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Background: Acanthamoeba keratitis is challenging to treat and thought to result in poor outcomes, but very few comparative studies exist to assess whether ulcers caused by Acanthamoeba are worse than those caused by bacteria or fungus. Methods: In a retrospective cohort study, all cases of smear- or culture-proven Acanthamoeba keratitis diagnosed from January 2006 to June 2011 at an eye hospital in South India were identified from the microbiology database. Random samples of the same number of cases of bacterial and fungal keratitis, matched by year, were identified from the same database in order to compare outcomes between the three types of organism. The main outcomes were the time until the following events: re-epithelialization, discontinuation of antimicrobials, perforation/keratoplasty, elevated intraocular pressure, and new cataract. Results: The median time until re-epithelialization was 113 days for Acanthamoeba keratitis, 30 days for fungal keratitis, and 25 days for bacterial keratitis, and the median time until discontinuation of antimicrobial therapy was 100 days for Acanthamoeba keratitis, 49 days for fungal keratitis, and 40 days for bacterial keratitis. Compared to the other two organisms, Acanthamoeba ulcers took significantly longer to re-epithelialize (adjusted HR 0.4, 95% CI 0.3 to 0.6 relative to bacterial ulcers and HR 0.3, 95% CI 0.2 to 0.5 relative to fungal ulcers; overall p<0.001) and had significantly longer courses of antimicrobials (adjusted HR 0.3, 95% CI 0.2 to 0.6 relative to bacterial ulcers and HR 0.5, 95%CI 0.3 to 0.8 relative to fungal ulcers; overall p<0.001). No statistically significant difference was observed between the three organisms for the other time-to-event outcomes. Conclusions: Acanthamoeba keratitis was more difficult to treat and had worse clinical outcomes than bacterial or fungal ulcers, highlighting the lack of adequate treatment regimens for this infection. [ABSTRACT FROM AUTHOR]

Neutrophils are innate immune response cells designed to kill invading microorganisms. One of the mechanisms neutrophils use to kill bacteria is generation of damaging reactive oxygen species (ROS) via the respiratory burst. However, during enteric salmonellosis, neutrophil-derived ROS actually facilitates Salmonella expansion and survival in the gut. This seeming paradox led us to hypothesize that Salmonella may possess mechanisms to influence the neutrophil respiratory burst. In this work, we used an in vitro Salmonella-neutrophil co-culture model to examine the impact of enteric infection relevant virulence factors on the respiratory burst of human neutrophils. We report that neutrophils primed with granulocyte-macrophage colony stimulating factor and suspended in serum containing complement produce a robust respiratory burst when stimulated with viable STm. The magnitude of the respiratory burst increases when STm are grown under conditions to induce the expression of the type-3 secretion system-1. STm mutants lacking the type-3 secretion system-1 induce less neutrophil ROS than the virulent WT. In addition, we demonstrate that flagellar motility is a significant agonist of the neutrophil respiratory burst. Together our data demonstrate that both the type-3 secretion system-1 and flagellar motility, which are established virulence factors in enteric salmonellosis, also appear to directly influence the magnitude of the neutrophil respiratory burst in response to STm in vitro. [ABSTRACT FROM AUTHOR]

The worldwide need for human cornea equivalents continues to grow. Few clinical options are limited to allogenic and synthetic material replacements. We hypothesized that tissue engineered human cornea systems based on mechanically robust, patterned, porous, thin, optically clear silk protein films, in combination with human corneal stromal stem cells (hCSSCs), would generate 3D functional corneal stroma tissue equivalents, in comparison to previously developed 2D approaches. Silk film contact guidance was used to control the alignment and distribution of hCSSCs on RGD-treated single porous silk films, which were then stacked in an orthogonally, multi-layered architecture and cultured for 9 weeks. These systems were compared similar systems generated with human corneal fibroblasts (hCFs). Both cell types were viable and preferentially aligned along the biomaterial patterns for up to 9 weeks in culture. H&E histological sections showed that the systems seeded with the hCSSCs displayed ECM production throughout the entire thickness of the constructs. In addition, the ECM proteins tested positive for keratocyte-specific tissue markers, including keratan sulfate, lumican, and keratocan. The quantification of hCSSC gene expression of keratocyte-tissue markers, including keratocan, lumican, human aldehyde dehydrogenase 3A1 (ALDH3A1), prostaglandin D2 synthase (PTDGS), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4), within the 3D tissue systems demonstrated upregulation when compared to 2D single silk films and to the systems generated with the hCFs. Furthermore, the production of ECM from the hCSSC seeded systems and subsequent remodeling of the initial matrix significantly improved cohesiveness and mechanical performance of the constructs, while maintaining transparency after 9 weeks. [ABSTRACT FROM AUTHOR]

The study of ocular drug delivery systems has been one of the most covered topics in drug delivery research. One potential drug carrier solution is the use of materials that are already commercially available in ophthalmic lenses for the correction of refractive errors. In this study, we present a diffusion-based mathematical model in which the parameters can be adjusted based on experimental results obtained under controlled conditions. The model allows for the design of multi-layered therapeutic ophthalmic lenses for controlled drug delivery. We show that the proper combination of materials with adequate drug diffusion coefficients, thicknesses and interfacial transport characteristics allows for the control of the delivery of drugs from multi-layered ophthalmic lenses, such that drug bursts can be minimized, and the release time can be maximized. As far as we know, this combination of a mathematical modelling approach with experimental validation of non-constant activity source lamellar structures, made of layers of different materials, accounting for the interface resistance to the drug diffusion, is a novel approach to the design of drug loaded multi-layered contact lenses. [ABSTRACT FROM AUTHOR]

Understanding immune responses elicited by vaccines, together with immune responses required for protection, is fundamental to designing effective vaccines and immunisation programs. This study examines the effects of the route of administration of a live attenuated vaccine on its interactions with, and stimulation of, the murine immune system as well as its ability to increase survival and provide protection from colonisation by a virulent challenge strain. We assess the effect of administration method using the murine model for typhoid, where animals are infected with S. Typhimurium. Mice were vaccinated either intravenously or orally with the same live attenuated S. Typhimurium strain and data were collected on vaccine strain growth, shedding and stimulation of antibodies and cytokines. Following vaccination, mice were challenged with a virulent strain of S. Typhimurium and the protection conferred by the different vaccination routes was measured in terms of challenge suppression and animal survival. The main difference in immune stimulation found in this study was the development of a secretory IgA response in orally-vaccinated mice, which was absent in IV vaccinated mice. While both strains showed similar protection in terms of challenge suppression in systemic organs (spleen and liver) as well as survival, they differed in terms of challenge suppression of virulent pathogens in gut-associated organs. This difference in gut colonisation presents important questions around the ability of vaccines to prevent shedding and transmission. These findings demonstrate that while protection conferred by two vaccines can appear to be the same, the mechanisms controlling the protection can differ and have important implications for infection dynamics within a population. [ABSTRACT FROM AUTHOR]

Purpose: To compare estimates of 24-hour intraocular pressure (IOP) peak timing and variation obtained using a contact lens sensor (CLS) and using a pneumatonometer. Methods: Laboratory data collected from 30 healthy volunteers (ages, 20-66 years) in a randomized, controlled clinical trial were analyzed. Participants were housed for 24 hours in a sleep laboratory. One randomly selected right or left eye was fitted with a CLS that monitored circumferential curvature in the corneoscleral region related to the change of IOP. Electronic output signals of 30 seconds were averaged and recorded every 5 minutes. In the contralateral eye, habitual IOP measurements were taken using a pneumatonometer once every two hours. Simulated 24-hour rhythms in both eyes were determined by cosinor fitting. Simulated peak timings (acrophases) and simulated data variations (amplitudes) were compared between the paired eyes. Results: Bilateral change patterns of average 24-hour data for the group were in parallel. The simulated peak timing in the CLS fitted eye occurred at 4:44 AM ± 210 min (mean ± SD) and the IOP peak timing in the contralateral eye at 4:11 AM ± 120 min (P=0.256, Wilcoxon signed-rank test). There was no significant correlation between the simulated data variations in the paired eyes (P=0.820, linear regression). Conclusions: The 24-hour CLS data showed a simulated peak timing close to the 24-hour IOP peak timing obtained using the pneumatonometer. However, the simulated variations of 24-hour data in the paired eyes were not correlated. Estimated 24-hour IOP rhythms using the two devices should not be considered interchangeable. [ABSTRACT FROM AUTHOR]

Polydopamine (PDA) coating provides a promising approach for immobilization of biomolecules onto almost all kinds of solid substrates. However, the deposition kinetics of PDA coating as a function of temperature and reaction method is not well elucidated. Since dopamine self-polymerization usually takes a long time, therefore, rapid-formation of PDA film becomes imperative for surface modification of biomaterials and medical devices. In the present study, a practical method for preparation of rapidly-deposited PDA coating was developed using a uniquely designed device, and the kinetics of dopamine self-polymerization was investigated by QCM sensor system. It was found that high temperature and vigorous stirring could dramatically speed up the formation of PDA film on QCM chip surface. Surface characterization, BSA binding study, cell viability assay and antibacterial test demonstrates that the polydopamine coating after polymerization for 30 min by our approach exhibits similar properties to those of 24 h counterpart. The method has a great potential for rapid-deposition of polydopamine films to modify biomaterial surfaces. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the treatment outcomes and costs of early keratectomy in the management of moderate Fusarium keratitis. Methodology/Principal Findings: Consecutive cases of culture proven Fusarium keratitis treated at our hospital between January 2004 to December 2010 were included in this retrospective study. There were 38 cases of moderate keratitis with infiltrates between 3 to 6 mm in diameter and depth of infiltration not exceeding the inner 1/3 of the cornea. After excluding 5 patients with incomplete follow-up data, 13 patients who received early keratectomy within 1 week of admission were compared with a group of 20 patients treated medically. The significance of the association between early keratectomy and visual acuity, progression to perforation, secondary glaucoma and cataract formation, adjuvant therapy, hospitalization days and cost were assessed. There were no differences between the keratectomy and medication groups in regards to age, sex, presence of systemic diseases, and hypopyon formation on presentation. The early keratectomy group had a shorter hospital stay than the medical therapy group. Disease duration was significantly lower in the early keratectomy group (median: 29.0 vs. 54.5 days, P<0.001). Median hospitalization costs per patient were lower with early keratectomy (mean ward fee: 15175.4 vs. 44159.5 NTD, P<0.001; mean donor fee: 0 vs. 900.0 NTD, P<0.001), primarily because of reductions in hospital stay. More patients in the medication group developed perforations than in the keratectomy group (20% vs. 0%, respectively) and the perforation-free rate was higher in those with early keratectomy, but the results were not statistically significant. Conclusions/Significance: Early keratectomy in moderate Fusarium keratitis may reduce length of hospital stay, hospital costs, and perforation rates. [ABSTRACT FROM AUTHOR]

Salmonella pathogenicity island 1 (SPI1), comprising a type III section system that translocates effector proteins into host cells, is essential for the enteric pathogen Salmonella to penetrate the intestinal epithelium and subsequently to cause disease. Using random transposon mutagenesis, we found that a Tn10 disruption in the flagellar fliDST operon induced SPI1 expression when the strain was grown under conditions designed to repress SPI1, by mimicking the environment of the large intestine through the use of the intestinal fatty acid butyrate. Our genetic studies showed that only fliT within this operon was required for this effect, and that exogenous over-expression of fliT alone significantly reduced the expression of SPI1 genes, including the invasion regulator hilA and the sipBCDA operon, encoding type III section system effector proteins, and Salmonella invasion of cultured epithelial cells. fliT has been known to inhibit the flagellar machinery through repression of the flagellar master regulator flhDC. We found that the repressive effect of fliT on invasion genes was completely abolished in the absence of flhDC or fliZ, the latter previously shown to induce SPI1, indicating that this regulatory pathway is required for invasion control by fliT. Although this flhDC-fliZ pathway was necessary for fliT to negatively control invasion genes, fliZ was not essential for the repressive effect of fliT on motility, placing fliT high in the regulatory cascade for both invasion and motility. [ABSTRACT FROM AUTHOR]

Purpose: Fungi are a major cause of keratitis, although few medications are licensed for their treatment. The aim of this study is to observe the variation in commercialisation of antifungal eye drops, and to predict the seasonal distribution of fungal keratitis in Brazil. Methods: Data from a retrospective study of antifungal eye drops sales from the only pharmaceutical ophthalmologic laboratory, authorized to dispense them in Brazil (Opthalmos) were gathered. These data were correlated with geographic and seasonal distribution of fungal keratitis in Brazil between July 2002 and June 2008. Results: A total of 26,087 antifungal eye drop units were sold, with a mean of 2.3 per patient. There was significant variation in antifungal sales during the year (p,<0.01). A linear regression model displayed a significant association between reduced relative humidity and antifungal drug sales (R2 = 0.17,p<,0.01). Conclusions: Antifungal eye drops sales suggest that there is a seasonal distribution of fungal keratitis. A possible interpretation is that the third quarter of the year (a period when the climate is drier), when agricultural activity is more intense in Brazil, suggests a correlation with a higher incidence of fungal keratitis. A similar model could be applied to other diseases, that are managed with unique, or few, and monitorable medications to predict epidemiological aspects. [ABSTRACT FROM AUTHOR]

Previous studies have established that the expression of Salmonella enterica pathogenicity island 1 (SPI1), which is essential for epithelial invasion, is mainly regulated by the HilD protein. The ferric uptake regulator, Fur, in turn modulates the expression of the S. enterica hilD gene, albeit through an unknown mechanism. Here we report that S. enterica Fur, in its metal-bound form, specifically binds to an AT-rich region (BoxA), located upstream of the hilD promoter (PhilD), at position -191 to -163 relative to the hilD transcription start site. Furthermore, in a PhilD variant with mutations in BoxA, PhilD*, Fur·Mn2+ binding is impaired. In vivo experiments using S. enterica strains carrying wild-type PhilD or the mutant variant PhilD* showed that Fur activates hilD expression, while in vitro experiments revealed that the Fur·Mn2+ protein is sufficient to increase hilD transcription. Together, these results present the first evidence that Fur·Mn2+, by binding to the upstream BoxA sequence, directly stimulates the expression of hilD in S. enterica. [ABSTRACT FROM AUTHOR]

Curcumin has gained immense importance for its vast therapeutic and prophylactic applications. Contrary to this, our study reveals that it regulates the defense pathways of Salmonella enterica serovar Typhimurium (S. Typhimurium) to enhance its pathogenicity. In a murine model of typhoid fever, we observed higher bacterial load in Peyer's patches, mesenteric lymph node, spleen and liver, when infected with curcumin-treated Salmonella. Curcumin increased the resistance of S. Typhimurium against antimicrobial agents like antimicrobial peptides, reactive oxygen and nitrogen species. This increased tolerance might be attributed to the up-regulation of genes involved in resistance against antimicrobial peptides - pmrD and pmrHFIJKLM and genes with antioxidant function - mntH, sodA and sitA. We implicate that iron chelation property of curcumin have a role in regulating mntH and sitA. Interestingly, we see that the curcumin-mediated modulation of pmr genes is through the PhoPQ regulatory system. Curcumin downregulates SPI1 genes, required for entry into epithelial cells and upregulates SPI2 genes required to intracellular survival. Since it is known that the SPI1 and SPI2 system can be regulated by the PhoPQ system, this common regulator could explain curcumin's mode of action. This data urges us to rethink the indiscriminate use of curcumin especially during Salmonella outbreaks. [ABSTRACT FROM AUTHOR]

Background: S. Typhi, a human-restricted Salmonella enterica serovar, causes a systemic intracellular infection in humans (typhoid fever). In comparison, S. Typhimurium causes gastroenteritis in humans, but causes a systemic typhoidal illness in mice. The PhoP regulon is a well studied two component (PhoP/Q) coordinately regulated network of genes whose expression is required for intracellular survival of S. enterica. Methodology/Principal Findings: Using high performance liquid chromatography mass spectrometry (HPLC-MS/MS), we examined the protein expression profiles of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro and compared these results to profiles of phoP-/Q- mutants derived from S. Typhimurium LT2 and S. Typhi Ty2. Our analysis identified 53 proteins in S. Typhimurium LT2 and 56 proteins in S. Typhi that were regulated in a PhoP-dependent manner. As expected, many proteins identified in S. Typhi demonstrated concordant differential expression with a homologous protein in S. Typhimurium. However, three proteins (HlyE, STY1499, and CdtB) had no homolog in S. Typhimurium. HlyE is a pore-forming toxin. STY1499 encodes a stably expressed protein of unknown function transcribed in the same operon as HlyE. CdtB is a cytolethal distending toxin associated with DNA damage, cell cycle arrest, and cellular distension. Gene expression studies confirmed up-regulation of mRNA of HlyE, STY1499, and CdtB in S. Typhi in PhoP-inducing conditions. Conclusions/Significance: This study is the first protein expression study of the PhoP virulence associated regulon using strains of Salmonella mutant in PhoP, has identified three Typhi-unique proteins (CdtB, HlyE and STY1499) that are not present in the genome of the wide host-range Typhimurium, and includes the first protein expression profiling of a live attenuated bacterial vaccine studied in humans (Ty800). [ABSTRACT FROM AUTHOR]

Background: Mice do not get diarrhea when orally infected with S. enterica, but pre-treatment with oral aminoglycosides makes them susceptible to Salmonella colitis. However, genetically susceptible ItyS mice (Nramp1G169D allele) die from systemic infection before they develop diarrhea, so a new model is needed to study the pathogenesis of diarrhea. We pretreated ItyR mice (Nramp1G169) with oral kanamycin prior to infecting them with virulent S. Typhimurium strain 14028s in order to study Salmonella-induced diarrhea. We used both a visual scoring system and the measurement of fecal water content to measure diarrhea. BALB/c.D2Nramp1 congenic started losing weight 5 days post-infection and they began to die from colitis 10-14 days after infection. A SPI-1 (invA) mutant caused cecal, but not colonic inflammation and did not cause diarrhea. A phoP- mutant did not cause manifestations of diarrhea in either normal or NADPH-deficient (gp91phox) mice. However, strain 14028s caused severe colitis and diarrhea in gp91phox-deficient mice on an ItyR background. pmr A and F mutants, which are less virulent in orally infected BALB/c mice, were fully virulent in this model of colitis. Conclusions: S. enterica must be able to invade the colonic epithelium and to persist in the colon in order to cause colitis with manifestations of diarrhea. The NADPH oxidase is not required for diarrhea in Salmonella colitis. Furthermore, a Salmonella phoP mutant can be cleared from the colon by non-oxidative host defenses. [ABSTRACT FROM AUTHOR]