Covalent and Non-covalent In-Flow Biofunctionalization for Capture Assays on Silicon Chips: White Light Reflectance Spectroscopy Immunosensor Combined with TOF-SIMS Resolves Immobilization Stability and Binding Stoichiometry.

Immunosensors that combine planar transducers with microfluidics to achieve in-flow biofunctionalization and assay were analyzed here regarding surface binding capacity, immobilization stability, binding stoichiometry, and amount and orientation of surface-bound IgG antibodies. Two IgG immobilization schemes, by physical adsorption [3-aminopropyltriethoxysilane (APTES)] and glutaraldehyde covalent coupling (APTES/GA), followed by blocking with bovine serum albumin (BSA) and streptavidin (STR) capture, are monitored with white light reflectance spectroscopy (WLRS) sensors as thickness dΓ of the adlayer formed on top of aminosilanized silicon chips. Multi-protein surface composition (IgG, BSA, and STR) is determined by time of flight secondary ion mass spectrometry (TOF-SIMS) combined with principal component analysis (applying barycentric coordinates to the score plot). In-flow immobilization shows at least 1.7 times higher surface binding capacity than static adsorption. In contrast to physical immobilization, which is unstable during blocking with BSA, chemisorbed antibodies desorb (reducing dΓ) only when the bilayer is formed. Also, TOF-SIMS data show that IgG molecules are partially exchanged with BSA on APTES but not on APTES/GA modified chips. This is confirmed by the WLRS data that show different binding stoichiometry between the two immobilization schemes for the direct binding IgG/anti-IgG assay. The identical binding stoichiometry for STR capture results from partial replacement with BSA of vertically aligned antibodies on APTES, with fraction of exposed Fab domains higher than on APTES/GA.

Theory of Mind and Parental Mental-State Talk in Children with CIs.

Previous studies have suggested that parents may support the development of theory of mind (ToM) in their child by talking about mental states (mental state talk; MST). However, MST has not been sufficiently explored in deaf children with cochlear implants (CIs). This study investigated ToM and availability of parental MST in deaf children with CIs (n = 39, Mage = 62.92, SD = 15.23) in comparison with their peers with typical hearing (TH; n = 52, Mage = 52.48, SD = 1.07). MST was measured during shared storybook reading. Parents' narratives were coded for cognitive, emotional, literal, and non-mental references. ToM was measured with a parental questionnaire. Children with CIs had lower ToM scores than their peers with TH, and their parents used more literal references during shared storybook reading. There were no significant differences in the frequencies of cognitive and emotional references between groups. Parental emotional references contributed positively to children's ToM scores when controlling for the child's age and receptive grammar only in the CI group. These results indicated some distinctive features in parents of deaf children with CIs' MST and highlighted the role of MST in the development of ToM abilities in this group.

The Effect of the Topmost Layer and the Type of Bone Morphogenetic Protein-2 Immobilization on the Mesenchymal Stem Cell Response.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) plays a key role in the stem cell response, not only via its influence on osteogenesis, but also on cellular adhesion, migration, and proliferation. However, when applied clinically, its supra-physiological levels cause many adverse effects. Therefore, there is a need to concomitantly retain the biological activity of BMP-2 and reduce its doses. Currently, the most promising strategies involve site-specific and site-directed immobilization of rhBMP-2. This work investigated the covalent and electrostatic binding of rhBMP-2 to ultrathin-multilayers with chondroitin sulfate (CS) or diazoresin (DR) as the topmost layer. Angle-resolved X-ray photoelectron spectroscopy was used to study the exposed chemical groups. The rhBMP-2 binding efficiency and protein state were studied with time-of-flight secondary ion mass spectrometry. Quartz crystal microbalance, atomic force microscopy, and enzyme-linked immunosorbent assay were used to analyze protein-substrate interactions. The effect of the topmost layer was tested on initial cell adhesion and short-term osteogenesis marker expression. The results show the highest expression of selected osteomarkers in cells cultured on the DR-ended layer, while the cellular flattening was rather poor compared to the CS-ended system. rhBMP-2 adhesion was observed only on negatively charged layers. Cell flattening became more prominent in the presence of the protein, even though the osteogenic gene expression decreased.

Comparison of Physical Adsorption and Covalent Coupling Methods for Surface Density-Dependent Orientation of Antibody on Silicon.

The orientation of antibodies, employed as capture molecules on biosensors, determines biorecognition efficiency and bioassay performance. In a previous publication we demonstrated for antibodies attached covalently to silicon that an increase in their surface amount Γ, evaluated with ellipsometry, induces changes in their orientation, which is traced directly using Time-of-Flight Secondary Ion Mass Spectroscopy combined with Principal Component Analysis. Here, we extend the above studies to antibodies adsorbed physically on a 3-aminopropyltriethoxysilane (APTES) monolayer. Antibodies physisorbed on APTES (0 ≤ Γ ≤ 3.5 mg/m2) reveal the Γ ranges for flat-on, side-on, and vertical orientation consistent with random molecular packing. The relation between orientation and Γ is juxtaposed for silicon functionalized with APTES, APTES modified with glutaraldehyde (APTES/GA) and N-hydroxysuccinimide-silane (NHS-silane). Antibody reorientation occurs at lower Γ values when physisorption (APTES) is involved rather than chemisorption (APTES/GA, NHS-silane). At high Γ values, comparable proportions of molecules adapting head-on and tail-on vertical alignment are concluded for APTES and the NHS-silane monolayer, and they are related to intermolecular dipole-dipole interactions. Intermolecular forces seem to be less decisive than covalent binding for antibodies on the APTES/GA surface, with dominant head-on orientation. Independently, the impact of glutaraldehyde activation of APTES on vertical orientation is confirmed by separate TOF-SIMS measurements.

Dewetting of Polymer Films Controlled by Protein Adsorption.

The stability of the film poly(n-butyl methacrylate) (PnBMA) with different tacticities, prepared on silicon oxide and exposed to aqueous phosphate-buffered saline with different concentrations of bovine serum albumin (CBSA between 0 and 4.5 mg/mL), was examined at temperatures close to the physiological limit (between 4 and 37 °C) with optical microscopy, contact angle measurements, atomic force microscopy, and time-of-flight secondary ion mass spectrometry. For PBS solutions with CBSA = 0, the stability of atactic PnBMA and dewetting of isotactic PnBMA was observed, caused by the interplay between the stabilizing long-range dispersion forces and the destabilizing short-range polar interactions. Analogous considerations of excess free energy cannot explain the retardation of dewetting observed for isotactic PnBMA in PBS solutions with higher CBSA. Instead, formation of a BSA overlayer, adsorbed preferentially but not exclusively to uncovered SiOx regions, is evidenced and postulated to hinder polymer dewetting. Polymer dewetting and protein patterning are obtained in one step, suggesting a simple approach to fabricate biomaterials with micropatterned proteins.

Using a lactadherin-immobilized silicon surface for capturing and monitoring plasma microvesicles as a foundation for diagnostic device development.

Microvesicles (MVs) are found in several types of body fluids and are promising disease biomarkers and therapeutic targets. This study aimed to develop a novel biofunctionalized surface for binding plasma microvesicles (PMVs) based on a lab-on-a-chip (LOC) approach. A new lactadherin (LACT)-functionalized surface was prepared and examined for monitoring PMVs. Moreover, two different strategies of LACT immobilization on a silicon surface were applied to compare different LACT orientations. A higher PMV to LACT binding efficiency was observed for LACT bonded to an αvß3 integrin-functionalized surface compared with that for LACT directly bonded to a glutaraldehyde-modified surface. Effective binding of PMVs and its components for both LACT immobilization strategies was confirmed using spectral ellipsometry and time-of-flight secondary ion mass spectrometry methods. The proposed PMV capturing system can be used as a foundation to design novel point-of-care (POC) diagnostic devices to detect and characterize PMVs in clinical samples. Graphical Abstract.

Immobilization and detection of platelet-derived extracellular vesicles on functionalized silicon substrate: cytometric and spectrometric approach.

Among the various biomarkers that are used to diagnose or monitor disease, extracellular vesicles (EVs) represent one of the most promising targets in the development of new therapeutic strategies and the application of new diagnostic methods. The detection of circulating platelet-derived microvesicles (PMVs) is a considerable challenge for laboratory diagnostics, especially in the preliminary phase of a disease. In this study, we present a multistep approach to immobilizing and detecting PMVs in biological samples (microvesicles generated from activated platelets and human platelet-poor plasma) on functionalized silicon substrate. We describe the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and spectroscopic ellipsometry methods to the detection of immobilized PMVs in the context of a novel imaging flow cytometry (ISX) technique and atomic force microscopy (AFM). This novel approach allowed us to confirm the presence of the abundant microvesicle phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) on a surface with immobilized PMVs. Phosphatidylcholine groups (C5H12N+; C5H15PNO4+) were also detected. Moreover, we were able to show that ellipsometry permitted the immobilization of PMVs on a functionalized surface to be evaluated. The sensitivity of the ISX technique depends on the size and refractive index of the analyzed microvesicles. Graphical abstract Human platelets activated with thrombin (in concentration 1IU/mL) generate population of PMVs (platelet derived microvesicles), which can be detected and enumerated with fluorescent-label method (imaging cytometry). Alternatively, PMVs can be immobilized on the modified silicon substrate which is functionalized with a specific IgM murine monoclonal antibody against human glycoprotein IIb/IIIa complex (PAC-1). Immobilized PMVs can be subjected to label-free analyses by means ellipsometry, atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS).

Electron-Beam Lithographic Grafting of Functional Polymer Structures from Fluoropolymer Substrates.

Well-defined submicrometer structures of poly(dimethylaminoethyl methacrylate) (PDMAEMA) were grafted from 100 µm thick films of poly(ethene-alt-tetrafluoroethene) after electron-beam lithographic exposure. To explore the possibilities and limits of the method under different exposure conditions, two different acceleration voltages (2.5 and 100 keV) were employed. First, the influence of electron energy and dose on the extent of grafting and on the structure's morphology was determined via atomic force microscopy. The surface grafting with PDMAEMA was confirmed by advanced surface analytical techniques such as time-of-flight secondary ion mass spectrometry and X-ray photoelectron spectroscopy. Additionally, the possibility of effective postpolymerization modification of grafted structures was demonstrated by quaternization of the grafted PDMAEMA to the polycationic QPDMAEMA form and by exploiting electrostatic interactions to bind charged organic dyes and functional proteins.

Imaging and spectroscopic comparison of multi-step methods to form DNA arrays based on the biotin-streptavidin system.

Three multi-step multi-molecular approaches using the biotin-streptavidin system to contact-print DNA arrays on SiO2 surfaces modified with (3-glycidoxypropyl)trimethoxysilane are examined after each deposition/reaction step by atomic force microscopy, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry. Surface modification involves the spotting of preformed conjugates of biotinylated oligonucleotides with streptavidin onto surfaces coated with biotinylated bovine serum albumin b-BSA (approach I) or the spotting of biotinylated oligonucleotides onto a streptavidin coating, the latter prepared through a reaction with immobilized b-BSA (approach II) or direct adsorption (approach III). AFM micrographs, quantified by autocorrelation and height histogram parameters (e.g. roughness), reveal uniform coverage after each modification step with distinct nanostructures after the reaction of biotinylated BSA with streptavidin or of a streptavidin conjugate with biotinylated oligonucleotides. XPS relates the immobilization of biomolecules with covalent binding to the epoxy-silanized surface. Protein coverage, estimated from photoelectron attenuation, shows that regarding streptavidin the highest and the lowest immobilization efficiency is achieved by following approaches I and III, respectively, as confirmed by TOF-SIMS microanalysis. The size of the DNA spot reflects the contact radius of the printed droplet and increases with protein coverage (and roughness) prior to the spotting, as epoxy-silanized surfaces are hardly hydrophilic. Representative TOF-SIMS images show sub-millimeter spots: uniform for approach I, doughnut-like (with a small non-zero minimum) for approach II, both with coffee-rings or peak-shaped for approach III. Spot features, originating from pinned contact lines and DNA surface binding and revealed by complementary molecular distributions (all material, DNA, streptavidin, BSA, epoxy, SiO2), indicate two modes of droplet evaporation depending on the details of each applied approach.

Post-Polymerization Modification of Fluoropolymers via UV Irradiation in the Presence of a Photoacid Generator.

Fluorinated polymers have unique wettability and protein adsorption properties. The site-specific alteration of these properties could expand their application to different research areas. In this work, a fluorinated homopolymer and two of its copolymers with 4-vinylbenzyl glycidyl ether (VBGE) are synthesized by free radical polymerization. The produced polymers are then used to develop resist formulations by the addition of a photoacid generator. Films of these formulations are exposed to ultraviolet radiation through a binary mask and heated to create the pattern. It is found that the water contact angle values of the exposed films areas are reduced compared to those of the unexposed ones, with the exception of pentafluorophenyl methacrylate (PFMA) homopolymer film. This is attributed to the reaction of the epoxy groups creating x-links and producing hydroxyl groups and the cleavage of the pentafluorophenyl group from the ester group leading to carboxylic acid groups. Both modifications on the exposed areas are verified by FTIR spectroscopy and ToF-SIMS analysis. In addition, the biomolecules adsorption ability of the exposed area is increasing 10-15 times compared to the unexposed one for the PFMA homopolymer and the PFMA/VBGE 1:1 copolymer. Thus, the proposed polymers and patterning procedure could find application to spatially directed immobilization of biomolecules and/or cells onto a surface for both biosensing and tissue engineering purposes.

Plasma Treatment of PDMS for Microcontact Printing (µCP) of Lectins Decreases Silicone Transfer and Increases the Adhesion of Bladder Cancer Cells.

The present study investigates silicone transfer occurring during microcontact printing (µCP) of lectins with polydimethylsiloxane (PDMS) stamps and its impact on the adhesion of cells. Static adhesion assays and single-cell force spectroscopy (SCFS) are used to compare adhesion of nonmalignant (HCV29) and cancer (HT1376) bladder cells, respectively, to high-affinity lectin layers (PHA-L and WGA, respectively) prepared by physical adsorption and µCP. The chemical composition of the µCP lectin patterns was monitored by time-of-flight secondary ion mass spectrometry (ToF-SIMS). We show that the amount of transferred silicone in the µCP process depends on the preprocessing of the PDMS stamps. It is revealed that silicone contamination within the patterned lectin layers inhibits the adhesion of bladder cells, and the work of adhesion is lower for µCP lectins than for drop-cast lectins. The binding capacity of microcontact printed lectins was larger when the PDMS stamps were treated with UV ozone plasma as compared to sonication in ethanol and deionized water. ToF-SIMS data show that ozone-based treatment of PDMS stamps used for µCP of lectin reduces the silicone contamination in the imprinting protocol regardless of stamp geometry (flat vs microstructured). The role of other possible contributors, such as the lectin conformation and organization of lectin layers, is also discussed.

Effect of poly(tert-butyl methacrylate) stereoregularity on polymer film interactions with peptides, proteins, and bacteria.

The impact of polymer stereoregularity on its interactions with peptides, proteins and bacteria strains was studied for three stereoregular forms of poly(tert-butyl methacrylate) (PtBMA): isotactic (iso), atactic (at) and syndiotactic (syn) PtBMA. Principal component analysis of the time-of-flight secondary ion mass spectrometry data recorded for thin polymer films indicated a different orientation of ester groups, which in the case of iso-PtBMA are exposed away from the surface whereas for at-PtBMA and syn-PtBMA these are located deeper within the film. This arrangement of chemical groups modified the interactions of iso-PtBMA with biomolecules when compared to at-PtBMA and syn-PtBMA. For peptides, the affected interactions were explained by the preferential hydrogen bonding and electrostatic interaction between the exposed polar ester groups of iso-PtBMA and positively charged peptides. In turn, for protein adsorption no impact on the amount of adsorbed proteins was observed. However, the polymer stereoregularity influenced the orientation of immunoglobulin G and induced conformational changes in bovine serum albumin structure. Moreover, the impact of polymer stereoregularity occurred equally for their interactions with Gram-positive bacteria (S. aureus), which absorbed preferentially onto iso-PtBMA films as compared to two other stereoregularities.

Sequential binary protein patterning on surface domains of thermo-responsive polymer blends cast by horizontal-dipping.

We characterize an approach enabling dual protein positioning over broad polymer areas based on subsequent selective adsorption of two fluorescently labelled lectins, Concanavalin A (Con A) and Lentil Lectin (LcH), on self-assembled gradient patterns of thermoresponsive poly(N-isopropyl acrylamide) (PNIPAM) and polystyrene (PS) polymers blend, prepared by horizontal dipping technique. The film morphologies of gradient samples prior dual selective protein adsorption are mapped with scanning microscopy (AFM) and secondary ion mass spectrometry (ToF-SIMS), whereas adsorbed proteins are imaged with fluorescence microscope. ToF-SIMS analysis reveals surface composition consisting of PNIPAM-rich domains in PS-rich matrix. The two-step protein adsorption experiment results in selective adsorption of Con A and LcH to PNIPAM- and PS-rich phases, respectively. Integral geometry approach is used to compare quantitatively morphology of polymer patterns varied in domain size due to horizontal dipping casting. Minkowski measures are also used to compare quantitatively fluorescence micrographs of protein patches with SIMS images of original isotropic polymer patterns. It confirms that PNIPAM domains size increases with increasing speed. Further, Minkowski analysis unveiled that adsorbed proteins cover about 60-70% of polymer surface. What is more fluorescence micrographs acknowledge both no lectins contamination and no adsorption to interphase areas. Additionally, protein displacement effect is observed.

Simultaneous determination of aflatoxin B1, fumonisin B1 and deoxynivalenol in beer samples with a label-free monolithically integrated optoelectronic biosensor.

A label-free optical biosensor for the fast simultaneous determination of three mycotoxins, aflatoxin B1 (AFB1), fumonisin B1 (FB1) and deoxynivalenol (DON), in beer samples is presented. The biosensor is based on an array of ten Mach-Zehnder interferometers (MZIs) monolithically integrated along with their respective broad-band silicon light sources onto a single chip. Multi-analyte determination is accomplished by functionalizing the sensing arms of individual MZIs with mycotoxin-protein conjugates. Assay is performed by pumping over the chip mixtures of calibrators or samples with a mixture of specific monoclonal antibodies, followed by reaction with a secondary anti-mouse IgG antibody. Reactions are monitored in real-time by continuously recording the MZI output spectra, which are then subjected to Discrete Fourier Transform to convert spectrum shifts to phase shifts. The detection limits achieved for AFB1, FB1 and DON were 0.8, 5.6 and 24 ng/ml, respectively, while the assay duration was 12 min. Recovery values ranging from 85 to 115% were determined in beer samples spiked with known concentrations of the three mycotoxins. In addition, beers of different types and origin were analysed with the biosensor developed and the results were compared with those provided by established laboratory methods, further supporting the accuracy of the proposed device.

Advanced age in mares affects endometrial secretion of arachidonic acid metabolites during equine subclinical endometritis.

Even if mares continue to breed up to an advanced age, in aging mares reproductive failure is quite common. Subclinical endometritis, which occurs more often in aging mares than in younger counterparts, may cause prolongation or shortening of the inter-estrus period or the corpus luteum lifespan. We hypothesized that during subclinical endometritis the secretion of selected arachidonic acid metabolites may differ in aging mares compared to younger females. To verify this thesis, ex vivo organ cultures of endometrium were established with subsequent measurements of concentrations of prostaglandin E2 (PGE2), 6-keto-PGF1α and both leukotrienes (LTs), LTB4 and LTC4 in the culture supernatants. The endometrial biopsies were obtained from 82 mares of known breeding history. This study revealed that the concentrations of the selected arachidonic acid metabolites, which act both as immunological mediators and endocrine modulators in the reproductive organs, depends on the mares' ages. Spontaneous endometrial secretion of PGE2, 6-keto-PGF1α and LTC4 was increased in mares aged 16-23 years that suffered from subclinical endometritis, compared with control counterparts. Moreover, secretion of these metabolites was higher in endometritis-positive mares aged 16-23 years than in younger females. We conclude that advanced age in mares further disturbs the immuno-endocrine balance in endometritis-positive mares.

Indirect immunoassay on functionalized silicon surface: Molecular arrangement, composition and orientation examined step-by-step with multi-technique and multivariate analysis.

The arrangement, composition and orientation of immunoreagents employed in an indirect immunoassay for determination of mycotoxin OchraToxin A (OTA) are specified for Si3N4 substrate, aiming to imitate biosensor transducers made of the same material. Si3N4 surfaces are examined after modification with (3-aminopropyl)triethoxysilane, spotting with OTA-ovalbumin conjugate (probe), blocking with bovine serum albumin, reaction with a mouse monoclonal antibody against OTA and, finally, reaction with a goat anti-mouse secondary antibody. Atomic force micrographs, their autocorrelation and height histogram parameters, show the stepwise development of a multi-component monolayer covered by groups of secondary antibody molecules. Time-Of-Flight Secondary Ion Mass Spectrometry reveals the composition of probe and blocking protein, as well as their partial desorption during the primary immunoreaction. Ellipsometry provides surface amount of all proteins, increasing step-by-step from 0.7 to 6.9mg/m2. In addition, ellipsometry combined with TOF-SIMS reveals the mass loadings of different molecules in the intermediate and the final overlayer. Based on this, some orientations of the immobilized molecules are proposed and a molar ratio of ∼2.5 for secondary to primary antibody is calculated. The orientations of the primary and secondary antibody are further clarified by Principal Component Analysis of TOF-SIMS data, through which a side-on and a head-on orientation is deduced for the primary and the secondary antibody, respectively. These findings demonstrate how the combination of multiple surface analysis techniques can provide insight on the arrangement, composition and orientation of biomolecules in the course of multi-step procedures employed in biosensors.

Type of Inflammation Differentially Affects Expression of Interleukin 1ß and 6, Tumor Necrosis Factor-α and Toll-Like Receptors in Subclinical Endometritis in Mares.

Mares that fail to conceive or lose their embryos, without showing typical signs of clinical endometritis, should be suspected of subclinical endometritis (SE). In this study, the question was addressed: does SE fully activate selected mechanisms of innate immunity in mares? For this aim, expression of mRNAs for Toll-like Receptor 2 and 4 (TLR 2/4), interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor α (TNF) was examined in control mares versus either mares suffering from chronic endometritis (ChE) or subacute suppurative endometritis (SSE). The concentrations of IL-1ß, IL-6 and TNF-α in supernatants from endometrial tissue cultures after 4 h incubation were measured using the enzyme immunoassay (EIA) method. Eighty-two warmblood mares, of known breeding history, were enrolled in this study. Based on histopathological assessment, mares were classified as suffering from ChE, SSE or as being healthy. In addition, immuno-localization of both TLR2 and TLR4 as well as TNF-α was investigated in the equine endometria. The mRNA expression of TLR2 (P < 0.01), IL-1ß (P < 0.0001), IL-6 (P < 0.0001) and TLR4 and TNF (P < 0.05) was up-regulated in endometria of mares suffering from SSE compared with unaffected mares. Concentrations of IL-6 and TNF-α were increased only in mares exhibiting SSE, compared with unaffected (P < 0.01 for both) and ChE mares (P < 0.05 for both). Immuno-localization of TNF-α and TLRs was confirmed, both in unaffected and SE-affected endometria, and was present in the luminal and glandular epithelia and stromal cells. The severity of inflammation impacts the immune response and fosters activation of innate immunity mechanisms, as observed in the endometria of mares. The intracellular localization of TLRs and TNF-α in the endometria indicates a key role of endometrial epithelial and stromal cells in the immune response and inflammation.

Altered secretion of selected arachidonic acid metabolites during subclinical endometritis relative to estrous cycle stage and grade of fibrosis in mares.

Mares that fail to become pregnant after repeated breeding, without showing typical signs of clinical endometritis, should be suspected of subclinical endometritis (SE). Contact with infectious agents results in altered synthesis and secretion of inflammatory mediators, including cytokines and arachidonic acid metabolites, and disturbs endometrial functional balance. To address the hypothesis that SE affects the immune endocrine status of the equine endometrium, spontaneous secretion of prostaglandin E(2) (PGE(2)), prostaglandin F(2α) (PGF(2α)), 6-keto-PGF(1α )(a metabolite of prostacyclin I(2)), leukotriene B(4) (LTB(4)), and leukotriene C(4) (LTC(4)) was examined. In addition, secretion of these factors was examined relative to the grade of inflammation, fibrosis, and estrous cycle stage. Eighty-two warmblood mares, of known breeding history, were enrolled in this study. On the basis of histopathologic assessment, mares were classified as suffering from first-grade SE, second-grade SE, or being healthy. The grade of fibrosis and the infiltration of endometrial tissue with polymorphonuclear leukocytes were examined by routine hematoxylin-eosin staining. In mares suffering from SE, the secretion profiles of PGE(2), 6-keto-PGF(1α), LTB(4), and LTC(4) were changed compared to mares that did not suffer from endometritis. The secretion of PGE(2) and 6-keto-PGF1α was increased, whereas that of LTB(4) and LTC(4) was decreased. Secretion of 6-keto-PGF(1α) was increased in first- and second-grade SE (P < 0.01). The concentration of PGI(2) metabolite was increased only in inflamed endometrium, independently of the inflammation grade, but was not affected by fibrosis. Prostaglandin E(2) secretion was increased in second-grade SE (P < 0.05). The secretion of LTB(4) decreased in both first- and second-grade SE (P < 0.05), whereas secretion of LTC(4) was decreased only in second-grade SE (P < 0.05). Fibrosis did not change the secretion profile of PGE(2), PGF(2α), and 6-keto-PGF(1α) during the course of SE. However, the secretion profile of LTB(4) was affected during the course of fibrosis. Evident divergences between PGE(2) and PGF(2α) profiles and in PGE2:PGF(2α) ratios in the control versus SE mares observed during the course of diestrus contribute to shortened or prolonged interestrous intervals observed clinically in SE mares.