Quantification of a panel for dry-eye protein biomarkers in tears: A comparative pilot study using standard ELISA and customized microarrays.

Purpose: This paper examines the tear concentration of cystatin S (CST4), calcyclin (S100A6), calgranulin A (S100A8), and matrix metalloproteinase 9 (MMP9), and the correlation between biomarker expression, clinical parameters, and disease severity in patients suffering from dry eye (DE). A comparison of the results is obtained via ELISA tests and customized antibody microarrays for protein quantification. Methods: This single-center, observational study recruited 59 participants (45 DE and 14 controls). Clinical evaluation included an Ocular Surface Disease Index (OSDI) questionnaire, a tear osmolarity (OSM) test, the Schirmer test (SCH), tear breakup time (TBUT), fluorescein (FLUO) and lissamine green (LG) corneal staining, and meibomian gland evaluation (MGE). Tear concentrations of CST4, S100A6, S100A8, and MMP9 were measured using standard individual ELISA assays. The levels of CST4, S100A6, and MMP9 were also measured using customized multiplexed antibody microarrays. Correlations between variables were evaluated, and a significance level was p value <0.05. Results: The quantification of tear protein biomarkers with ELISA showed that the concentration of CST4 was significantly (2.14-fold) reduced in tears of DE patients in comparison with control (CT) subjects (p < 0.001). S100A6 and S100A8 concentrations were significantly higher in the tears of DE patients (1.36- and 2.29-fold; p < 0.001 and 0.025, respectively) in comparison with CT. The MMP9 level was also higher in DE patients (5.83-fold), but not significantly (p = 0.22). The changes in CST4 and S100A6 concentrations were significantly correlated with dry eye disease (DED) severity. Quantification of CST4, S100A6, and MMP9, using antibody microarrays, confirmed the ELISA results. Similar trends were observed: 1.83-fold reduction for CST4 (p value 0.01), 8.63-fold increase for S100A6 (p value <0.001) and 9.67-fold increase for MMP9 (p value 0.94), but with higher sensitivity. The biomarker concentrations were significantly associated with the signs and symptoms related with DED. Conclusions: S100A6, S100A8, and CST4 diagnostic biomarkers strongly correlate with DED clinical parameters. S100A6 and CST4 are also useful for grading DE severity. The multiplexed antibody microarray technique, used here for tear multi-marker quantification, appears more sensitive than standard ELISA tests.

Asymmetric, visible light-mediated radical sulfinyl-Smiles rearrangement to access all-carbon quaternary stereocentres.

The asymmetric construction of all-carbon quaternary centres within acyclic settings represents a long-standing challenge for synthetic chemists. Alongside polar and radical methods, rearrangement reactions represent an attractive platform, but still broadly applicable methods are in high demand. Here we report an asymmetric, radical sulfinyl-Smiles rearrangement to access acyclic amides that bear an α-all-carbon quaternary centre. Our strategy uses enantioenriched N-arylsulfinyl acrylamides as acceptors for a variety of radicals produced in situ under mild photoredox conditions. The sulfinamido group not only directs the 1,4-migration of the aryl moiety onto the α-carbon of the amide, which thus governs its absolute configuration, but also functions as a traceless chiral auxiliary. The amides obtained in this multicomponent process are prevalent in pharmaceuticals, agrochemicals and bioactive natural products, and can be transformed into valuable chiral α,α-disubstituted acids, oxindoles as well as into ß,ß-disubstituted amines, highlighting the synthetic potential of this transformation.

Lacritin proteoforms prevent tear film collapse and maintain epithelial homeostasis.

Lipids in complex, protein-enriched films at air/liquid interfaces reduce surface tension. In the absence of this benefit, the light refracting and immunoprotective tear film on eyes would collapse. Premature collapse, coupled with chronic inflammation compromising visual acuity, is a hallmark of dry eye disease affecting 7 to 10% of individuals worldwide. Although collapse seems independent of mutation (unlike newborn lung alveoli), selective proteome and possible lipidome changes have been noted. These include elevated tissue transglutaminase and consequent inactivation through C-terminal cross-linking of the tear mitogen lacritin, leading to significant loss of lacritin monomer. Lacritin monomer restores homeostasis via autophagy and mitochondrial fusion and promotes basal tearing. Here, we discover that lacritin monomer C-terminal processing, inclusive of cysteine, serine, and metalloproteinase activity, generates cationic amphipathic α-helical proteoforms. Such proteoforms (using synthetic peptide surrogates) act like alveolar surfactant proteins to rapidly bind and stabilize the tear lipid layer. Immunodepletion of C- but not N-terminal proteoforms nor intact lacritin, from normal human tears promotes loss of stability akin to human dry eye tears. Stability of these and dry eye tears is rescuable with C- but not N-terminal proteoforms. Repeated topical application in rabbits reveals a proteoform turnover time of 7 to 33 h with gradual loss from human tear lipid that retains bioactivity without further processing. Thus, the processed C-terminus of lacritin that is deficient or absent in dry eye tears appears to play a key role in preventing tear film collapse and as a natural slow release mechanism that restores epithelial homeostasis.

Inmunoexpresión de biomarcadores Bax, Bcl-2, CD138, H3, Ki-67, MCM3 y p53 en liquen plano oral / Immunoexpression of biomarkeres BAX, BCL-2, CD-138, H3, Ki-67, MCM3 AND p53 in oral lichen planus

El objetivo del presente trabajo fue determinar la expresión de diversos biomarcadores moleculares en liquen plano oral para ayudar a comprender su conducta biológica. Materiales y métodos: Se realizó un estudio inmunohistoquímico en 40 casos de liquen plano oral contra BAX, BCL-2, CD-138, Histona 3, Ki-67, MCM3 y p53, en el Área de Patología Molecular Estomatológica de la Facultad de Odontología, UDELAR, Uruguay. Resultados: Se observó mayor expresión de BAX en contraposición con BCL-2, sugiriendo un comportamiento proapoptótico, respaldado a su vez por la ausencia de expresión de p53. La expresión de los marcadores de proliferación celular fue en todo el tejido lesional observado, sugiriendo así alteraciones de la proliferación. CD-138 se expresó de manera intensa y uniforme, determinando una baja alteración de las uniones intercelulares para estos casos. Conclusiones: La alteración en la expresión de las proteínas estudiadas sugiere un trastorno en los mecanismos proliferativos y apoptóticos, los cuales se asocian con una conducta patológica de la mucosa oral.

This study aims to establish an association of the expression of specific biomarkers in oral lichen planus to understandits biological behavior. Materials and methods: An immunohistochemistry study was conducted in 40 cases of oral lichen planus against BAX, BCL-2, CD138, Histone 3, Ki-67, MCM3 and p53 at the Molecular Pathology Area of the School of Dentistry, UDELAR, Uruguay. Results: A greater expression of BAX was detected compared to BCL-2, suggesting a pro-apoptotic behavior, supported by the absence of p53 expression. MCM3 expression was more sensitive than Ki-67, considering proliferation alterations. CD-138 had a more intense and uniform expression, determining fewer intercellular adhesion alterations. Conclusions: The expression of the proteins studied suggests an alteration in proliferative and apoptotic mechanisms, associated with a pathological behavior of the oral mucosa.

O objetivo deste trabalho foi determinar a expressão de vários biomarcadores moleculares no líquen plano oral para ajudar a compreender seu comportamento biológico. Materiais emétodos: Foi realizado um estudo imunohistoquímico em 40 casos de líquen plano oral contra BAX, BCL-2, CD-138, Histona 3, Ki-67, MCM3 e p53, na área de Patologia Molecular Estomatológica da Faculdade de Odontologia , UDELAR, Uruguai. Resultados: Observou-se aumento da expressão de BAX em contraste com BCL-2, sugerindo um comportamento proapoptótico, apoiado por sua vez pela ausência da expressão de p53. A expressão de marcadores de proliferação celular foi observada em todo o tecido da lesão, sugerindo alterações na proliferação. CD-138 foi expressado de maneira intensa e uniforme, determinando uma baixa alteração das junções intercelulares para esses casos. Conclusões: A alteração na expressão das proteínas estudadas sugere um distúrbio nos mecanismos proliferativos e apoptóticos, os quais estão associados a um comportamento patológico da mucosa oral.

Changes in tear biomarker levels in keratoconus after corneal collagen crosslinking.

Purpose: The purpose of this work was to analyze the expressions of matrix metalloproteinase 9 (MMP-9), calcyclin (S100A6), and cystatin S (CST4) in the tears of keratoconus (KC) patients. The correlations between the expressions of these proteins and the values of various ocular surface parameters were examined after accelerated corneal crosslinking (A-CXL) with pulsed ultraviolet light. Methods: This prospective, observational study enrolled patients with different grades of KC, scheduled to undergo the A-CXL procedure, as well as healthy subjects. Tear samples were analyzed by employing customized antibody microarray assays for MMP-9, S100A6, and CST4 proteins. The keratometry readings at the maximum keratometry (Kmax) and the simulated keratometry (SimK) values were obtained for examining the postoperative evolution of corneal topography. The state of the ocular surface was evaluated using the results of the Ocular Surface Disease Index (OSDI) questionnaire, tear osmolarity (OSM) test, Schirmer test (SCH), Tear Break Up Time (TBUT), tear clearance (CLR), and fluorescein (FLUO) and lissamine green (LG) corneal staining. Results: A total of 18 patients (22 eyes) and 10 healthy subjects were studied. The concentrations of MMP-9 and S100A6 decreased in tears, from 104.5 ± 78.98 ng/ml and 350.20 ± 478.08 ng/ml before the surgery to 48.7 ± 24.20 ng/ml and 55.70 ± 103.62 ng/ml, respectively, after 12 months of follow up. There were no changes in the CST4 concentration after 12 months of follow up (2202.75 ± 2863.70 versus 2139.68 ±2719.89 ng/ml). When the patients were divided into three groups according to the evolutive stage of KC, the trends for the three biomarkers in each group were the same as in the general group. Basal concentrations of MMP-9 and S100A6 from healthy subjects and KC patients were compared. The levels of MMP-9 and S100A6 in tears were (9.8 ± 5.11 and 104.55 ± 78.98 ng/ml, p<0.01; and 11.35 ± 3.18 and 350.26 ± 478.06 ng/ml, respectively, p<0.01). This was not the case for CST4, which did not exhibit statistically significant differences between the two groups (2261.94 ± 510.65 and 2176.73 ± 2916.27 ng/ml respectively, p=0.07). Conclusions: A-CXL promoted a decrease in the concentrations of MMP-9 and S100A6 in the tear film. This effect may be related to the restoration of corneal homeostasis and the consequent repair of the tissue damage caused by KC. Moreover, the A-CXL treatment did not produce lasting alterations in the ocular surface, and the values of the evaluated clinical parameters did not change significantly.

The analysis of human conjunctival epithelium proteome in ocular surface diseases using impression cytology and 2D-DIGE.

Conjunctival impression cytology samples from patients with meibomian gland dysfunction (MGD), dry eye (DE), and healthy subjects (CT) were collected for determination of the degree of squamous metaplasia (SM) by PAS-hematoxylin staining and for comparative proteomic analyses by 2D-DIGE. The protein spots with discriminant expression were identified by MALDI-TOF/TOF mass spectrometry. Three independent statistical studies were conducted: i). Analysis of differential protein expression between study groups: We observed increased expression of proteins S100A4, S100A8, retinal dehydrogenase-1, peroxiredoxin-1, annexin-A1, annexin-A2, α-enolase, and glutathione S-transferase-P in DE, whereas the highest expression of peroxiredoxin-6, actin cytoplasmic-1, peroxiredoxin-2, and heat shock protein HSP-90-α was observed in MGD; ii). Correlation between changes in the proteome profile and the grade of SM: The expression of 5 different cytokeratins (KRT1, KRT4, KRT8, KRT10, and KRT13) correlated with the degree of SM; iii). Proteome profile differences between pathological and CT groups: An overall proteome analysis revealed upregulation of 9 proteins in the pathological groups (Annexin-A1, α-enolase, Annexin-A2, S100A8, cytokeratin-1, Peroxiredoxin-2 and Leukocyte elastase inhibitor) and downregulation of 2 proteins (Galectin-3 and Lipocalin-1). In conclusion, a sensitive proteomic approach to study conjunctival tissue collected from minimally invasive impression cytology was implemented. Differential proteomics analyses showed that in comparison with the MGD, the DE patients presented higher overexpression of proteins related to antimicrobial defense, tissue-damage response, and regulation of body fluid secretions. Changes in MGD proteome were associated with oxidative stress and anti-apoptotic processes. We found a correlation between the grade of SM and expression of proteins associated with cytoskeleton and keratinization. The studied pathological groups shared elements related to the defense and inflammatory responses. Dot blot assays of proteins ANXA1, S100A8, and S100A4 validated the proteomic results obtained from 2D-DIGE experiments and confirmed the correlation between the expression of these proteins and the clinical parameters.

Tear proteome analysis in ocular surface diseases using label-free LC-MS/MS and multiplexed-microarray biomarker validation.

We analyzed the tear film proteome of patients with dry eye (DE), meibomian gland dysfunction (MGD), and normal volunteers (CT). Tear samples were collected from 70 individuals. Of these, 37 samples were analyzed using spectral-counting-based LC-MS/MS label-free quantitation, and 33 samples were evaluated in the validation of candidate biomarkers employing customized antibody microarray assays. Comparative analysis of tear protein profiles revealed differences in the expression levels of 26 proteins, including protein S100A6, annexin A1, cystatin-S, thioredoxin, phospholipase A2, antileukoproteinase, and lactoperoxidase. Antibody microarray validation of CST4, S100A6, and MMP9 confirmed the accuracy of previously reported ELISA assays, with an area under ROC curve (AUC) of 87.5%. Clinical endpoint analysis showed a good correlation between biomarker concentrations and clinical parameters. In conclusion, different sets of proteins differentiate between the groups. Apolipoprotein D, S100A6, S100A8, and ceruloplasmin discriminate best between the DE and CT groups. The differences between antileukoproteinase, phospholipase A2, and lactoperoxidase levels allow the distinction between MGD and DE, and the changes in the levels of annexin A1, clusterin, and alpha-1-acid glycoprotein 1, between MGD and CT groups. The functional network analysis revealed the main biological processes that should be examined to identify new candidate biomarkers and therapeutic targets.

TFOS DEWS II Tear Film Report.

The members of the Tear Film Subcommittee reviewed the role of the tear film in dry eye disease (DED). The Subcommittee reviewed biophysical and biochemical aspects of tears and how these change in DED. Clinically, DED is characterized by loss of tear volume, more rapid breakup of the tear film and increased evaporation of tears from the ocular surface. The tear film is composed of many substances including lipids, proteins, mucins and electrolytes. All of these contribute to the integrity of the tear film but exactly how they interact is still an area of active research. Tear film osmolarity increases in DED. Changes to other components such as proteins and mucins can be used as biomarkers for DED. The Subcommittee recommended areas for future research to advance our understanding of the tear film and how this changes with DED. The final report was written after review by all Subcommittee members and the entire TFOS DEWS II membership.

Custom RT-qPCR-array for glaucoma filtering surgery prognosis.

Excessive subconjunctival scarring is the main reason of failure of glaucoma filtration surgery. We analyzed conjunctival and systemic gene expression patterns after non penetrating deep sclerectomy (NPDS). To find expression patterns related to surgical failure and their correlation with the clinical outcomes. This study consisted of two consecutive stages. The first was a prospective analysis of wound-healing gene expression profile of six patients after NPDS. Conjunctival samples and peripheral blood samples were collected before and 15, 90,180, and 360 days after surgery. In the second stage, we conducted a retrospective analysis correlating the late conjunctival gene expression and the outcome of the NPDS for 11 patients. We developed a RT-qPCR Array for 88 key genes associated to wound healing. RT-qPCR Array analysis of conjunctiva samples showed statistically significant differences in 29/88 genes in the early stages after surgery, 20/88 genes between 90 and 180 days after surgery, and only 2/88 genes one year after surgery. In the blood samples, the most important changes occurred in 12/88 genes in the first 15 days after surgery. Correspondence analyses (COA) revealed significant differences between the expression of 20/88 genes in patients with surgical success and failure one year after surgery. Different expression patterns of mediators of the bleb wound healing were identified. Examination of such patterns might be used in surgery prognosis. RT-qPCR Array provides a powerful tool for investigation of differential gene expression wound healing after glaucoma surgery.

Antigen-Antibody Affinity for Dry Eye Biomarkers by Label Free Biosensing. Comparison with the ELISA Technique.

The specificity and affinity of antibody-antigen interactions is a fundamental way to achieve reliable biosensing responses. Different proteins involved with dry eye dysfunction: ANXA1, ANXA11, CST4, PRDX5, PLAA and S100A6; were validated as biomarkers. In this work several antibodies were tested for ANXA1, ANXA11 and PRDX5 to select the best candidates for each biomarker. The results were obtained by using Biophotonic Sensing Cells (BICELLs) as an efficient methodology for label-free biosensing and compared with the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique.

Solid lipid nanoparticles for delivery of Calendula officinalis extract.

Solid lipid nanoparticles (SLN) composed of long-chain fatty acids (palmitic acid, stearic acid or arachidic acid), Epikuron 200 (purified phosphatidylcholine), and bile salts (cholate, taurocholate or taurodeoxycholate) have been prepared by dilution of a microemulsion. A total of five different systems were prepared, and characterized by photon correlation spectroscopy, transmission electron microscopy, differential scanning calorimetry, and infrared spectroscopy. The SLN formulation showing optimal properties (lowest size and polydispersity index and highest zeta potential) was obtained with stearic acid and taurodeoxycholate as cosurfactant. This formulation was loaded with Calendula officinalis extract, a natural compound used on ophthalmic formulations given its anti-inflammatory, emollient, and wound repairing activity. Calendula-loaded SLN preparations were characterized in order to determine loading capacity and entrapment efficiency. In vitro cytotoxicity and wound healing efficacy of Calendula-loaded SLN compared to that of a free plant extract were evaluated on a conjunctival epithelium cell line WKD. Our results suggest that this SLN formulation is a safe and solvent-free Calendula extract delivery system which could provide a controlled therapeutic alternative for reducing disease-related symptoms and improving epithelium repair in ocular surface.

Human Basal Tear Peptidome Characterization by CID, HCD, and ETD Followed by in Silico and in Vitro Analyses for Antimicrobial Peptide Identification.

Endogenous peptides are valuable targets in the analysis of biological processes. The tear film contains proteins and peptides released by the tear duct mucosal cells, including antimicrobial peptides involved in the protection against exogenous pathogens; however, the peptide content of the tear liquid remains poorly characterized. We analyzed naturally occurring peptides isolated from human basal tears. Mass spectrometry analysis of endogenous peptides presents a number of drawbacks, including size heterogeneity and nonpredictable fragmentation patterns, among others. Therefore, CID, ETD, and HCD methods were used for the characterization of the tear peptide content. The contribution of DMSO as an additive of the chromatographic solvents was also evaluated. We identified 157, 131, and 122 peptides using CID-, ETD-, and HCD-based methods, respectively. Altogether, 234 different peptides were identified, leading to the generation of the biggest data set of endogenous tear peptides to date. The antimicrobial activity prediction analysis performed in silico revealed different putative antimicrobial peptides. Two of the extracellular glycoprotein lacritin peptides were de novo synthesized, and their antimicrobial activity was confirmed in vitro. Our findings demonstrate the benefits of using different fragmentation methods for the analysis of endogenous peptides and provide a useful approach for the discovery of peptides with antimicrobial activity.

Label-free LC-MS/MS quantitative analysis of aqueous humor from keratoconic and normal eyes.

PURPOSE: The etiology of keratoconus (KC) and the factors governing its progression are not well understood. It has been suggested that this disease might be caused by biochemical alterations in the cornea; changes in the expression profiles of human aqueous humor (hAH) proteins have been observed in some diseases. To gain a new insight into the molecular mechanisms of KC pathology, we examined the hAH proteomes of those in the advanced stages of this disease. We used a high-throughput mass spectrometry approach to compare hAH protein expression in patients with KC and in control subjects. METHODS: Aqueous humor samples were acquired from five keratoconus patients during keratoplasty surgery and from five myopic control subjects during phakic intraocular lens implantation. Quantitative mass spectrometry analysis using spectral counting was performed to determine the relative amounts of hAH proteins in the samples from KC patients and control individuals. RESULTS: All KC patients included in the study presented severe keratoconus (K2 >52 D), and slit-lamp examination revealed microfolds in Descemet's membrane, without clinical signs of hydrops. We found significant differences between the expression levels of 16 proteins in the two groups. In KC samples, seven proteins were overexpressed and nine were underexpressed in comparison with the control group. Gene ontology analysis revealed that these deregulated proteins are implicated in several biologic processes, such as the regulation of proteolysis, responses to hypoxia, and responses to hydrogen peroxide, among others. CONCLUSIONS: The protein expression profiles in hAH from KC patients and myopic control subjects differ significantly. This result suggests that some components of the hAH proteome are involved in this disease. Further in-depth analysis of the hAH proteome should provide a better understanding of the mechanisms governing the pathophysiology of KC.

Functional consequences of radiation-induced oxidative stress in cultured neural stem cells and the brain exposed to charged particle irradiation.

AIMS: Redox homeostasis is critical in regulating the fate and function of multipotent cells in the central nervous system (CNS). Here, we investigated whether low dose charged particle irradiation could elicit oxidative stress in neural stem and precursor cells and whether radiation-induced changes in redox metabolism would coincide with cognitive impairment. RESULTS: Low doses (<1 Gy) of charged particles caused an acute and persistent oxidative stress. Early after (<1 week) irradiation, increased levels of reactive oxygen and nitrogen species were generally dose responsive, but were less dependent on dose weeks to months thereafter. Exposure to ion fluences resulting in less than one ion traversal per cell was sufficient to elicit radiation-induced oxidative stress. Whole body irradiation triggered a compensatory response in the rodent brain that led to a significant increase in antioxidant capacity 2 weeks following exposure, before returning to background levels at week 4. Low dose irradiation was also found to significantly impair novel object recognition in mice 2 and 12 weeks following irradiation. INNOVATION: Data provide evidence that acute exposure of neural stem cells and the CNS to very low doses and fluences of charged particles can elicit a persisting oxidative stress lasting weeks to months that is associated with impaired cognition. CONCLUSIONS: Exposure to low doses of charged particles causes a persistent oxidative stress and cognitive impairment over protracted times. Data suggest that astronauts subjected to space radiation may develop a heightened risk for mission critical performance decrements in space, along with a risk of developing long-term neurocognitive sequelae.

Hemangiomas mesentéricos: presentación de cuatro casos / Mesenteric hemangiomas: report of four cases

Los hemangiomas son tumores vasculares benignos comunes, encontrados principalmente en la piel y el tejido celular subcutáneo. Su localización en el mesenterio es muy poco frecuente. En este artículo se reportan cuatro casos de hemangiomas mesentéricos, y uno de ellos que se presentó con hemoperitoneo significativo.

Evaluation of inter-day and inter-individual variability of tear peptide/protein profiles by MALDI-TOF MS analyses.

PURPOSE: To characterize the tear film peptidome and low molecular weight protein profiles of healthy control individuals, and to evaluate changes due to day-to-day and individual variation and tear collection methods, by using solid phase extraction coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling. METHODS: The tear protein profiles of six healthy volunteers were analyzed over seven days and inter-day and inter-individual variability was evaluated. The bilaterality of tear film and the effect of tear collection methods on protein profiles were also analyzed in some of these patients. MALDI-TOF MS analyses were performed on tear samples purified by using a solid phase extraction (SPE) method based on C18 functionalized magnetic beads for peptide and low molecular weight protein enrichment, focusing spectra acquisition on the 1 to 20 kDa range. Spectra were analyzed using principal component analysis (PCA) with MultiExperiment Viewer (TMeV) software. Volunteers were examined in terms of tear production status (Schirmer I test), clinical assessment of palpebral lids and meibomian glands, and a subjective OSD questionnaire before tear collection by a glass micro-capillary. RESULTS: Analysis of peptides and proteins in the 1-20 kDa range showed no significant inter-day differences in tear samples collected from six healthy individuals during seven days of monitoring, but revealed subtle intrinsic inter-individual differences. Profile analyses of tears collected from the right and left eyes confirmed tear bilaterality in four healthy patients. The addition of physiologic serum for tear sample collection did not affect the peptide and small protein profiles with respect to the number of resolved peaks, but it did reduce the signal intensity of the peaks, and increased variability. Magnetic beads were found to be a suitable method for tear film purification for the profiling study. CONCLUSIONS: No significant variability in tear peptide and protein profiles below 20 kDa was found in healthy controls over a seven day period, nor in right versus left eye profiles from the same individual. Subtle inter-individual differences can be observed upon tear profiling analysis and confirm intrinsic variability between control subjects. Addition of physiologic serum for tear collection affects the proteome and peptidome in terms of peak intensities, but not in the composition of the profiles themselves. This work shows that MALDI-TOF MS coupled with C18 magnetic beads is an effective and reproducible methodology for tear profiling studies in the clinical monitoring of patients.

Comparative study of limbal stem cell deficiency diagnosis methods: detection of MUC5AC mRNA and goblet cells in corneal epithelium.

PURPOSE: To evaluate a limbal stem cell deficiency (LSCD) diagnosis method based on the detection of the MUC5AC transcript by reverse transcription-polymerase chain reaction (RT-PCR) in comparison with the standard diagnostic method based on goblet cell detection by periodic acid-Schiff (PAS)-hematoxylin staining, using samples obtained from corneal epithelium impression cytology (IC). DESIGN: Transversal, comparative case series. PARTICIPANTS: We studied 59 eyes from 43 patients clinically diagnosed with LSCD. METHODS: Impression cytology was used to gather cells from corneal and conjunctival epithelium from the same eye. The presence of goblet cells in the cornea was determined by PAS-hematoxylin staining, whereas the presence of the MUC5AC transcript was detected by RT-PCR using a custom-designed primer pair. MAIN OUTCOME MEASURES: Goblet cells in the corneal epithelium were detected by light microscopy, and the MUC5AC transcript was detected as the corresponding PCR amplicon in agarose gels. RESULTS: Our study included 59 corneal samples, together with their respective conjunctival samples for RT-PCR assays. Of these, 47 samples were also available for comparative PAS-hematoxylin staining. The MUC5AC amplicon was detected in 56 of 59 (94.9%) corneal epithelium samples. In contrast, conventional IC staining detected goblet cells in only 17 of 47 (36.2%) samples; these were not found in 27 of 47 (57.4%) samples (negative results), and 3 of 47 (6.4%) showed inconclusive results. CONCLUSIONS: The detection of the MUC5AC transcript in corneal epithelium is a more sensitive method to diagnose LSCD than the conventional PAS-hematoxylin method, although a minimum RNA concentration of 1.2 ng/µl is required for negative results to be reliable. Moreover, RT-PCR is a highly specific and more objective technique. Overall, these findings indicate that molecular analysis facilitates a more precise clinical diagnosis of LSCD, thereby reducing the risk of surgical failure.

Reading and interpretation of chest X-ray in adults with community-acquired pneumonia

INTRODUCTION: Traditional reading of chest X-rays usually has a low prognostic value and poor agreement. OBJECTIVE: This study aimed to determine the interobserver and intraobserver agreement using two reading formats in patients with community-acquired pneumonia, and to explore their association with etiology and clinical outcomes. METHODS: A pulmonologist and a radiologist, who were blind to clinical data, interpreted 211 radiographs using a traditional analysis format (type and location of pulmonary infiltrates and pleural findings), and a quantitative analysis (pulmonary damage categorized from 0 to 10). For both, the interobserver and intraobserver agreement was estimated (Kappa statistic and intraclass correlation coefficient). The latter was assessed in a subsample of 25 radiographs three months after the initial reading. Finally, the observers made a joint reading to explore its prognostic usefulness via multivariate analysis. RESULTS: Seventy-four chest radiographs were discarded due to poor quality. With the traditional reading, the mean interobserver agreement was moderate (0.43). It was considered good when the presence of pleural effusion, and the location of the infiltrates in the right upper lobe and both lower lobes, were evaluated; moderate for multilobar pneumonia; and poor for the type of infiltrates. The mean intraobserver agreement for each reviewer was 0.71 and 0.5 respectively. The quantitative reading had an agreement between good and excellent (interobserver 0.72, intraobserver 0.85 and 0.61). Radiological findings were neither associated to a specific pathogen nor to mortality. CONCLUSION: In patients with pneumonia, the interpretation of the chest X-ray, especially the smallest of details, depends solely on the reader.

Changes in tear protein profile in patients with conjunctivochalasis.

PURPOSE: To compare the protein profiles of tears from normal volunteers and patients with conjunctivochalasis (CCH), with a view to identifying proteins whose expression is altered in this pathology. METHODS: Tears from 8 normal subjects and 6 patients with CCH were analyzed by 2-dimensional electrophoresis. Total protein from tears was separated in the first dimension by isoelectric focusing, and the second dimension was carried out using 8%-16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel images were analyzed using Progenesis SameSpot software. Those spots of interest were manually cut out from the gels, and the corresponding proteins were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF). Expression levels of proteins that had been found to be significantly altered were further verified by Western blot. RESULTS: Approximately 250 spot proteins were detected in the whole tear proteome. Twenty-four spots were significantly upregulated in CCH compared with that in controls. Eleven protein spots were identified, which included proteins belonging to the S100 family (A8, A9, A4; 2.44, 1.71, and 2.82 fold upregulation, respectively), guanosine triphosphate-binding protein 2 (1.95 fold), l-lactate dehydrogenase A-like 6B (2.32 fold), fatty acid-binding protein (2.01 fold), keratin type I cytoskeletal 10 (1.81 fold), glutathione S-transferase P (2.27 fold), peroxiredoxin-1, peroxiredoxin-5 (1.79- and 1.92 fold, respectively), and cullin-4B+ glyceraldehyde 3-phosphate dehydrogenase (1.96 fold). CONCLUSIONS: We have identified a group of proteins, which is upregulated in CCH tears. Although some of them, such as S100A4, S100A8, and peroxiredoxin-5, are markers of inflammation and oxidative processes, monitoring their levels in CCH might be useful for assessing the severity and progression of the disease.

Reading and interpretation of chest X-ray in adults with community-acquired pneumonia.

INTRODUCTION: Traditional reading of chest X-rays usually has a low prognostic value and poor agreement. OBJECTIVE: This study aimed to determine the interobserver and intraobserver agreement using two reading formats in patients with community-acquired pneumonia, and to explore their association with etiology and clinical outcomes. METHODS: A pulmonologist and a radiologist, who were blind to clinical data, interpreted 211 radiographs using a traditional analysis format (type and location of pulmonary infiltrates and pleural findings), and a quantitative analysis (pulmonary damage categorized from 0 to 10). For both, the interobserver and intraobserver agreement was estimated (Kappa statistic and intraclass correlation coefficient). The latter was assessed in a subsample of 25 radiographs three months after the initial reading. Finally, the observers made a joint reading to explore its prognostic usefulness via multivariate analysis. RESULTS: Seventy-four chest radiographs were discarded due to poor quality. With the traditional reading, the mean interobserver agreement was moderate (0.43). It was considered good when the presence of pleural effusion, and the location of the infiltrates in the right upper lobe and both lower lobes, were evaluated; moderate for multilobar pneumonia; and poor for the type of infiltrates. The mean intraobserver agreement for each reviewer was 0.71 and 0.5 respectively. The quantitative reading had an agreement between good and excellent (interobserver 0.72, intraobserver 0.85 and 0.61). Radiological findings were neither associated to a specific pathogen nor to mortality. CONCLUSION: In patients with pneumonia, the interpretation of the chest X-ray, especially the smallest of details, depends solely on the reader.