A Soluble, Minimalistic Glycosylphosphatidylinositol Transamidase (GPI-T) Retains Transamidation Activity.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a eukaryotic, post-translational modification catalyzed by GPI transamidase (GPI-T). The Saccharomyces cerevisiae GPI-T is composed of five membrane-bound subunits: Gpi8, Gaa1, Gpi16, Gpi17, and Gab1. GPI-T has been recalcitrant to in vitro structure and function studies because of its complexity and membrane-solubility. Furthermore, a reliable, quantitative, in vitro assay for this important post-translational modification has remained elusive despite its discovery more than three decades ago.Three recent reports describe the structure of GPI-T from S. cerevisiae and humans, shedding critical light on this important enzyme and offering insight into the functions of its different subunits. Here, we present the purification and characterization of a truncated soluble GPI-T heterotrimer complex (Gpi823-306, Gaa150-343, and Gpi1620-551) without transmembrane domains. Using this simplified heterotrimer, we report the first quantitative method to measure GPI-T activity in vitro and demonstrate that this soluble, minimalistic GPI-T retains transamidase activity. These results contribute to our understanding of how this enzyme is organized and functions, and provide a method to screen potential GPI-T inhibitors.

Airborne Particulates Affect Corneal Homeostasis and Immunity.

Purpose: To determine the effects of airborne particulate matter (PM) <2.5 µm in vitro and on the normal and Pseudomonas aeruginosa (PA)-infected cornea. Methods: An MTT viability assay tested the effects of PM2.5 on mouse corneal epithelial cells (MCEC) and human corneal epithelial cells (HCET). MCEC were tested for reactive oxygen species using a 2',7'-dichlorodihydrofluorescein assay; RT-PCR determined mRNA levels of inflammatory and oxidative stress markers in MCEC (HMGB1, toll-like receptor 2, IL-1ß, CXCL2, GPX1, GPX2, GR1, superoxide dismutase 2, and heme oxygenase 1) and HCET (high mobility group box 1, CXCL2, and IL-1ß). C57BL/6 mice also were infected and after 6 hours, the PM2.5 was topically applied. Disease was graded by clinical score and evaluated by histology, plate count, myeloperoxidase assay, RT-PCR, ELISA, and Western blot. Results: After PM2.5 (25-200 µg/mL), 80% to 90% of MCEC and HCET were viable and PM exposure increased reactive oxygen species in MCEC and mRNA expression levels for inflammatory and oxidative stress markers in mouse and human cells. In vivo, the cornea of PA+PM2.5 exposed mice exhibited earlier perforation over PA alone (confirmed histologically). In cornea, plate counts were increased after PA+PM2.5, whereas myeloperoxidase activity was significantly increased after PA+PM2.5 over other groups. The mRNA levels for several proinflammatory and oxidative stress markers were increased in the cornea in the PA+PM2.5 over other groups; protein levels were elevated for high mobility group box 1, but not toll-like receptor 4 or glutathione reductase 1. Uninfected corneas treated with PM2.5 did not differ from normal. Conclusions: PM2.5 triggers reactive oxygen species, upregulates mRNA levels of oxidative stress, inflammatory markers, and high mobility group box 1 protein, contributing to perforation in PA-infected corneas.

Glycyrrhizin Use for Multi-Drug Resistant Pseudomonas aeruginosa: In Vitro and In Vivo Studies.

Purpose: Our purpose was to test glycyrrhizin (GLY) effects and ciprofloxacin interactions on multidrug resistant (MDR) isolates of Pseudomonas aeruginosa in vitro and in vivo in a mouse model of keratitis. Methods: A Hardy-disk tested antibiotic sensitivity of isolates MDR9 (nonocular) and B1045 (ocular). GLY MIC (both isolates) and ciprofloxacin was determined spectrophotometrically. A live/dead assay using confocal microscopy and plate count, tested GLY effects on bacterial permeabilization/viability. Proteomics profiled bacterial efflux pumps (MDR9 vs. PAO1); RT-PCR comparatively tested GLY effects on their mRNA expression levels. The activity of efflux pumps was tested using ethidium bromide (EB); and scanning electron microscopy (SEM) visualized the effects of GLY treatment of bacteria. A combination of GLY and ciprofloxacin was tested in C57BL/6 mice (begun 18 hours after infection) and disease scored, photographed and MPO and plate counts done. Results: MDR9 was resistant to 6/12 and B1045 to 7/12 antibiotics (both to ciprofloxacin). MIC GLY for MDR9 was 40 mg/mL and 15 mg/mL for B1045. Ciprofloxacin MIC (32 µg/mL) was reduced 2-fold to 16 µg/mL when ciprofloxacin and GLY were combined. GLY altered bacterial membrane permeability and reduced viability. Proteomics revealed increased efflux pumps in MDR9 versus PAO1; GLY reduced their mRNA expression levels and EB suggested decreased activity. In C57BL/6 mice, treatment with GLY and ciprofloxacin versus ciprofloxacin, significantly reduced clinical scores, plate count, and MPO. Conclusions: GLY decreases MDR by: altering bacterial parameters, including viability and efflux pump activity. In vivo, it increases the effectiveness of ciprofloxacin, reducing ocular disease, plate count, and MPO activity.

The miR-183/96/182 Cluster Regulates Macrophage Functions in Response to Pseudomonas aeruginosa.

Macrophages (Mϕ) are an important component of the innate immune system; they play critical roles in the first line of defense to pathogen invasion and modulate adaptive immunity. MicroRNAs (miRNAs) are a newly recognized, important level of gene expression regulation. However, their roles in the regulation of Mϕ and the immune system are still not fully understood. In this report, we provide evidence that the conserved miR-183/96/182 cluster (miR-183/96/182) modulates Mϕ function in their production of reactive nitrogen (RNS) and oxygen species (ROS) and their inflammatory response to Pseudomonas aeruginosa (PA) infection and/or lipopolysaccharide (LPS) treatment. We show that knockdown of miR-183/96/182 results in decreased production of multiple proinflammatory cytokines in response to PA or LPS treatment in Mϕ-like Raw264.7 cells. Consistently, peritoneal Mϕ from miR-183/96/182-knockout versus wild-type mice are less responsive to PA or LPS, although their basal levels of proinflammatory cytokines are increased. In addition, overexpression of miR-183/96/182 results in decreased production of nitrite and ROS in Raw264.7 cells. We also provide evidence that DAP12 and Nox2 are downstream target genes of miR-183/96/182. These data suggest that miR-183/96/182 imposes global regulation on various aspects of Mϕ function through different downstream target genes.

Synthetic and immunological studies of 5'-N-phenylacetyl sTn to develop carbohydrate-based cancer vaccines and to explore the impacts of linkage between carbohydrate antigens and carrier proteins.

5'- N-Phenylacetyl sTn (sTnNPhAc), an unnatural derivative of sTn antigen expressed by many tumors, and its alpha-linked protein conjugates were prepared and investigated to explore glycoconjugate cancer vaccines. sTnNPhAcalpha-KLH elicited a robust T cell dependent immunity. The antiserum derived from sTnNPhAcalpha- or sTnNPhAcbeta-KLH-inoculated mice was similarly reactive to sTnNPhAcalpha and sTnNPhAcbeta but showed very little reactivity to sTn, NeuNPhAcalpha(2,3)GalNAc--a regioisomer of sTnNPhAc, isolated phenylacetyl group, and the linker employed to conjugate sTnNPhAc and carrier protein. It was concluded that the sTnNPhAc-elicited immunity was specific for the whole antigen rather than the phenylacetyl group or other partial structures of sTnNPhAc and that the reducing end configuration or linkage of sTnNPhAc did not affect its immunological identity. It was also concluded that a new linker designed to conjugate carbohydrates and proteins did not provoke any immune reaction and that the linker, as well as the associated new and convenient coupling strategy, can be safely used for the development of glycoconjugate vaccines.

Topical Glycyrrhizin Is Therapeutic for Pseudomonas aeruginosa Keratitis.

PURPOSE: Glycyrrhizin (GLY), an inhibitor of high-mobility group box 1 (HMGB1) protects prophylactically against Pseudomonas aeruginosa keratitis. However, the therapeutic potential of GLY to enhance an antibiotic has not been tested and is our purpose. METHODS: C57BL/6 mice (B6) were infected with a clinical isolate (KEI 1025) of P. aeruginosa and treated topically at 6 h postinfection (p.i.) with GLY or phosphate-buffered saline (PBS). Clinical scores, photography with a slit lamp, enzyme-linked immunosorbent assay, myeloperoxidase assay, bacterial plate counts, histopathology, reactive oxygen/nitrogen species (ROS/RNS) assays, and in vitro macrophage (Mφ) stimulation assays were used to assess effects of GLY treatment. In separate similar experiments, the ability of GLY to bioenhance the antibiotic, tobramycin (TOB), was assessed. RESULTS: In vivo, GLY versus PBS topical treatment began at 6 h p.i., improved disease outcome by significantly reducing clinical scores, proinflammatory proteins (HMGB1, RAGE, TLR4, TNF-α, and CXCL2), neutrophil infiltrate, bacterial load, ROS/RNS, and nitric oxide. In vitro, GLY downregulated iNOS and COX-2 expression (mRNA) in both mouse and human (THP-1) Mφ. At 6 and 24 h p.i., treatment with GLY enhanced the effects of TOB compared with TOB alone by significantly reducing corneal bacterial load and/or protein levels of cytokines CXCL2 and IL-1ß. CONCLUSIONS: Data provide evidence that GLY is not only therapeutic for Pseudomonas keratitis through its ability to reduce HMGB1, bacterial load, and oxidative damage but also through its bioenhancement of an antibiotic, even when treatment is initiated at 24 h after infection.

HMGB1 Antagonist, Box A, Reduces TLR4, RAGE, and Inflammatory Cytokines in the Cornea of P. aeruginosa-Infected Mice.

PURPOSE: High mobility group box 1 (HMGB1) contributes to adverse disease outcome in Pseudomonas aeruginosa keratitis. This study tests Box A, an HMGB1 antagonist, in a model of the disease. METHODS: C57BL/6 mice (B6) were injected subconjunctivally (1 day before infection) with Box A or phosphate-buffered saline (PBS), infected with P. aeruginosa strain ATCC 19660, and injected intraperitoneally with Box A or PBS at 1 and 3 days postinfection (p.i.). Clinical scores, photographs with a slit lamp camera, real-time polymerase chain reaction (RT-PCR), western blot, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), myeloperoxidase (MPO), and bacterial plate count were used to assess disease outcome. In separate experiments, the therapeutic potential of Box A was tested as described above, but with treatment begun at 6 h p.i. RESULTS: Box A versus PBS prophylactic treatment significantly reduced clinical scores, MPO activity, bacterial load, and expression of TLR4, RAGE, IL-1ß, CXCL2, and TNF-α in the infected cornea. Box A blocked co-localization of HMGB1/TLR4 in infiltrated cells in the stroma at 3 and 5 days p.i., but only at 5 days p.i. for HMGB1/RAGE. Box A versus PBS therapeutic treatment significantly reduced clinical scores, MPO activity, bacterial load, and protein levels of IL-1ß, CXCL2, and IL-6 in the infected cornea. CONCLUSION: Overall, Box A lessens the severity of Pseudomonas keratitis in mice by decreasing expression of TLR4, RAGE (their interaction with HMGB1), IL-1ß, CXCL2 (decreasing neutrophil infiltrate), and bacterial plate count when given prophylactically. Therapeutic treatment was not as effective at reducing opacity (disease), but shared similar features with pretreatment of the mice.

Characterization of Three Ocular Clinical Isolates of P. aeruginosa: Viability, Biofilm Formation, Adherence, Infectivity, and Effects of Glycyrrhizin.

We selectively characterized three isolates from Pseudomonas aeruginosa keratitis patients and how glycyrrhizin (GLY) affected them. Type III toxins were determined using polymerase chain reaction (PCR). Minimum Inhibitory Concentration (MIC) of GLY and assays for its effects on: time kill, bacterial permeability, and biofilm/adhesion were done. In vivo, C57BL/6 (B6) mice were treated topically with GLY after G81007 infection. Clinical score, photography with a slit lamp and RT-PCR were used to assess treatment effects. Isolates expressed exoS and exoT, but not exoU. MIC for all isolates was 40 mg/mL GLY and bacteriostatic effects were seen for G81007 after treatment using time kill assays. From viability testing, GLY treatment significantly increased the number of permeabilized bacteria (live/dead assay). Isolates 070490 and G81007 formed more biofilms compared with R59733 and PAO1 (control). GLY-treated bacteria had diminished biofilm compared with controls for all isolates. GLY reduced adherence of the G81007 isolate to cultured cells and affected specific biofilm associated systems tested by reverse transcription PCR (RT-PCR). In vivo, after G81007 infection, GLY treatment reduced clinical score and messenger RNA (mRNA) expression of IL-1ß, TNF-α, CXCL2 and HMGB1. This study provides evidence that GLY is bacteriostatic for G81007. It also affects biofilm production, adherence to cultured cells, and an improved keratitis outcome.

Challenges of corneal infections.

INTRODUCTION: Ocular infections remain an important cause of blindness worldwide and represent a challenging public health concern. In this regard, microbial keratitis due to fungal, bacterial, or viral infection can result in significant vision loss secondary to corneal scarring or surface irregularity. Left untreated corneal perforation and endophthalmitis can result, leading to loss of the eye. Rigorously studied animal models of disease pathogenesis have provided novel information that suggests new modes of treatment that may be efficacious clinically and emerging clinical data is supportive of some of these discoveries. AREAS COVERED: This review focuses on advances in our understanding of disease pathogenesis in animal models and clinical studies and how these relate to improved clinical treatment. We also discuss a novel approach to treatment of microbial keratitis due to infection with these bacterial pathogens using PACK-CXL and recommend increased basic and clinical studies to address and refine the efficacy of this procedure. EXPERT COMMENTARY: Because resistance to antibiotics has developed over time to these bacterial pathogens, caution must be exercised in treatment. Attractive novel modes of treatment that hold new promise for further investigation include lipid based therapy, as well as use of small molecules that bind deleterious specific host responsive molecules and use of microRNA based therapies.

Decreasing HMGB1 levels improves outcome of Pseudomonas aeruginosa keratitis in mice.

Pseudomonas (P.) aeruginosa is a Gram negative bacterium widely dispersed in the environment which can cause acute and chronic infections in humans. According to the Centers for Disease Control and Prevention (CDC), the overall incidence of P. aeruginosa infections in USA hospitals averages about 0.4% (4/1000 discharges), and the bacterium is the fourth most commonly-isolated nosocomial pathogen accounting for 10.1% of all hospital-acquired infections. P. aeruginosa keratitis is a severe infection of the eye, progresses rapidly and remains a leading cause of corneal ulcers worldwide. Use of contact lenses is the major risk factor in the USA, while in less industrialized countries, trauma from agricultural accidents are of importance. Animal models of bacterial keratitis are of value in the study of this disease and suggest potential alternative therapeutic targets that are needed urgently due to increasing antibiotic resistance. Recently we have shown success and improved disease outcome after down-regulation of one promising target, high mobility group box1 (HMGB1) using small interfering RNA (siRNA). Testing more clinically relevant approaches are underway to reduce HMGB1 levels in P. aeruginosa keratitis which may hold promise for its treatment.

Glycyrrhizin Reduces HMGB1 and Bacterial Load in Pseudomonas aeruginosa Keratitis.

PURPOSE: High mobility group box 1 (HMGB1) contributes to poor disease outcome in Pseudomonas aeruginosa keratitis. This study tests the prophylactic effect of treatment with HMGB1 inhibitors, glycyrrhizin (GLY) and its derivative, carbenoxolone (CBX), for Pseudomonas keratitis. METHODS: We treated C57BL/6 (B6) mice subconjunctivally with GLY or CBX, infected with a noncytotoxic clinical isolate (KEI 1025) or a cytotoxic strain (ATCC 19660) of P. aeruginosa, and injected intraperitoneally with either agent. Clinical score, photography with a slit lamp, real-time RT-PCR, ELISA, myeloperoxidase (MPO) assay, bacterial plate count, histopathology, and absorbance assays were used to assess treatment efficacy and bacteriostatic activity. RESULTS: After KEI 1025 infection, GLY treatment reduced HMGB1 (mRNA and protein levels) and improved disease outcome with significant reduction in mRNA levels of IL-1ß, TLR4, CXCL2, and IL-12; protein expression (IL-1ß, CXCL2); neutrophil infiltrate; and bacterial load. Treatment with GLY enhanced antimicrobial proteins, including CRAMP and mBD2, but not mBD3. Glycyrrhizin also reduced clinical scores and improved disease outcome in corneas infected with strain 19660. However, neither HMGB1 mRNA or protein levels were reduced, but rather, CXCL2 expression (mRNA and protein), neutrophil infiltrate, and bacterial load were reduced statistically. Treatment with GLY initiated 6 hours after infection reduced plate count; GLY also was bacteriostatic for KEI 1025 and ATCC 19660. CONCLUSIONS: Glycyrrhizin reduces HMGB1 and is protective against P. aeruginosa-induced keratitis with a clinical isolate that is noncytotoxic. It was similar, but less effective when used after infection with a cytotoxic strain, which did not reduce HMGB1.

Thrombomodulin Protects Against Bacterial Keratitis, Is Anti-Inflammatory, but Not Angiogenic.

PURPOSE: Thrombomodulin (TM) is a multidomain, transmembrane protein with anti-inflammatory properties. Thrombomodulin domain (D) 1 is lectin-like, interacting with Lewis Y antigen on lipopolysaccharide, and with HMGB1, while TMD23 is associated with angiogenic and anti-inflammatory functions. Thus, we tested if TM is protective against Pseudomonas aeruginosa keratitis and whether it enhanced corneal vascularity. METHODS: Eyes of C57BL/6 (B6) mice were injected with recombinant TM (rTM), rTMD1, or PBS subconjunctivally before and intraperitoneally after infection with P. aeruginosa. Clinical scores, photography with a slit lamp, RT-PCR, ELISA, myeloperoxidase (MPO) assay, viable bacterial plate counts, and India ink perfusion were used to assess the disease response and corneal vascularity (rTM only). RESULTS: Recombinant TM versus PBS treatment reduced clinical scores and corneal opacity. Corneal mRNA levels for HMGB1 were unchanged, but proinflammatory molecules IL-1ß, CXCL2, NF-κB, TLR4, and RAGE were decreased; anti-inflammatory molecules SIGIRR and ST2 were increased. ELISA confirmed the mRNA data for HMGB1, IL-1ß, and CXCL2 proteins. Both neutrophil influx and viable bacterial plate counts also were decreased after rTM treatment. Protein levels for angiogenic molecules VEGF, VEGFR-1, and VEGFR-2 were measured at 5 days post infection and were not different or reduced significantly after rTM treatment. Further, perfusion with India ink revealed similar vessel ingrowth between the two groups. Similar studies were performed with rTMD1, but disease severity, mRNA, proteins, MPO, and plate counts were not changed from controls. CONCLUSIONS: These data provide evidence that rTM treatment is protective against bacterial keratitis, does not reduce HMGB1, and is not angiogenic.