Topical TSG-6 Administration Protects the Ocular Surface in Two Mouse Models of Inflammation-Related Dry Eye.

PURPOSE: To investigate the therapeutic potential of TNF-α stimulated gene/protein (TSG)-6 in two mouse models of inflammation-mediated dry eye syndrome (DES). METHODS: We created inflammation-mediated DES in mice by injecting concanavalin A (ConA; 10 mg/mL) into intraorbital and extraorbital lacrimal glands. Recombinant TSG-6 (1 µg in phosphate-buffered solution [PBS]) or the same volume of PBS was administered topically to eyes of the mice four times a day (QID) for 1 week. In parallel experiments, we topically applied TSG-6 (1 µg) or PBS QID to eyes of 12-week-old NOD.B10.H2b mice, a model for primary Sjögren's syndrome. Seven days later, tear production was measured, and the corneal surface was observed for epithelial defects. The number of goblet cells was evaluated in the forniceal conjunctiva. The levels of proinflammatory cytokines were analyzed in the cornea, conjunctiva, and lacrimal glands. Also, in vitro experiments were performed using cultures of corneal epithelial cells (CECs) to test the effects of TSG-6 on cell proliferation and migration. RESULTS: Topical TSG-6 administration improved tear production and reduced corneal epithelial defects both in ConA-injected mice and NOD.B10.H2b mice. The conjunctival goblet cell density was higher in TSG-6-treated eyes than in PBS-treated eyes. The expression of proinflammatory cytokines in the cornea, conjunctiva, and intraorbital gland was repressed by TSG-6, while the levels of proinflammatory cytokines in the extraorbital gland were not changed. In vitro experiments revealed that TSG-6 promoted the migration of CECs, but did not affect the proliferation. CONCLUSIONS: Topical TSG-6 protected the ocular surface by suppressing inflammation and promoting corneal epithelial wound healing.

Mesenchymal stem/stromal cells protect against autoimmunity via CCL2-dependent recruitment of myeloid-derived suppressor cells.

Exogenously administered mesenchymal stem/stromal cells (MSCs) suppress autoimmunity despite transient engraftment. However, the mechanism is unclear. In this study, we report a novel mechanism by which MSCs modulate the immune system by recruiting myeloid-derived suppressor cells in a mouse model of experimental autoimmune uveitis (EAU). Intravenous infusion of MSCs blocked EAU development and reduced Th1 and Th17 responses. Time course analysis revealed an increase of MHC class II(lo)Ly6G(-)Ly6C(hi)CD11b(+) cells in draining lymph nodes by MSCs. These Ly6C(hi)CD11b(+) cells suppressed CD4(+) cell proliferation and Th1/Th17 differentiation and induced CD4(+) cell apoptosis. Adoptive transfer of Ly6C(hi)CD11b(+) cells ameliorated EAU, whereas depletion of Ly6C(hi)CD11b(+) cells abrogated the effects of MSCs. 1.8% of MSCs were present in draining lymph nodes 1 d after infusion, and MSCs with CCL2 knockdown did not increase MHC class II(lo)Ly6G(-)Ly6C(hi)CD11b(+) cells and failed to attenuate EAU. Therefore, our findings demonstrate that MSCs suppress autoimmunity by recruiting myeloid-derived suppressor cells into sites of inflammation in a CCL2-dependent manner.

Mesenchymal stem/stromal cells protect the ocular surface by suppressing inflammation in an experimental dry eye.

Dry eye syndrome (DES) is one of the most common ocular diseases affecting nearly 10% of the US population. Most of the currently available treatments are palliative, and few therapeutic agents target biological pathway of DES. Although DES is a multifactorial disease, it is well-known that inflammation in the ocular surface plays an important role in the pathogenesis of DES. Mesenchymal stem/stromal cells (MSCs) have been shown to repair tissues by modulating excessive immune responses in various diseases. Therefore, we here investigated the therapeutic potential of MSCs in a murine model of an inflammation-mediated dry eye that was induced by an intraorbital injection of concanavalin A. We found that a periorbital administration of MSCs reduced the infiltration of CD4(+) T cells and the levels of inflammatory cytokines in the intraorbital gland and ocular surface. Also, MSCs significantly increased aqueous tear production and the number of conjunctival goblet cells. Subsequently, corneal epithelial integrity was well-preserved by MSCs. Together, the results demonstrate that MSCs protect the ocular surface by suppressing inflammation in DES, and suggest that MSCs may offer a therapy for a number of ocular surface diseases where inflammation plays a key role.

TSG-6 protects corneal endothelium from transcorneal cryoinjury in rabbits.

PURPOSE: To investigate the effect of an anti-inflammatory protein, TNF-α stimulated gene/protein (TSG)-6 and an antiapoptotic protein, stanniocalcin (STC)-1 on corneal endothelium in rabbits with transcorneal cryoinjury. METHODS: Transcorneal freezing (-80°C) was applied to rabbit corneas for 30 seconds. Immediately post injury, either TSG-6 (10 µg/100 µL), STC-1 (10 µg/100 µL), or the same volume of balanced salt solution (BSS) was injected into the anterior chamber. Each eye was examined for corneal opacity, corneal thickness, endothelial cell density, and endothelial hexagonality every 2 to 6 hours for 48 hours post injury. The concentrations of myeloperoxidase (MPO) and IL-1ß were measured in the aqueous humor every 6 hours. At 48 hours post injury, each cornea was assayed for TNF-α, IL-1ß, IL-6, and MPO, and histologically evaluated with alizarin red-trypan blue staining, hematoxylin-eosin staining, and immunostaining for neutrophils. RESULTS: Tumor necrosis factor-α stimulated gene/protein-6 significantly decreased the development of corneal opacity and edema after cryoinjury compared with STC-1 or BSS. The corneal endothelial cell density and hexagonality were markedly preserved by TSG-6. The mRNA levels of TNF-α, IL-1ß, and IL-6 in the cornea and the protein levels of MPO and IL-1ß in the aqueous humor and cornea were significantly lower in TSG-6-treated eyes than BSS-treated controls. Similarly, the expression of fibroblast growth factor-2 was reduced by TSG-6 treatment. Histologic evaluation demonstrated that neutrophil infiltration of the cornea was decreased in TSG-6-treated eyes. CONCLUSIONS: Tumor necrosis factor-α stimulated gene/protein-6 protected corneal endothelial cells from transcorneal cryoinjury through suppression of inflammation.

Analysis of etoxazole in red pepper after major modification of QuEChERS for gas chromatography-nitrogen phosphorus detection.

A major modification to the QuEChERS (quick, easy, cheap, effective, rugged and safe) method was developed for the analysis of etoxazole in red pepper using gas chromatography coupled with a nitrogen-phosphorus detector. Etoxazole was extracted with acetonitrile, partitioned with magnesium sulfate and purified with a solid-phase extraction cartridge. The method showed good linearity with a determination coefficient (R(2) ) of 0.998 for the 0.02-2.0 mg/L concentration range. The method was validated using blank red pepper spiked at 0.2 and 1.0 mg/kg, and the average recovery rate was 74.4-79.1% with relative standard deviations <5% for intra- and inter-day precision. The limits of detection and quantification were 0.007 and 0.02 mg/kg, respectively. The developed method was successfully applied to field-incurred samples, and the presence of etoxazole residues was confirmed using gas chromatography/mass spectrometry.

Optimization of supercritical fluid extraction method for detection of fluquinconazole and tetraconazole in soil using gas chromatography and confirmation using GC-MS: application to dissipation kinetics.

The aim of this study was to establish an analytical method to detect fluquinconazole and tetraconazole in soil using supercritical fluid extraction (SFE) and gas chromatography (GC). The optimal extraction conditions for SFE were: temperature, 60 °C; pressure, 280 kg/cm(2) ; extraction time, 50 min; and a 10% modifier ratio. The linearity of the calibration curves was good and yielded a determination coefficient (R(2) ) ≥ 0.995. The soil samples were fortified with known quantities of the analytes at three different concentrations (0.01, 0.02 and 0.1 µg/g for fluquinconazole; 0.05, 0.1 and 0.5 µg/g for tetraconazole), and the recoveries ranged between 83.7 and 94.1%. The intra- and inter-day relative standard deviations were 1.3-10.6 and 2.2-11.9% for fluquinconazole and tetraconazole, respectively. The limit of detection and limit of quantitation were 0.002 and 0.01 µg/g for fluquinconazole and 0.01 and 0.05 for tetraconazole, respectively. The method was successfully applied to the analysis of soil residues collected from an onion field. The results show that a combination of SFE and GC can be used as an environmentally friendly technique to detect fungicides in soil.

A modified QuEChERS method for simultaneous determination of flonicamid and its metabolites in paprika using tandem mass spectrometry.

A modified quick, easy, cheap, effective, rugged and safe (QuEChERS) acetate-buffered sample preparation method was developed to improve extraction recovery of flonicamid and its two metabolites (4-trifluoromethylnicotinic acid and N-(4-trifluoromethylnicotinoyl)glycine) in paprika followed by analysis using tandem mass spectrometry. Acidified acetonitrile (containing 5% acetic acid) was used as an extraction solvent and partitioning was carried out using sodium chloride. The extract was then cleaned up using C18. The linearity over a concentration range of 0.005-1 µg/mL was good with a determination coefficient (R(2))>0.9997. Recovery at three different fortification levels was 82.2-101.7% with a relative standard deviation <10 for all analytes. The limit of quantitation of 0.01 mg/kg was quite lower than the maximum residue level set by the Korea Food and Drug Administration (2mg/kg). The method was successfully applied to determine flonicamid and its metabolites from field incurred samples. The undulating residue pattern observed for the parent analyte together with its metabolites could explain the movement behavior of systemic pesticides into plants over time.

Identification of volatile organic compounds generated from healthy and infected powdered chili using solvent-free solid injection coupled with GC/MS: application to adulteration.

To investigate adulteration in commercial chili powder, the volatile organic compounds of healthy and infected powdered chili pepper were characterized using a solvent-free solid injector (SFSI) coupled with gas chromatography/mass spectrometry (GC/MS). Except for one compound (capillary compound for blank), 43 compounds were identified in healthy and infected chili powder. Specifically, 31, 36, and 41 compounds were identified in healthy, medium-infected, and severely infected chili powder. Among these compounds, acetic acid (13.77%), propanal (2.477%), N-methylpyrrole (1.986%), and 2-methyl-propanal (1.768%) were leading volatiles in the healthy chili powder. In contrast, infected chili powder contained 9,12-octadecadienoic acid, ethyl ester (15.984%), acetic acid (11.249%), hexadecanoic acid, methyl ester (3.3%), N-methylpyrrole (3.221%), and 2-furanmethanol (2.629%) as major compounds. Trimethylamine and isosorbide were detected in both medium and severely infected chili, but not in healthy chili. This means that these compounds could be used as biomarkers to distinguish between healthy and infected chili. The proposed technique was applied to 12 commercial chili powders, and trimethylamine and isosorbide were detected in six samples. These results suggest that a contaminated chili that was added to a healthy one could be successfully identified by a combination of the SFSI and GC/MS.

Intravenous infusion of mesenchymal stem/stromal cells decreased CCR7(+) antigen presenting cells in mice with corneal allotransplantation.

PURPOSE: To investigate the effects of intravenous (IV) infusion of human mesenchymal stem/stromal cells (hMSCs) on activation and migration of CCR7(+) antigen presenting cells (APCs) in allogeneic corneal transplantation. MATERIALS AND METHODS: We first analyzed the cellular and molecular profiles of draining lymph nodes (DLNs) in early and late phases after syngeneic or allogeneic corneal transplantation in mice, and then investigated the effects of hMSCs on APCs expressing CCR7, a key molecule implicated in APC migration to DLNs. RESULTS: After early transplantation, the numbers of MHC class II(+)CD11b(+)CD11c(-), MHC class II(+)CD11b(-)CD11c(+), and MHC II(+)CD11b(+)CD11c(+) cells as well as the levels of APC-derived cytokines (IL-12a and IL-12b) driving the Th1 response were increased in both syngeneic and allogeneic transplants indicating activation of APCs. In late phase, the numbers of CD3(+)CD4(+)CD8(-) and CD3(+)CD4(-)CD8(+) cells and the levels of T cell-derived cytokines were increased in allogeneic transplants, but not in syngeneic transplants indicating immune rejection. The peri-transplant infusion of IV hMSCs significantly reduced the numbers of CCR7(+)CD11b(+) or CCR7(+)CD11c(+) cells in DLNs and the cornea in the early phase. Also, the expression of CCR7 and its ligands, CCL19, CCL21, and CXC3R as well as IL-12 were markedly decreased by hMSCs in the cornea and DLNs. CONCLUSIONS: IV hMSCs reduced the activation and migration of CCR7(+) APCs in the cornea and DLNs in allogeneic corneal transplantation.

Determination of kresoxim-methyl and its thermolabile metabolites in pear utilizing pepper leaf matrix as a protectant using gas chromatography.

Kresoxim-methyl and its two thermolabile metabolites, BF 490-2 and BF 490-9, were analyzed in pear using a pepper leaf matrix protection to maintain the metabolites inside the gas chromatography system. Samples were extracted with a mixture of ethyl acetate and n-hexane (1:1, v/v) and purified and/or separated using a solid phase extraction procedure. The pepper leaf matrix was added and optimized with cleaned pear extract to enhance metabolite sensitivity. Matrix matched calibration was used for kresoxim-methyl in the pear matrix and for metabolites in the pear mixed with pepper leaf matrix. Good linearity was obtained for all analytes with a coefficient of determination, r (2) â©¾ 0.992. Limits of detection (LOD) and quantification (LOQ) were 0.006 and 0.02 mg kg(-1) and 0.02 and 0.065 mg kg(-1) for kresoxim-methyl and the metabolites, respectively. Recoveries were carried out at two concentration levels and were 85.6-97.9% with a relative standard deviation <2.5%. The method was successfully applied to field incurred pear samples, and only kresoxim-methyl was detected at a concentration of 0.03 mg kg(-1).

Development of a simple extraction and oxidation procedure for the residue analysis of imidacloprid and its metabolites in lettuce using gas chromatography.

Simple extraction and optimised oxidation procedures were developed for the determination of the total residues of imidacloprid and its metabolites (containing the 6-chloropicolyl moiety) in lettuce using a gas chromatography-micro electron capture detector (GC-µECD). Samples were extracted with acetonitrile, and the extract was then evaporated. The remaining residues were dissolved in water and oxidised with potassium permanganate to yield 6-chloronicotinic acid (6-CAN). The acid residues were further dissolved in n-hexane:acetone (8:2, v/v) and then silylated with MSTFA (N-methyl-N-(trimethylsilyl)trifluoroacetamide) to 6-chloronicotinic acid trimethylsilyl ester. Calibration curves were linear over the concentration ranges (0.025-5 µg mL(-1)) with a determination coefficient (r(2)) of 0.991. The limits of detection and quantification were 0.015 and 0.05 mg kg(-1), respectively. Recoveries at two fortification levels ranged between 72.8% and 108.3% with relative standard deviation (RSD) lower than 8%. The method was effective, and sensitive enough to determine the total residues of imidacloprid and its metabolites in field-incurred lettuce samples. The identity of the analyte was confirmed using gas chromatography-tandem mass spectrometry (GC-MS/MS).

Cross-reactivity between decellularized porcine corneal lamellae for corneal xenobridging and subsequent corneal allotransplants.

The purpose of this study is to investigate cross-reactivity between hypertonic saline-treated decellularized porcine corneal lamellae for corneal xenobridging and subsequent corneal allotransplants. Five Chinese rhesus macaques, which had undergone anterior partial thickness corneal transplantation using hypertonic saline-treated decellularized porcine corneal lamellae in preceding experiments, were used as recipients for subsequent full-thickness corneal allografts. To determine whether sensitization of recipients to xenoantigens leads to cross-reactivity against alloantigens, we compared; (i) allogeneic one-way mixed lymphocyte reaction (MLR) of peripheral blood mononuclear cells (PBMCs) from xeno-sensitized recipients with that of PBMCs from naïve rhesus macaques, and (ii) amounts of IgG antibodies that bound to the PBMCs of a rhesus panel (five monkeys) before and after xeno-sensitization. Graft survival and immunologic profiles including memory T-cell subsets and donor rhesus-specific antibodies were also evaluated. No hyperacute or acute rejection was observed within a month of subsequent allotransplantation in any recipient. Alloreactivity by MLR was not different between xeno-sensitized rhesus recipients and naïve rhesus monkeys. Panel-reactive IgG antibodies were unchanged after xeno-sensitization, and no change in donor rhesus-specific antibodies was observed in any recipient. No significant changes in memory T-cell subsets were observed during the early post-operative period in any recipient. Decellularized porcine corneal lamellae may not increase cross-reactivity to alloantigens, and thus, porcine corneal lamellae may be used as a bridge to subsequent corneal allografting.

A matrix sensitive gas chromatography method for the analysis of pymetrozine in red pepper: application to dissipation pattern and PHRL.

A gas chromatography (GC) method for the analysis of pymetrozine was developed after utilizing matrix enhancement effect of pymetrozine to nitrogen phosphorus detector (NPD). Samples were extracted with acetonitrile and purified through primary secondary amine (PSA) and C18 dispersive sorbent. Matrix-matched calibration curve prepared after spiking standard pymetrozine across the studied range of concentrations (0.003-1.0mg/L) into blank red pepper extract was excellent with a determination coefficients (R(2))=1. Recovery studies were carried out at three concentration levels (0.04, 0.4, and 2.0mg/kg, n=3) and the rates were ranged between 77.2% and 109.1%, with relative standard deviations ranged from 1.3% to 16.4%. The developed method was applied to field samples to characterize the dissipation pattern, half life, and pre-harvest residue limits (PHRL). The dissipation rates of the analyte were ascribed to first-order kinetics with half-life of 2.7 and 2.5days for recommended and double the recommended doses. From the PHRL curve, we could predict that if the residue level of pymetrozine is below the 1.23mg/kg at 10days or 0.71mg/kg at 7days before harvest, then the residues will be below the maximum residue limits (MRL=0.2mg/kg) established by the Korea Food and Drug Administration (KFDA).

Analysis of macrophage phenotype in rejected corneal allografts.

PURPOSE: We investigated the phenotype of macrophages infiltrating rejected corneal allografts. METHODS: We performed allogeneic or syngeneic corneal transplantation in mice, and humanely killed animals at day 28 during allograft rejection when 60% of corneal allografts were rejected. We divided allografts into two groups: grafts with rejection as rejectors and grafts without rejection as nonrejectors, and analyzed for macrophage infiltration and their phenotype using immunohistochemistry. In addition, we investigated the time course of proinflammatory cytokines and chemokines by analyzing corneal grafts at days 7, 28, and 42 using real-time RT-PCR. Also, we assayed human corneal allografts with chronic graft failure. RESULTS: We found that a large number of CD11b(+), F4/80(+), or inducible nitrous oxide synthase cells (iNOS(+)) infiltrated corneal allografts during rejection in mice, while the cells were found rarely in syngeneic or allogeneic grafts that were not rejected. There were rare CD11c(+) cells in rejectors and nonrejectors. Many mannose receptor cells (MRC(+)) were present in nonrejectors, but not in rejectors. The levels of Th1 cytokines, IFN-γ, and IL-2 were highly increased in rejectors at day 28, indicating immune rejection. Also, the levels of IL-12a, IL-1ß, TNF-α, CCL3, and iNOS that are produced by activated macrophages were markedly increased in rejectors at day 28, compared to syngeneic grafts or nonrejectors. Similarly, human corneal allografts with chronic graft failure had higher levels of IL-12a, IL-1ß, CCL3, and iNOS than controls. CONCLUSIONS: Increased numbers of macrophages in rejected corneal allografts implicate that these cells might contribute to the immunopathogenesis of corneal graft rejection.

Effect of electrocauterization on the inflammation of the conjunctiva in experimental animal model.

PURPOSE: Recently, conjunctivochalasis repair surgery using electrocauterization has been gaining popularity. However, patients with electrocauterized conjunctivoplasty tend to complain of more postoperative pain than patients undergoing simple excision with suturing. Therefore, we investigated the effects of electrocauterization on inflammation of the conjunctiva using an experimental animal model and compared these with the effects of simple excision with suturing. METHODS: Ten New Zealand white rabbits underwent cauterization in the right eyes and excision and suturing in the left eyes. For each eye, we excised or electrocauterized the inferior bulbar conjunctiva, 1 mm in width and 6 mm in length, 2 mm from the limbus. A fine-needle electrode was inserted subconjunctivally, and electrocauterization was performed. In the contralateral eye, the corresponding area was excised and re-approximated with 10-0 nylon sutures. Sutures were removed after 14 days. Tissue samples were obtained at 21 days post-procedure, and inflammatory cells were counted in five randomly selected fields (×200) on hematoxylin-eosin stained slides. Tumor necrosis factor (TNF)-α and interleukin (IL)-1ß concentrations in tears were measured using enzyme linked immunosorbent assays. RESULTS: All cauterized eyes demonstrated smooth surface healing without scarring after 5 days, whereas sutured eyes presented with mild edema with some scarring until the suture was removed. The number of inflammatory cells was significantly greater in sutured eyes compared with cauterized eyes (p = 0.035, Mann-Whitney U-test) at 21 days post-procedure. Tear TNF-α and IL-1ß concentrations at 21 days were similar in both groups. CONCLUSIONS: Electrocauterization for conjunctivoplasty seems to be advantageous in terms of inflammation compared with simple suturing and excision.

Quantifying fenobucarb residue levels in beef muscles using liquid chromatography-tandem mass spectrometry and QuEChERS sample preparation.

This paper describes a comparison of the properties of the three versions of the QuEChERS method (quick, easy, cheap, effective, rugged and safe) - the original (unbuffered), acetate-buffered, and citrate-buffered methods - for the determination of fenobucarb residues in beef muscles via liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI(+)-MS/MS). The recovery results were good for all the versions; however, the acetate-buffered version gave higher and more consistent recoveries for fenobucarb than the other versions. Performance characteristics, such as linearity, accuracy, and precision were determined. Matrix-matched standard calibration was used for quantification, obtaining recoveries in the range of 83.7-93.4% with relative standard deviations of <5%, at two spiking levels: 10 and 40 µg/kg. The limits of detection (LOD) and quantification (LOQ) were estimated to be 1.5 and 5 µg/kg, respectively. Finally, the method was applied to the analysis of 15 market samples, and no residues were found over the limit of quantification. The method developed was found able to determine the analyte with satisfactory intensity and accuracy.

Intravenous mesenchymal stem cells prevented rejection of allogeneic corneal transplants by aborting the early inflammatory response.

Mesenchymal stem/progenitor cells (MSCs) were reported to enhance the survival of cellular and organ transplants. However, their mode of action was not established. We here used a mouse model of corneal allotransplantation and demonstrated that peri-transplant intravenous (i.v.) infusion of human MSCs (hMSCs) decreased the early surgically induced inflammation and reduced the activation of antigen-presenting cells (APCs) in the cornea and draining lymph nodes (DLNs). Subsequently, immune rejection was decreased, and allograft survival was prolonged. Quantitative assays for human GAPDH revealed that <10 hMSCs out of 1 × 10(6) injected cells were recovered in the cornea 10 hours to 28 days after i.v. infusion. Most of hMSCs were trapped in lungs where they were activated to increase expression of the gene for a multifunctional anti-inflammatory protein tumor necrosis factor-α stimulated gene/protein 6 (TSG-6). i.v. hMSCs with a knockdown of TSG-6 did not suppress the early inflammation and failed to prolong the allograft survival. Also, i.v. infusion of recombinant TSG-6 reproduced the effects of hMSCs. Results suggest that hMSCs improve the survival of corneal allografts without engraftment and primarily by secreting TSG-6 that acts by aborting early inflammatory responses. The same mechanism may explain previous reports that MSCs decrease rejection of other organ transplants.