to investigate the correlation with serum HBV DNA levels. METHODS: A total of 645 treatment-naïve Korean CHB patients were included in this retrospective cross-sectional study. They were categorized into immune tolerance (IT, n=56), HBeAg-positive hepatitis (EPH, n=150), inactive carrier (IC, n=274) and HBeAg-negative hepatitis (ENH, n=165). The baseline HBsAg and HBV DNA levels were measured. RESULTS: The mean HBsAg titres (log IU/ml) differed (P<0.001): IT 4.29, EPH 3.64, IC 2.05 and ENH 3.23. In 645 patients, HBsAg and HBV DNA showed a significant correlation (r=0.693, P<0.001), and this was also observed in the IT, EPH and IC groups (r=0.664, r=0.541, r=0.505, respectively, all P<0.001), but not in the ENH group (r=0.093, P=0.321). Age had a negative correlation with HBsAg (r=-0.451, P<0.001). The cirrhotic patients had a significantly lower HBsAg level than the non-cirrhotic patients (2.41 ± 1.36 vs. 3.02 ± 1.21 log IU/ml, P<0.001). CONCLUSIONS: The HBsAg level varied significantly in different phases of CHB and was correlated with HBV DNA during the IT, EPH and IC phases. These findings can provide additional information to understand the natural course and pathogenesis of CHB.

Association of a single nucleotide polymorphism near the interleukin-28B gene with response to hepatitis C therapy in Asian patients.

BACKGROUND AND AIMS: A single nucleotide polymorphism near the interleukin-28B (IL28B) gene has been shown to predict hepatitis C virus (HCV) treatment response. We aim to determine the role of the IL28B genotype in Asian patients. METHODS: A total of 118 patients (all Korean, 55 patients with genotype 1 infection and 63 patients with genotype 2 infection) were consecutively enrolled and analyzed. RESULTS: The sustained virological response (SVR) rate was 74% (87/118), while 26 patients (22%) relapsed and five patients were non-responders (4%). For rs8099917, the frequencies of major homozygotes (TT), heterozygotes (GT), and minor homozygotes (GG) were 0.85, 0.14 and 0.01, respectively. Of the 55 patients with HCV genotype 1 infection, the SVR rate was 67% and 44% (P = 0.19) and the non-response rate was 2% and 22% (P = 0.015) for the major allele and minor or hetero allele, respectively. Of the 63 patients with HCV genotype 2 infection, the SVR rate was 80% and 100% (P = 0.13) and the non-response rate was 4% and 0% (P = 0.55) for major allele and hetero allele, respectively. CONCLUSIONS: The IL28B genotype may help identify non-responding patients in HCV genotype 1, but not in HCV genotype 2. Because of the high frequency of favorable alleles and the low frequency of non-response, the IL28B polymorphism may play a smaller role in Asian patients.

JNK/FOXO-mediated neuronal expression of fly homologue of peroxiredoxin II reduces oxidative stress and extends life span.

Activation of c-Jun N-terminal kinase (JNK) signaling in neurons increases stress resistance and extends life span, in part through FOXO-mediated transcription in Drosophila. However, the JNK/FOXO target genes are unknown. Here, we identified Jafrac1, a Drosophila homolog of human Peroxiredoxin II (hPrxII), as a downstream effecter of JNK/FOXO signaling in neurons that enhances stress resistance and extends life span. We found that Jafrac1 was expressed in the adult brain and induced by paraquat, a reactive oxygen species-generating chemical. RNA interference-mediated neuronal knockdown of Jafrac1 enhanced, while neuronal overexpression of Jafrac1 and hPrxII suppressed, paraquat-induced lethality in flies. Neuronal expression of Jafrac1 also significantly reduced ROS levels, restored mitochondrial function, and attenuated JNK activation caused by paraquat. Activation of JNK/FOXO signaling in neurons increased the Jafrac1 expression level under both normal and oxidative stressed conditions. Moreover, neuronal knockdown of Jafrac1 shortened, while overexpression of Jafrac1 and hPrxII extended, the life span in flies. These results support the hypothesis that JNK/FOXO signaling extends life span via amelioration of oxidative damage and mitochondrial dysfunction in neurons.

Ocular surface inflammation, and nerve growth factor level in tears in active thyroid-associated ophthalmopathy.

PURPOSE: To measure tear nerve growth factor (NGF) concentrations in cases of active thyroid-associated ophthalmopathy (TAO) before and after glucocorticoid treatment, and to correlate NGF levels with disease inflammatory activity and thyroid autoantibody concentration. METHODS: The study involved 20 patients with active TAO and 20 age- and gender-matched controls. Tear break-up time (BUT) was obtained, the Schirmer test was performed, and tear NGF/total protein ratio was measured in control subjects and patients with active TAO before, and 2 and 4 weeks after, steroid treatment. RESULTS: Tear BUT and Schirmer values significantly increased after 2 and 4 weeks of steroid treatment (p < 0.001 and p = 0.004 respectively). Baseline tear NGF/total protein ratio was higher in patients with active TAO than in control subjects, and the ratio significantly decreased after 2 and 4 weeks of steroid treatment (p < 0.001). Tear NGF/total protein ratio did not correlate with inflammatory activity score, exophthalmos value and thyroid binding inhibiting immunoglobulin (TBII) level (p > 0.05). CONCLUSIONS: Tear NGF may have a specific role in ocular surface inflammation, which protects against ocular surface damage in patients with active TAO. Anti-inflammatory treatment significantly reduced the level of NGF in tears, increased tear film stability and production, and decreased congestive symptoms.

HIF1α-mediated TRAIL Expression Regulates Lacrimal Gland Inflammation in Dry Eye Disease.

PURPOSE: The purpose of this study was to investigate the expression of death ligands in the lacrimal glands (LGs), identify upstream factors that regulate their expression, and determine the functional roles of these factors in the pathogenesis of dry eye disease (DED). METHODS: For DED experiment, ex vivo coculture system with LG and in vivo murine model using a controlled environment chamber were utilized. C57BL/6 mice and hypoxia-inducible factor (HIF)-1α conditional knockout (CKO) mice were used. Immunohistochemical staining, polymerase chain reaction, and immunoblotting were performed to determine levels of death ligands including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in DED-induced LGs. Additionally, acinar cell and CD45+ cell apoptosis was determined with neutralizing TRAIL treatment. RESULTS: Desiccating stress significantly increased HIF-1α expression in LG-acinar cells. Furthermore, HIF-1α deficiency significantly enhanced the infiltration of CD45+ inflammatory cells in LG and induced LG-acinar cell death. Meanwhile, only TRAIL expression was increased in DED-LG, but abrogated in HIF-1α CKO. Interestingly, the main source of TRAIL was the CD45- LG-acinar cells, but not CD45+ immune cells after DED induction. Using ex vivo coculture system, we confirmed LG-induced apoptosis of immune cells via HIF-1α-mediated TRAIL secretion following DED. Consistent with ex vivo, the insufficiency of HIF-1α and TRAIL enhanced recruitment of inflammatory cells to the LG and subsequently exacerbated ocular surface damage in DED mice. CONCLUSIONS: Our findings offer novel insight into the regulatory function of acinar cell-derived TRAIL in limiting inflammatory damage and could be implicated in the development of potential therapeutic strategies for DED.

PDGF stimulation of Müller cell proliferation: Contributions of c-JNK and the PI3K/Akt pathway.

Platelet-derived growth factor (PDGF) has a critical role in proliferative vitreoretinopathy (PVR) as a chemoattractant and mitogen for retinal pigment epithelial cells and retinal glial cells. Here, we investigated the potential effects of PDGF on the proliferation of Müller cells and the intracellular signaling pathway mediating these changes. PDGF induced Müller cell proliferation and increased phosphorylation of the PDGF receptor (PDGFR), as shown by an MTT assay and immunoprecipitation analyses. Both effects were blocked by JNJ, a PDGFR-selective tyrosine kinase inhibitor. PDGF also stimulated phosphorylation of c-JNK and Akt. PDGF-induced Müller cell proliferation was significantly reduced by pre-treatment with SP600125 and LY294002, inhibitors of c-JNK and Akt phosphorylation, respectively. Our findings collectively indicate that PDGF-stimulated Müller cell proliferation occurs via activation of the c-JNK and PI3K/Akt signaling pathways. These data provide useful information in establishing the role of Müller cells in the development of proliferative vitreoretinopathy.