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PURPOSE: Contact lens wear can induce corneal parainflammation involving CD11c+ cell responses (24 hours), γδ T cell responses (24 hours and 6 days), and IL-17-dependent Ly6G+ cell responses (6 days). Topical antibiotics blocked these CD11c+ responses. Because corneal CD11c+ responses to bacteria require transient receptor potential (TRP) ion-channels (TRPA1/TRPV1), we determined if these channels mediate lens-induced corneal parainflammation. METHODS: Wild-type mice were fitted with contact lenses for 24 hours or 6 days and compared to lens wearing TRPA1 (-/-) or TRPV1 (-/-) mice or resiniferatoxin (RTX)-treated mice. Contralateral eyes were not fitted with lenses. Corneas were examined for major histocompatibility complex (MHC) class II+, CD45+, γδ T, or TNF-α+ cell responses (24 hours) or Ly6G+ responses (6 days) by quantitative imaging. The quantitative PCR (qPCR) determined cytokine gene expression. RESULTS: Lens-induced increases in MHC class II+ cells after 24 hours were abrogated in TRPV1 (-/-) but not TRPA1 (-/-) mice. Increases in CD45+ cells were unaffected. Increases in γδ T cells after 24 hours of wear were abrogated in TRPA1 (-/-) and TRPV1 (-/-) mice, as were 6 day Ly6G+ cell responses. Contralateral corneas of TRPA1 (-/-) and TRPV1 (-/-) mice showed reduced MHC class II+ and γδ T cells at 24 hours. RTX inhibited lens-induced parainflammatory phenotypes (24 hours and 6 days), blocked lens-induced TNF-α and IL-18 gene expression, TNF-α+ cell infiltration (24 hours), and reduced baseline MHC class II+ cells. CONCLUSIONS: TRPA1 and TRPV1 mediate contact lens-induced corneal parainflammation after 24 hours and 6 days of wear and can modulate baseline levels of resident corneal immune cells.

PURPOSE: Previously, using a murine model, we reported that contact lens (CL) wear induced corneal parainflammation involving CD11c+ cells after 24 h and Ly6G+ cells (neutrophils) after 5-6 days. Here, we investigated the role of IL-17 and γδ T cells in the CL-induced neutrophil response. METHODS: CL-wearing C57BL/6 wild-type (WT) mice were compared to lens-wearing IL-17A/F single or double gene knock-out mice, or mice treated with UC7-13D5 monoclonal antibody to functionally deplete γδ T cells. Contralateral eyes served as no lens wear controls. Corneal Ly6G+ and γδ T cell responses were quantified as was expression of genes encoding pro-inflammatory cytokines IL-17A/F, IL-β, IL-18 and expression of IL-17A/F protein. RESULTS: After 6 days lens wear, WT corneas showed Ly6G+ cell infiltration while remaining free of visible pathology. In contrast, lens-wearing corneas of IL-17AF (-/-), IL-17A (-/-) mice and γδ T cell-depleted mice showed little or no Ly6G+ cell infiltration. No Ly6G+ cell infiltration was detected in contralateral eye controls. Lens-wearing WT corneas also showed a significant increase in γδ T cells after 24 h that was maintained after 6 days of wear, and significantly increased cytokine gene expression after 6 days versus contralateral controls: IL-18 & IL-17A (∼3.9 fold) and IL-23 (∼6.5-fold). Increased IL-17A protein (∼4-fold) was detected after 6 days lens wear. γδ T cell-depletion abrogated these lens-induced changes in cytokine gene and protein expression. CONCLUSION: Together, these data show that IL-17A and γδ T cells are required for Ly6G+ cell (neutrophil) infiltration of the cornea during contact lens-induced parainflammation.

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

ObjectiveTo determine patterns of respiratory medications used in neonatal intensive care unit graduates.Study designThe Prematurity Respiratory Outcomes Program enrolled 835 babies

BackgroundMany premature infants with respiratory failure are deficient in surfactant, but the relationship to occurrence of bronchopulmonary dysplasia (BPD) is uncertain.MethodsTracheal aspirates were collected from 209 treated and control infants enrolled at 7-14 days in the Trial of Late Surfactant. The content of phospholipid, surfactant protein B, and total protein were determined in large aggregate (active) surfactant.ResultsAt 24 h, surfactant treatment transiently increased surfactant protein B content (70%, p

PurposeContact lens wear carries a risk of complications, including corneal infection. Solving these complications has been hindered by limitations of existing animal models. Here, we report development of a new murine model of contact lens wear.MethodsC57BL/6 mice were fitted with custom-made silicone-hydrogel contact lenses with or without prior inoculation with Pseudomonas aeruginosa (PAO1-GFP). Contralateral eyes served as controls. Corneas were monitored for pathology, and examined ex vivo using high-magnification, time-lapse imaging. Fluorescent reporter mice allowed visualization of host cell membranes and immune cells. Lens-colonizing bacteria were detected by viable counts and FISH. Direct-colony PCR was used for bacterial identification.ResultsWithout deliberate inoculation, lens-wearing corneas remained free of visible pathology, and retained a clarity similar to non-lens wearing controls. CD11c-YFP reporter mice revealed altered numbers, and distribution, of CD11c-positive cells in lens-wearing corneas after 24 h. Worn lenses showed bacterial colonization, primarily by known conjunctival or skin commensals. Corneal epithelial cells showed vacuolization during lens wear, and after 5 days, cells with phagocyte morphology appeared in the stroma that actively migrated over resident keratocytes that showed altered morphology. Immunofluorescence confirmed stromal Ly6G-positive cells after 5 days of lens wear, but not in MyD88 or IL-1R gene-knockout mice. P. aeruginosa-contaminated lenses caused infectious pathology in most mice from 1 to 13 days.ConclusionsThis murine model of contact lens wear appears to faithfully mimic events occurring during human lens wear, and could be valuable for experiments, not possible in humans, that help solve the pathogenesis of lens-related complications.

OBJECTIVE:To assess the utility of clinical predictors of persistent respiratory morbidity in extremely low gestational age newborns (ELGANs). STUDY DESIGN:We enrolled ELGANs (

BackgroundPulmonary surfactant provides an alveolar surface-active film that is critical for normal lung function. Our objective was to determine in vitro film formation properties of therapeutic and infant surfactants and the influence of surfactant protein (SP)-B content.MethodsWe used a multiwell fluorescent assay measuring maximum phospholipid surface accumulation (Max), phospholipid concentration required for half-maximal film formation (½Max), and time for maximal accumulation (tMax).ResultsAmong five therapeutic surfactants, calfactant (highest SP-B content) had film formation values similar to natural surfactant, and addition of SP-B to beractant (lowest SP-B) normalized its Max value. Addition of budesonide to calfactant did not adversely affect film formation. In tracheal aspirates of preterm infants with evolving chronic lung disease, SP-B content correlated with ½Max and tMax values, and SP-B supplementation of SP-B-deficient infant surfactant restored normal film formation. Reconstitution of normal surfactant indicated a role for both SP-B and SP-C in film formation.ConclusionFilm formation in vitro differs among therapeutic surfactants and is highly dependent on SP-B content in infant surfactant. The results support a critical role of SP-B for promoting surface film formation.

ObjectiveInhaled nitric oxide (iNO) has been tested to prevent bronchopulmonary dysplasia (BPD) in premature infants, however, the role of cyclic guanosine monophosphate (cGMP) is not known. We hypothesized that levels of NO metabolites (NOx) and cGMP in urine, as a noninvasive source for biospecimen collection, would reflect the dose of iNO and relate to pulmonary outcome.Study designStudies were performed on 125 infants who required mechanical ventilation at 7 to 14 days and received 24 days of iNO at 20-2 ppm. A control group of 19 infants did not receive iNO.ResultsIn NO-treated infants there was a dose-dependent increase of both NOx and cGMP per creatinine (maximal 3.1- and 2-fold, respectively, at 10-20 ppm iNO) compared with off iNO. NOx and cGMP concentrations at both 2 ppm and off iNO were inversely related to severity of lung disease during the 1st month, and the NOx levels were lower in infants who died or developed BPD at term. NOx was higher in Caucasian compared with other infants at all iNO doses.ConclusionUrinary NOx and cGMP are biomarkers of endogenous NO production and lung uptake of iNO, and some levels reflect the severity of lung disease. These results support a role of the NO-cGMP pathway in lung development.

Corticosterone and insulin play complex roles in the amount and composition of calories ingested, and the utilization and deposition of this energy. Understanding the interplay of these two hormones is complicated because increasing concentrations of corticosterone dose-dependently increase circulating insulin levels. We addressed individual contributions of each hormone by controlling, at steady-state levels, corticosterone (by adrenalectomy and exogenous replacement) and insulin (by streptozotocin-induced destruction of pancreatic beta-cells and exogenous replacement) across a spectrum of concentrations in rats, creating 8 hormonal combinations. For 5 days after surgery, all rats received chow. At day 5, they were subdivided into those that continued to receive chow and those that had a choice between chow, lard, and 32% sucrose for a further 5 days. During the choice/chow period, total calories ingested were stimulated by corticosterone and choice diet, and subject to a corticosterone-insulin interaction. Sucrose, but not lard, intake was stimulated by insulin. Body weight was increased by insulin, decreased by high corticosterone, and unaffected by diet. White adipose tissue depot weights were stimulated by insulin, corticosterone, and diet. Plasma triglycerides, free fatty acids, total ketone bodies, glucose, and glycerol were all significantly increased by corticosterone and the choice diet but inhibited by insulin. In contrast, plasma leptin was only increased by insulin and diet, plasma glucagon and liver glycogen was only affected by insulin and liver triglycerides, and arcuate nucleus proopiomelanocortin mRNA was only influenced by diet. Collectively, these data show that corticosterone and insulin determine the intake, form, and compartmentalization of energy both independently and interactively.

The common hepatic branch of the vagus nerve negatively regulates lard intake in rats with streptozotocin (STZ)-induced, insulin-dependent diabetes. However, this branch consists of two subbranches: the hepatic branch proper, which serves the liver, and the gastroduodenal branch, which serves the distal stomach, pancreas, and duodenum. The aim of this study was to determine whether the gastroduodenal branch specifically regulates voluntary lard intake. We performed a gastroduodenal branch vagotomy (GV) on nondiabetic, STZ-diabetic, and STZ-diabetic insulin-treated groups of rats and compared them with sham-operated counterparts. All rats had high steady-state corticosterone levels to maximize lard intake. Five days after surgery, all rats were provided with the choice of chow or lard to eat for another 5 days. STZ-diabetes resulted in a reduction in lard intake that was partially rescued by either GV or insulin treatment. Patterns of white adipose tissue (WAT) deposition differed after GV- and insulin-induced lard intake, with subcutaneous WAT increasing exclusively after the former and mesenteric WAT increasing exclusively in the latter. GV also prevented the insulin-induced reduction in the STZ-elevated plasma glucagon, triglycerides, free fatty acids, and total ketone bodies but did not alter the effect of insulin-induced reduction of plasma glucose levels. These data suggest that the gastroduodenal branch of the vagus inhibits lard intake and regulates WAT deposition and plasma metabolite levels in STZ-diabetic rats.