High-mobility group box 1: a novel target for treatment of Pseudomonas aeruginosa keratitis.

High-mobility group box 1 (HMGB1), a prototypic alarmin, mediates the systemic inflammatory response syndrome. Treatment with vasoactive intestinal peptide, an anti-inflammatory neuropeptide, downregulates proinflammatory cytokines and promotes healing in a susceptible (cornea perforates) model of Pseudomonas aeruginosa keratitis, and also significantly downregulates HMGB1 expression. Therefore, we examined targeting HMGB1 for the treatment of P. aeruginosa keratitis to avoid delivery and other issues associated with vasoactive intestinal peptide. For this, HMGB1 was silenced using small interfering RNA, whereas controls were treated with a nonspecific scrambled sequence small interfering RNA. Less disease was seen postinfection in siHMGB1 compared with control mice and was documented by clinical score and photographs with a slit lamp. Real-time RT-PCR and ELISA confirmed HMGB1 knockdown. RT-PCR analysis also revealed reduced mRNA levels of IL-1ß, MIP-2, TNF-α, TLR4, and receptor for advanced glycation end products, whereas mRNA levels of anti-inflammatory TLRs single Ig IL-1-related receptor and ST2 were increased significantly. HMGB1 knockdown also decreased IL-1ß and MIP-2 proteins, reducing polymorphonuclear cell number in the infected cornea. mRNA and protein levels of CXCL12 and CXCR4, as well as mononuclear cells, were reduced significantly after HMGB1 knockdown. Ab neutralization of HMGB1, infection with a clinical isolate, and recombinant HMGB1 treatment of resistant mice supported the silencing studies. These data provide evidence that silencing HMGB1 promotes better resolution of P. aeruginosa keratitis by decreasing levels of proinflammatory mediators (decreasing polymorphonuclear cell infiltration), increasing anti-inflammatory TLRs, reducing CXCL12 (preventing HMGB1/CXCL12 heterodimer formation), and signaling through CXCR4, reducing monocyte/macrophage infiltration.

Vasoactive intestinal peptide downregulates proinflammatory TLRs while upregulating anti-inflammatory TLRs in the infected cornea.

TLRs recognize microbial pathogens and trigger an immune response, but their regulation by neuropeptides, such as vasoactive intestinal peptide (VIP), during Pseudomonas aeruginosa corneal infection remains unexplored. Therefore, C57BL/6 (B6) mice were injected i.p. with VIP, and mRNA, protein, and immunostaining assays were performed. After VIP treatment, PCR array and real-time RT-PCR demonstrated that proinflammatory TLRs (conserved helix-loop-helix ubiquitous kinase, IRAK1, TLR1, TLR4, TLR6, TLR8, TLR9, and TNFR-associated factor 6) were downregulated, whereas anti-inflammatory TLRs (single Ig IL-1-related receptor [SIGIRR] and ST2) were upregulated. ELISA showed that VIP modestly downregulated phosphorylated inhibitor of NF-κB kinase subunit α but upregulated ST2 ~2-fold. SIGIRR was also upregulated, whereas TLR4 immunostaining was reduced in cornea; all confirmed the mRNA data. To determine whether VIP effects were cAMP dependent, mice were injected with small interfering RNA for type 7 adenylate cyclase (AC7), with or without VIP treatment. After silencing AC7, changes in mRNA levels of TLR1, TNFR-associated factor 6, and ST2 were seen and unchanged with addition of VIP, indicating that their regulation was cAMP dependent. In contrast, changes were seen in mRNA levels of conserved helix-loop-helix ubiquitous kinase, IRAK1, 2, TLR4, 9 and SIGIRR following AC7 silencing alone; these were modified by VIP addition, indicating their cAMP independence. In vitro studies assessed the effects of VIP on TLR regulation in macrophages and Langerhans cells. VIP downregulated mRNA expression of proinflammatory TLRs while upregulating anti-inflammatory TLRs in both cell types. Collectively, the data provide evidence that VIP downregulates proinflammatory TLRs and upregulates anti-inflammatory TLRs and that this regulation is both cAMP dependent and independent and involves immune cell types found in the infected cornea.

A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy.

Interleukin (IL)-7 is broadly active on T-cell populations, and modified versions have been clinically evaluated for a variety of therapeutic applications, including cancer, lymphopenia, and infectious diseases; and found to be relatively well-tolerated and biologically active. Here we describe novel IL-7R agonists that are unrelated in structure to IL-7, bind to the receptor subunits differently from IL-7, but closely emulate IL-7 biology. The small size, low structural complexity, and the natural amino acid composition of the pharmacologically active peptide MDK1472 allows facile incorporation into protein structures, such as the IgG2-Fc fusion MDK-703. This molecule possesses properties potentially better suited to therapeutic applications than native IL-7 or its derivatives. We compared these compounds with IL-7 for immune cell selectivity, induction of IL-7R signaling, receptor-mediated internalization, proliferation, and generation of immune cell phenotypes in human and non-human primate (NHP) peripheral blood cells in vitro; and found them to be similar in biological activity to IL-7. In cynomolgus macaques, MDK-703 exhibits a circulating half-life of 46 hr and produces sustained T-cell expansion characteristic of IL-7 treatment. In the huCD34+-engrafted NSG mouse model of the human immune system, MDK-703 induces an immune cell profile very similar to that generated by IL-7-derived compounds; including the pronounced expansion of memory T-cells, particularly the population of stem-like memory T-cells (Tscm) which may be important for anti-tumor activities reported with IL-7 treatment. Clinical administration of IL-7 and modified variants has been reported to induce anti-drug antibodies (ADAs), including IL-7 neutralizing antibodies. The novel peptide agonist reported here scores very low in predicted immunogenicity, and because the peptide lacks sequence similarity with IL-7, the problematic immunogenic neutralization of endogenous cytokine should not occur. The properties we report here implicate MDK-703 as a candidate for clinical evaluation in oncology, anti-viral and other infectious disease, vaccine enhancement, and treatment of lymphopenia.

Beta-defensin-2 promotes resistance against infection with P. aeruginosa.

Corneal infection with Pseudomonas aeruginosa results in corneal perforation in susceptible C57BL/6 (B6) mice, but not in resistant BALB/c mice. To explore the role of two important defensins, murine beta-defensin-1 (mBD1) and mBD2, in the ocular immune defense system, their mRNA and protein expression levels were tested by real-time RT-PCR and Western blot, respectively. mRNA, protein, and immunostaining data demonstrated that both mBD1 and mBD2 were constitutively expressed in normal BALB/c and B6 corneas, and they were disparately up-regulated in BALB/c (more) vs B6 (less) corneas after infection. To determine whether either defensin played a role in host resistance, BALB/c mice were treated with either mBD1 or mBD2 small interfering RNA by subconjunctival injection together with topical application. Increased corneal opacity and worsened disease were displayed after knockdown of mBD2 but not of mBD1. mBD2 silencing also increased bacterial counts and polymorphonuclear neutrophil infiltration in BALB/c corneas. Real-time RT-PCR data further demonstrated that mBD2, not mBD1, differentially modulated mRNA expression of proinflammatory cytokines/molecules such as IFN-gamma, MIP-2, IL-1beta, TNF-alpha, IL-6, and inducible NO synthase; TLR signaling molecules, including TLR2, TLR4, TLR9, and MyD88; and the transcription factor NF-kappaB. Additionally, in vivo studies indicated that mBD2 silencing enhanced corneal nitrite levels and NF-kappaB activation. Collectively, the data provide evidence that mBD2, but not mBD1, is required for host resistance against P. aeruginosa-induced corneal infection.

Beta-defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis.

Defensins play an important role in both innate and adaptive immunity due to their antimicrobial, regulatory, and chemotactic effects. Nonetheless, the role of murine beta-defensins (mBD) 3 and 4, the murine homologs of human beta-defensins (hBD) 2 and 3, remains unknown in Pseudomonas aeruginosa keratitis. This study explored their role in corneal infection and potential synergy with mBD2, a defensin associated with better outcome in this disease. Immunostaining and real-time RT-PCR data demonstrated that mBD3 and mBD4 expression was inducible and differentially regulated in the infected cornea of resistant BALB/c vs susceptible C57BL/6 (B6) mice. Knockdown studies using small interfering RNA treatment indicated that mBD3, but not mBD4, is required in ocular defense. Moreover, in vivo studies demonstrated individual and combined effects of mBD2 and mBD3 that modulate bacterial load, polymorphonuclear neutrophil (PMN) infiltration, and production of IFN-gamma, MIP-2, IL-1beta, TNF-alpha, inducible NO synthase (iNOS), TLR2, TLR4, MyD88, and NF-kappaB. Most notably, bacterial load was increased at 5 days postinfection by silencing either mBD2 or mBD3, but it was elevated at both 1 and 5 days postinfection when silencing both defensins. PMN infiltration was increased at 1 day postinfection by silencing both defensins or mBD3, but not mBD2 alone. iNOS expression was elevated by silencing mBD2, but it was reduced after silencing mBD3 or both defensins. Additionally, cell sources of mBD2 (macrophages, PMN and fibroblasts) and mBD3 (PMN) in corneal stroma were identified by dual label immunostaining after infection. Collectively, the data provide evidence that mBD2 and mBD3 together promote resistance against corneal infection.

Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.

A randomized, double-blind, placebo-controlled, crossover study of XP13512/GSK1838262 in the treatment of patients with primary restless legs syndrome.

STUDY OBJECTIVE: To evaluate the efficacy and tolerability of XP13512/ GSK1838262, an investigational nondopaminergic agent for the treatment of moderate-to-severe primary restless legs syndrome (RLS). DESIGN: Randomized, double-blind, placebo-controlled, crossover trial. SETTING: Nine US clinical sites. PATIENTS: Thirty-eight treatment-naive subjects with RLS (mean +/- SD age 50.1 +/- 13.2 years). INTERVENTIONS: XP13512 1800 mg/day followed by placebo or placebo followed by XP13512 1800 mg/day for 14 days, with a 7-day washout between treatment periods. MEASUREMENTS AND RESULTS: The primary endpoint was mean change from baseline International RLS Study Group rating scale (IRLS) total score on Day 14, analyzed using analysis of variance with sequence, period, and treatment as fixed effects and subjects within sequence as a random effect. XP13512 significantly reduced IRLS total score on Day 14 compared with placebo (mean +/- SD: XP13512 -12.1 +/-6.5, placebo -1.9 +/- 6.3; P < 0.0001). Polysomnographic data showed that XP13512 significantly improved sleep architecture on Day 14 compared with placebo (mean +/- SD change from baseline sleep time [minutes]: stage 1: XP13512 -9.8 +/- 23.9, placebo 0.4 +/-23.2; adjusted P<0.0054, nominal P<0.0001; stage 3/4 (slow-wave sleep): XP13512 22.8 +/- 40.8, placebo 1.4 +/- 34.3; adjusted P=0.0092, nominal P=0.0002). The most frequently reported adverse events were somnolence (XP13512 30.6%, placebo 2.8%) and dizziness (XP13512 27.8%, placebo 5.6%). CONCLUSIONS: XP13512 1800 mg/day significantly reduced RLS symptoms, improved sleep, and was generally well tolerated in subjects with moderate-to-severe primary RLS across 14 days of treatment.

Constitutive excitation by Gly90Asp rhodopsin rescues rods from degeneration caused by elevated production of cGMP in the dark.

Previous experiments indicate that congenital human retinal degeneration caused by genetic mutations that change the Ca(2+) sensitivity of retinal guanylyl cyclase (retGC) can result from an increase in concentration of free intracellular cGMP and Ca(2+) in the photoreceptors. To rescue degeneration in transgenic mouse models having either the Y99C or E155G mutations of the retGC modulator guanylyl cyclase-activating protein 1 (GCAP-1), which produce elevated cGMP synthesis in the dark, we used the G90D rhodopsin mutation, which produces constitutive stimulation of cGMP hydrolysis. The effects of the G90D transgene were evaluated by measuring retGC activity biochemically, by recording single rod and electroretinogram (ERG) responses, by intracellular free Ca(2+) measurement, and by retinal morphological analysis. Although the G90D rhodopsin did not alter the abnormal Ca(2+) sensitivity of retGC in the double-mutant animals, the intracellular free cGMP and Ca(2+) concentrations returned close to normal levels, consistent with constitutive activation of the phosphodiesterase PDE6 cascade in darkness. G90D decreased the light sensitivity of rods but spared them from severe retinal degeneration in Y99C and E155G GCAP-1 mice. More than half of the photoreceptors remained alive, appeared morphologically normal, and produced electrical responses, at the time when their siblings lacking the G90D rhodopsin transgene lost the entire retinal outer nuclear layer and no longer responded to illumination. These experiments indicate that mutations that lead to increases in cGMP and Ca(2+) can trigger photoreceptor degeneration but that constitutive activation of the transduction cascade in these animals can greatly enhance cell survival.

Substance P promotes susceptibility to Pseudomonas aeruginosa keratitis in resistant mice: anti-inflammatory mediators downregulated.

PURPOSE: Studies have shown that blocking substance P (SP) binding to neurokinin 1 receptor with spantide I prevents Pseudomonas aeruginosa-induced corneal perforation in susceptible C57BL/6 mice. This study tested the effect of SP injection on the resistance response (cornea heals) of BALB/c mice. METHODS: The day before infection, mice were injected intraperitoneally with SP or PBS. Disease was graded by clinical score, slit lamp, plate count, real-time RT-PCR, and ELISA assays, and polymorphonuclear neutrophils (PMNs) were quantitated using a myeloperoxidase assay. In additional experiments, BALB/c mice were injected intraperitoneally with vasoactive intestinal peptide (VIP) antagonist and similarly analyzed. RESULTS: Mice injected with SP exhibited worsened disease on days 1 to 7 after infection compared with controls. SP injection resulted in elevated PMN levels and viable bacterial counts in the cornea 3 and 5 days after infection. mRNA expression for NFkappaB and type 1 cytokines (e.g., IFN-gamma), as well as for TNF-alpha, MIP-2, IL-18, IL-6, and IL-1beta, were significantly elevated, whereas VIP and cytokines TGF-beta and IL-10 were significantly reduced. Differences in mRNA expression were selectively confirmed at the protein level by ELISA for NFkappaB, IL-1beta, and IL-10. VIP antagonist treatment also resulted in exacerbated disease scores, elevated proinflammatory mediators, and reduced anti-inflammatory mediators. CONCLUSIONS: These data provide evidence that the neuropeptide SP, among its broad systemic effects, is a potent neuroimmunoregulator that promotes susceptibility in the resistant BALB/c mouse by overcoming the anti-inflammatory effects of VIP and IL-10 and that a balance between SP and VIP levels may be critical in disease resolution.

Rat silicone hydrogel contact lens model: effects of high- versus low-Dk lens wear.

OBJECTIVES: This study used a rat contact lens (CL) model to test if high- versus low-Dk lens wear caused changes in (1) conjunctival Langerhans cell (LC) number or location; (2) Bcl-2 expression; and (3) infection risk. METHODS: Female, Lewis rats wore a high- or low-Dk CL continuously for 2 weeks. Afterward, corneas were harvested and processed for ADPase activity to identify LCs, for immunostaining and for real time-polymerase chain reaction. Contact lens-wearing rats also were challenged with Pseudomonas aeruginosa by placing a bacterial-soaked CL on the eye followed by topical delivery of bacteria. After 48 hrs, slit lamp examination and real time-polymerase chain reaction were used to evaluate the corneal response. RESULTS: Conjunctival LC were significantly increased after low- versus high-Dk CL wear (P<0.0001). In contrast, conjunctival LC in non-lens wearing rats was not significantly different from the high-Dk lens wearing group. Bcl-2 mRNA levels were significantly decreased in low- versus high-Dk CL wearing rats, while Bax, FasL, caspase 3, and caspase 9 levels were unchanged. Immunostaining for Bcl-2 showed fewer positively stained epithelial cells in the low- versus high-Dk lens wearing group. After bacterial challenge, 30% of low- versus none of the high-Dk CL wearing corneas became infected and showed increased mRNA levels for several proinflammatory cytokines/chemokines, inducible nitric oxide synthase and matrix metalloproteinase-9. CONCLUSION: Low- versus high-Dk or non-CL wear led to an increased number of conjunctival LC, decreased Bcl-2 levels, and increased the risk of bacterial infection.

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.

ST2 is essential for Th2 responsiveness and resistance to pseudomonas aeruginosa keratitis.

PURPOSE: To elucidate the role of ST2, a member of the TLR/IL-1R (TIR) superfamily, in protecting against Pseudomonas aeruginosa keratitis in BALB/c mice. METHODS: ST2 mRNA and protein expression levels were tested by real-time PCR and Western-blot in C57BL/6 (B6; susceptible) versus BALB/c (resistant) mice before and after P. aeruginosa (strain 19660; American Type Culture Collection, Philadelphia, PA) challenge. Infected BALB/c mice also were tested after subconjunctival injection with recombinant murine (rm)ST2 or PBS. Disease was monitored by clinical score, slit lamp, bacterial plate count, a myeloperoxidase (MPO) assay to measure polymorphonuclear neutrophil (PMN) infiltrate, real-time RT-PCR, and ELISA. RESULTS: ST2 mRNA and protein were constitutively expressed in the uninfected normal corneas of both mouse groups. ST2 levels in the cornea of BALB/c compared with B6 mice were elevated significantly at 1 to 3 days post infection (PI), peaked at 3 and decreased at 5 days PI. BALB/c mice treated with rmST2 showed increased corneal opacity and perforation (at 5 days PI) when compared with PBS controls. rmST2- versus PBS-injected mice exhibited increased bacterial load, PMN infiltrate, and higher corneal mRNA levels for IL-1beta, MIP-2, IL-6, IL-1R1, and Th1-type cytokine such as IFN-gamma. Protein levels for IL-1beta, MIP-2, and IL-6 also were significantly upregulated, whereas the Th2 cytokines IL-4 (mRNA), IL-5 (mRNA), and IL-10 (mRNA and protein) were significantly reduced. CONCLUSIONS: ST2 is critical in resistance to P. aeruginosa keratitis, functioning to reduce corneal infection (bacterial load) and inflammation by negatively regulating proinflammatory cytokines and inhibiting type-1 immunity, but upregulating type-2 cytokine production, particularly IL-10.

Spantide I decreases type I cytokines, enhances IL-10, and reduces corneal perforation in susceptible mice after Pseudomonas aeruginosa infection.

PURPOSE: To determine the effects of blocking substance P (SP) interactions with its major receptor (NK1-R) using the antagonist spantide I in susceptible mice infected with Pseudomonas aeruginosa. METHODS: Immunohistochemistry and enzyme immunosorbent assay (EIA) tested levels of SP in the cornea of B6 and BALB/c mice. B6 mice were treated with spantide, and after infection, slit lamp examination; clinical score; bacterial counts; and myeloperoxidase (MPO), RT-PCR, ELISA, and polymorphonuclear (PMN) cell chemotaxis assays were performed. RESULTS: SP corneal levels were significantly elevated constitutively and after infection in the B6 more than in BALB/c mice. Spantide treatment of B6 mice significantly decreased the number of perforated corneas, bacterial counts, and PMNs. mRNA levels for type I cytokines (e.g., IFN-gamma) as well as MIP-2, IL-6, TNF-alpha, and IL-1beta (mRNA and protein) also were significantly reduced after spantide treatment. The type II cytokine IL-10 (mRNA and protein) was elevated, whereas TGF-beta mRNA levels were unchanged after spantide treatment. PMN chemotaxis was induced by SP and other neuropeptides in vitro, but was not affected by spantide I. mRNA for neurokinin-1-receptor-1 (NK-1R) was detected in the normal and infected corneas and on macrophages (Mphis), but not on PMNs (unstimulated or stimulated with endotoxin [LPS]). Spantide treatment of Mphis reduced IL-1beta after LPS+SP treatment but not after either alone. CONCLUSIONS: The SP antagonist Spantide provides a novel approach to reduce type 1 and enhance the type 2 cytokine IL-10 in the infected cornea of B6 mice, leading to a significant reduction in corneal perforation and improved disease outcome.

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.

Matrix metalloproteinase-9 amplifies the immune response to Pseudomonas aeruginosa corneal infection.

PURPOSE: The purpose of this study was to determine the role of matrix metalloproteinases (MMP) in Pseudomonas aeruginosa keratitis. METHODS: Gene array and selective real-time PCR examined MMP expression in the cornea of susceptible (C57BL/6, B6) versus resistant (BALB/c) mice before and after infection; zymography tested enzyme activity for MMP-2 and -9. Clinical score, Langerhans cell (LC), and Neutrophil (PMN) quantitation were done in recombinant (r) MMP-9, antibody neutralized, and MMP-9(-/-) mice. The chemotactic potential of MMP-9 was tested in a Boyden chamber assay; light and transmission microscopy and immunostaining for collagen IV and MMP-9 were used to examine the effects and the source of MMP-9 after infection. ELISA was used to assess IL-1beta and MIP-2 levels. RESULTS: Gene array (confirmed by PCR) revealed sixfold more MMP-9, and zymography showed greater enzyme activity in the infected cornea of B6 over BALB/c mice. rMMP-9 injection of BALB/c mice enhanced, whereas MMP-9 antibody neutralization in B6 mice and its absence in MMP-9(-/-) mice decreased corneal disease. MMP-9(-/-) and antibody neutralized mice had fewer LCs in cornea; rMMP-9-treated mice had more. A myeloperoxidase (MPO) assay showed a similar pattern for PMN. MMP-9 was not chemotactic for LC or PMN. The basement membrane was more intact in MMP-9(-/-) over wild-type infected mice and correlated with staining for collagen IV; PMN was a source of MMP-9. IL-1beta and MIP-2 were increased in rMMP-9 but decreased in MMP-9 antibody neutralized and MMP-9(-/-) over control groups. CONCLUSIONS: MMP-9 regulates immune function in cornea by proteolysis, potentiating P. aeruginosa keratitis by degrading collagen IV and upregulating chemotactic cytokines/chemokines IL-1beta and MIP-2.

TLR4 is required for host resistance in Pseudomonas aeruginosa keratitis.

PURPOSE: To determine the role of Toll-like receptor 4 (TLR4) in Pseudomonas aeruginosa (P. aeruginosa) keratitis in resistant (cornea-healing) BALB/c mice. METHODS: Corneal TLR4 mRNA levels were tested by real-time PCR in BALB/c mice before and after infection. Clinical score, slit lamp, histopathology, bacterial counts, and polymorphonuclear neutrophil (PMN) quantitation were performed in the infected cornea of TLR4-deficient (TLR4(lps-d)) and wild-type BALB/c mice. mRNA for IL-1beta, MIP-2, IFN-gamma, IL-18, inducible nitric oxide synthase (iNOS), and beta-defensin-2 levels were measured by real-time PCR. Protein levels for IL-1beta, MIP-2, and IFN-gamma were tested by ELISA. RESULTS: In resistant BALB/c mice, TLR4 mRNA expression was significantly upregulated in the cornea after P. aeruginosa infection. In contrast, TLR4-deficient mice were susceptible to infection with P. aeruginosa and showed increased corneal opacity, PMN infiltration, bacterial counts, and perforated infected corneas. After infection, TLR4-deficient mice also showed increased mRNA expression of proinflammatory cytokines (IL-1beta and MIP-2) and type-1-associated cytokines (IFN-gamma and IL-18) when compared with wild-type BALB/c mice. ELISA analyses showed that IL-1beta, MIP-2, and IFN-gamma protein levels also were significantly upregulated in the cornea of TLR4-deficient versus wild-type mice. In contrast, levels of iNOs and beta-defensin-2 were significantly decreased in TLR4-deficient compared with wild-type mice. CONCLUSIONS: TLR4 is critical in host resistance to P. aeruginosa, as its deficiency results in increased PMN infiltration and proinflammatory cytokine production, decreased iNOs and beta-defensin-2 production, impaired bacterial killing, and a susceptible phenotype.

Dark Winter and the spring of 1972: deflecting the social lessons of smallpox.

This article examines how the master status of bioterrorism has distracted professional and political attention from the social lessons of smallpox. I illustrate this by comparing an influential bioterrorism simulation known as Dark Winter with the social history surrounding the Yugoslavian smallpox epidemic of 1972. Dark Winter's epidemiological premises were largely based upon what was learned from the Yugoslavian outbreak. Yet, although this epidemic was non-deliberate, the exercise did not attend to the social conditions within which it developed. Most notably, it did not consider that this epidemic was mainly borne by marginalized communities of Kosovan Albanians and that difficulties in controlling it were linked to the relative lack of pre-existing public health infrastructure among these people; instead, the Dark Winter exercise mainly focused upon the proximate determinants of violence and its immediate management. This distraction from the social dynamics of infectious diseases has major implications for the prevention and management of future outbreaks, regardless of whether or not they are deliberately initiated.

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.

Inactivation of the miR-183/96/182 Cluster Decreases the Severity of Pseudomonas aeruginosa-Induced Keratitis.

PURPOSE: The microRNA-183/96/182 cluster (miR-183/96/182) plays important roles in sensory organs. Because the cornea is replete with sensory innervation, we hypothesized that miR-183/96/182 modulates the corneal response to bacterial infection through regulation of neuroimmune interactions. METHODS: Eight-week-old miR-183/96/182 knockout (ko) mice and their wild-type littermates (wt) were used. The central cornea of anesthetized mice was scarred and infected with Pseudomonas aeruginosa (PA), strain 19660. Corneal disease was graded at 1, 3, and 5 days postinfection (dpi). Corneal RNA was harvested for quantitative RT-PCR. Polymorphonuclear neutrophils (PMN) were enumerated by myeloperoxidase assays; the number of viable bacteria was determined by plate counts, and ELISA assays were performed to determine cytokine protein levels. A macrophage (Mϕ) cell line and elicited peritoneal PMN were used for in vitro functional assays. RESULTS: MicroRNA-183/96/182 is expressed in the cornea, and in Mϕ and PMN of both mice and humans. Inactivation of miR-183/96/182 resulted in decreased corneal nerve density compared with wt mice. Overexpression of miR-183/96/182 in Mϕ decreased, whereas knockdown or inactivation of miR-183/96/182 in Mϕ and PMN increased their capacity for phagocytosis and intracellular killing of PA. In PA-infected corneas, ko mice showed decreased proinflammatory neuropeptides such as substance P and chemoattractant molecules, MIP-2, MCP1, and ICAM1; decreased number of PMN at 1 and 5 dpi; increased viable bacterial load at 1 dpi, but decreased at 5 dpi; and markedly decreased corneal disease. CONCLUSIONS: MicroRNA-183/96/182 modulates the corneal response to bacterial infection through its regulation of corneal innervation and innate immunity.