46 research outputs found
Recommended from our members
Overexpression of MMPs in Corneas Requiring Penetrating and Deep Anterior Lamellar Keratoplasty.
PurposeMatrix metalloproteinases (MMPs) comprise a family of zinc-dependent endopeptidases involved in wound healing processes, including neovascularization and fibrosis. We assessed MMP protein expression levels in diseased corneas of patients requiring penetrating and deep anterior lamellar keratoplasty. The purpose of this study was to test the hypothesis that upregulation of MMPs in diseased corneas is positively associated with clinical levels of corneal neovascularization and fibrosis.MethodsProtein expression levels of nine individual MMPs were quantified simultaneously in human corneal lysates by using the Bio-Plex Pro Human MMP 9-Plex Panel and the MAGPIX technology. Measurements of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP12, and MMP13 were performed on diseased specimens from 21 patients undergoing corneal transplantation (17 for penetrating keratoplasty and 4 for deep anterior lamellar keratoplasty) and 6 normal control corneas.ResultsLuminex-based expression analysis revealed a significant overexpression of four of the nine MMPs tested (MMP2, MMP8, MMP12, and MMP13) in patient samples compared to control. Significant overexpression of MMP1, MMP2, MMP8, MMP12, and MMP13 was observed in diseased corneas with neovascularization compared with diseased corneas without neovascularization. Overexpression of MMP1, MMP2, MMP8, MMP12, and MMP13 also corresponded with the levels of corneal fibrosis. Finally, reduced expression of MMP3 was detected in keratoconus patients.ConclusionsMultiple MMPs are expressed in the corneas of patients with chronic disease requiring keratoplasty even when the pathologic process appears to be clinically inactive. In particular, the expression of several MMPs (MMP2, MMP8, MMP12, and MMP13) is positively associated with increased levels corneal fibrosis and neovascularization
Recommended from our members
Risk Factors Predicting Loss to Follow-Up, Medication Noncompliance, and Poor Visual Outcomes Among Patients With Infectious Keratitis at a Public County Hospital
PurposeInfectious keratitis is a vision-threatening condition requiring close follow-up and disciplined eye drop administration to achieve resolution. Although patients presenting to county hospitals often have more severe presentations, there is a paucity of risk and outcomes data in this setting. This study investigates risk factors predicting loss to follow-up (LTFU), medication noncompliance, and poor outcomes for infectious keratitis in the county hospital setting.MethodsThis was a retrospective case-control study at Zuckerberg San Francisco General Hospital and Trauma Center. Inclusion criteria were patients who had corneal cultures for suspected infectious bacterial or fungal keratitis between 2010 and 2021. Exclusion criteria were patients with viral keratitis only. Multivariable logistic regression was used to analyze the relationship of social and medical risk factors with LTFU, medication noncompliance, worsened visual acuity (VA), and delayed resolution time.ResultsOf 174 patients with infectious keratitis in this analysis, 69 (40.0%) had LTFU. Unemployment was associated with increased risk of LTFU (odds ratio 2.58, P = 0.049) and worse final VA ( P = 0.001). Noncompliance trended toward an association with homelessness (odds ratio 3.48, P = 0.095). Increasing age correlated with longer resolution time, with each 1-year increase associated with delayed resolution by 0.549 days ( P = 0.042).ConclusionsPatients experiencing unemployment, homelessness, or increased age demonstrate higher risk for treatment barriers including loss to follow-up and medication noncompliance, resulting in worse VA and delayed time to resolution. These risk factors should be considered when determining the need for more deliberate follow-up measures in patients with infectious keratitis
Immune responses to typhoid conjugate vaccine in a two dose schedule among Nepalese children <2 years of age
Background
Previously, the Vi-typhoid conjugate vaccine (Vi-TT) was found to be highly efficacious in Nepalese children under 16 years of age. We assessed the immunogenicity of Vi-TT at 9 and 12 months of age and response to a booster dose at 15 months of age.
Methods
Infants were recruited at Patan Hospital, Kathmandu and received an initial dose of Vi-TT at 9 or 12 months of age with a booster dose at 15 months of age. Blood was taken at four timepoints, and antibody titres were measured using a commercial ELISA kit. The primary study outcome was seroconversion (4-fold rise in antibody titre) of IgG one month after both the doses.
Findings
Fifty children were recruited to each study group.Some visits were disrupted by the COVID19 pandemic and occurred out of protocol windows. Both the study groups attained 100 % IgG seroconversion after the initial dose. IgG seroconversion in the 9-month group was significantly higher than in the 12-month group (68.42 % vs 25.8 %, p < 0.001). Among individuals who attended visits per protocol, IgG seroconversion after the first dose occurred in 100 % of individuals (n = 27/27 in 9-month and n = 32/32 in 12-month group). However, seroconversion rates after the second dose were 80 % in the 9-month and 0 % in the shorter dose-interval 12-month group (p < 0.001) (n = 16/20 and n = 0/8, respectively).
Interpretation
Vi-TT is highly immunogenic at both 9 and 12 months of age. Stronger response to a booster in the 9-month group is likely due to the longer interval between doses
sFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life In Vivo
We would like to thank Jonathan Winger and Xiao Zhu for guidance with the insect cell protein expression system and providing reagents. We would like to acknowledge Ann Fischer for help with expressing the sFlt protein in the Tissue Culture Facility at UC Berkeley and Dawn Spelke and Anusuya Ramasubramanian for help optimizing protein purification from insect cells. We are also grateful for the help from Leah Byrne and John Flannery at in the Helen Wills Neuroscience Institute at UC Berkeley for aiding us in the development of the rat intravitreal residence time model and for allowing us to use their facilities.Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.Yeshttp://www.plosone.org/static/editorial#pee
Robust estimation of bacterial cell count from optical density
Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data
Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19
IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19.
Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19.
DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 nonâcritically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022).
INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (nâ=â257), ARB (nâ=â248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; nâ=â10), or no RAS inhibitor (control; nâ=â264) for up to 10 days.
MAIN OUTCOMES AND MEASURES The primary outcome was organ supportâfree days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes.
RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ supportâfree days among critically ill patients was 10 (â1 to 16) in the ACE inhibitor group (nâ=â231), 8 (â1 to 17) in the ARB group (nâ=â217), and 12 (0 to 17) in the control group (nâ=â231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ supportâfree days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively).
CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes.
TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570
Confocal Imaging of Myeloid Cells in the Corneal Stroma of Live Mice.
The accessibility and transparency of the cornea makes it a good tissue model for monitoring immunological responses using in vivo real time imaging analysis (Lee et al., 2010; Tan et al., 2013). These corneal qualities have also allowed for high-resolution in vivo imaging of non-ocular tissue transplanted into the anterior chamber of the mouse eye (Speier et al., 2008a; Speier et al., 2008b). This protocol was adapted from Speier (2008) to successfully assess real-time in vivo myeloid cell dynamics in wounded corneas of c-fms-EGFP mice (Chan et al., 2013). Macrophage colony-stimulating factor (CSF-1) regulates the differentiation and proliferation of cells of the mononuclear phagocyte system. The activity of CSF-1 is mediated by the CSF-1 receptor that is encoded by c-fms (Csf1r) protooncogene. The c-fms gene is expressed in macrophage, trophoblast cell lineages, and to some extent granulocytes. In the c-fms-EGFP mice EGFP, enhanced green fluorescent protein, is driven under the Csf1r, colony stimulating factor 1 receptor, promoter and highlights myeloid cells (Sasmono et al., 2003). This protocol can be further adapted to image other transgenic mice expressing fluorescent proteins
Recommended from our members
Confocal Imaging of Myeloid Cells in the Corneal Stroma of Live Mice.
The accessibility and transparency of the cornea makes it a good tissue model for monitoring immunological responses using in vivo real time imaging analysis (Lee et al., 2010; Tan et al., 2013). These corneal qualities have also allowed for high-resolution in vivo imaging of non-ocular tissue transplanted into the anterior chamber of the mouse eye (Speier et al., 2008a; Speier et al., 2008b). This protocol was adapted from Speier (2008) to successfully assess real-time in vivo myeloid cell dynamics in wounded corneas of c-fms-EGFP mice (Chan et al., 2013). Macrophage colony-stimulating factor (CSF-1) regulates the differentiation and proliferation of cells of the mononuclear phagocyte system. The activity of CSF-1 is mediated by the CSF-1 receptor that is encoded by c-fms (Csf1r) protooncogene. The c-fms gene is expressed in macrophage, trophoblast cell lineages, and to some extent granulocytes. In the c-fms-EGFP mice EGFP, enhanced green fluorescent protein, is driven under the Csf1r, colony stimulating factor 1 receptor, promoter and highlights myeloid cells (Sasmono et al., 2003). This protocol can be further adapted to image other transgenic mice expressing fluorescent proteins
Recommended from our members
Animal Models of Corneal Injury.
The cornea is an excellent model system to use for the analysis of wound repair because of its accessibility, lack of vascularization, and simple anatomy. Corneal injuries may involve only the superficial epithelial layer or may penetrate deeper to involve both the epithelial and stromal layers. Here we describe two well-established in vivo corneal wound models: a mechanical wound model that allows for the study of re-epithelialization and a chemical wound model that may be used to study stromal activation in response to injury (Stepp et al., 2014; Carlson et al., 2003)