[Evolution of dry eye disease diagnostics]. / Evolyutsiya metodov diagnostiki sindroma sukhogo glaza.

This article reviews modern functional and instrumental examination methods included in the diagnostic algorithm for dry eye disease. The described methods can serve as an objective criterion for the effectiveness of the therapy.

Probing PIEZO1 Localization upon Activation Using High-Resolution Atomic Force and Confocal Microscopy.

PIEZO1 ion channels are activated by mechanical stimuli, triggering intracellular chemical signals. Recent structural studies suggest that plasma membrane tension or local curvature changes modulate PIEZO1 channel gating and activation. However, whether PIEZO1 localization is governed by tension gradients or long-range mechanical perturbations across the cells is still unclear. Here, we probe the nanoscale localization of PIEZO1 on red blood cells (RBCs) at high resolution (∼30 nm), and we report for the first time the existence of submicrometric PIEZO1 clusters in native conditions. Upon interaction with Yoda1, an allosteric modulator, PIEZO1 clusters increase in abundance in regions of higher membrane tension and lower curvature. We further show that PIEZO1 ion channels interact with the spectrin cytoskeleton in both resting and activated states. Our results point toward a strong interplay between plasma membrane tension gradients, curvature, and cytoskeleton association of PIEZO1.

Use of the Inferior Whorl for Detecting Age-Related Changes in Human Corneal Subbasal Nerve Plexus with Laser Scanning Confocal Microscopy.

PURPOSE: The purpose of this study was to determine the effect of aging on the corneal subbasal nerve plexus (SNP) by employing a wide-field mapping technique of composite images, scanned at the location of a distinctive spiraled subbasal nerve pattern located 1-2 mm inferior to the corneal apex (the inferior whorl) for SNP structural quantification. MATERIALS AND METHODS: Central corneal tactile sensitivity (CCTS) and inferior whorl length (IWL) were compared among individuals in 3 age-groups (20-39 years, 40-59 years, and 60-79 years). Statistical analyses constituted the Kruskal-Wallis test, one-way analysis of variance (with the post hoc least significant difference test), Spearman correlation coefficient, and linear regression analysis. RESULTS: CCTS remained stable until the age of 50 years, when it began to decrease; the mean CCTS was 58.15 ± 2.46 mm in the group aged 20-39 years, 55.74 ± 3.85 mm in the group aged 40-59 years, and 50.23 ± 3.27 mm in the group aged 60-79 years. IWL decreased with increasing age, with a corresponding linear decline of 0.2088 mm/mm2 per year, and the mean IWL was 25.43 ± 4.50 mm/mm2 in the group aged 20-39 years, 22.71 ± 6.19 mm/mm2 in the group aged 40-59 years, and 18.60 ± 4.21 mm/mm2 in the group aged 60-79 years. CONCLUSION: Our work provided a more accurate and repeatable method for corneal nerve analysis using laser scanning confocal microscopy. By using this technique, we confirmed that aging is associated with progressive reduction in subbasal nerve length.

Validation of laser scanning confocal microscopy as a diagnostic method for lymphedema using a rat model.

Most previous diagnostic methods for lymphedema are invasive. Laser scanning confocal microscopy (LSCM) combines laser and computer image processing technology, is capable of increasing the resolution of optical microscopy by 30-40%, and boasts a comparable resolution to that of histological examination. We constructed the rat tail lymphedema model to simulate secondary lymphedema and to validate the noninvasive technique of in vivo reflectance LSCM for the diagnosis of lymphedema. The rat tail lymphedema model was constructed by cutting and ligating the lymphatic vessels in the rat tail. Lymphedema in the postoperative rat tail was assessed by a comprehensive range of methods including the change of rat tail diameter, lymphocytic radionuclide imaging, LSCM, and immunohistochemistry using a specific lymphatic vessel marker, prospero homeobox protein 1 (PROX1). The noninvasive LSCM method along with other techniques validated the rat tail lymphedema model. LSCM was used to perform qualitative and quantitative evaluation of the state and extent of lymphedema in the rat tail model. Receiver operating characteristic (ROC) curve analysis, which provided an area under the curve (AUC) value of 0.861, supported the feasibility of using LSCM as a reliable diagnostic technique for lymphedema. The rat tail lymphedema model can be successfully constructed by cutting and ligating the lymphatic vessels in the rat tail. Although LSCM cannot replace the method of skin biopsy examination, it offers a painless and noninvasive alternative for diagnosing lymphedema. Thus, LSCM can potentially be adopted in clinical practice as a supporting method to be used in combination with other techniques.

[Morphofunctional substantiation of repeated invasive treatment of chronic blepharitis]. / Morfofunktsional'noe obosnovanie provedeniya povtornykh kursov invazivnogo lecheniya khronicheskogo blefarita.

The expanding range of diagnostic instrumental methods allows an in-depth study of the morphological and functional state of the eyelids, which is the basis for determining the strategy for the treatment of chronic blepharitis and subsequent timely supplementation and altering of its algorithm. PURPOSE: To substantiate the repeated courses of invasive treatment of chronic blepharitis based on morphological and functional studies. MATERIAL AND METHODS: The study included 45 patients (90 eyes) with chronic mixed blepharitis. Instrumental research methods - laser Doppler flowmetry, laser scanning confocal microscopy (LSCM), tiascopy, and optical coherence tomography - were used to assess the morphological and functional state of the eyelids during meibomian gland probing (MGP) and eyelid massage course. The therapy effectiveness was evaluated after 1 week, 1, 3 and 6 months. RESULTS: Using a complex of diagnostic methods for assessing the morphological and functional state of the eyelids, surgical invasive treatment for mixed chronic blepharitis was proved to have a significant positive clinical effect compared with the eyelid massage traditionally used in polyclinic practice. After 6 months from the start of treatment, in the absence of changes in the clinical picture in both groups, functional changes in the control group were recorded that were expressed as a decrease in the values of the Norn test, confirmed by the data of tiascopy, and the number of functioning meibomian glands. The indicators of lacrimal meniscus depth in both groups corresponded to the values of the monitoring stage after 3 months. According to LSCM, the control group had higher inflammatory activity: the heterogeneity of the interstitium and the walls of the acini of the meibomian glands increased, the acinar area decreased in comparison with the main group. Deterioration of the blood flow microcirculation in both groups was manifested as a decrease in neurogenic rhythms. Additionally, a 3.05% decrease in myogenic rhythms was recorded in the main group. The study of the microcirculation of lymph flow showed a decrease in neurogenic rhythms in the main group by 4.79%. CONCLUSION: The analysis of the morphological and functional state of the eyelids after MGP has shown that its results persisted for 6 months, and repeated probing was justified, while the interval before repeated course of eyelid massage averaged 1.5 months.

Three-dimensional imaging and quantification of real-time cytosolic calcium oscillations in microglial cells cultured on electrospun matrices using laser scanning confocal microscopy.

The development of a minimally invasive, robust, and inexpensive technique that permits real-time monitoring of cell responses on biomaterial scaffolds can improve the eventual outcomes of scaffold-based tissue engineering strategies. Towards establishing correlations between in situ biological activity and cell fate, we have developed a comprehensive workflow for real-time volumetric imaging of spatiotemporally varying cytosolic calcium oscillations in pure microglial cells cultured on electrospun meshes. Live HMC3 cells on randomly oriented electrospun fibers were stained with a fluorescent dye and imaged using a laser scanning confocal microscope. Resonance scanning provided high-resolution in obtaining the time-course of intracellular calcium levels without compromising spatial and temporal resolution. Three-dimensional reconstruction and depth-coding enabled the visualization of cell location and intracellular calcium levels as a function of sample thickness. Importantly, changes in cell morphology and in situ calcium spiking were quantified in response to a soluble biochemical cue and varying matrix architectures (i.e., randomly oriented and aligned fibers). Importantly, raster plots generated from spiking data revealed calcium signatures specific to culture conditions. In the future, our approach can be used to elucidate correlations between calcium signatures and cell phenotype/activation, and facilitate the rational design of scaffolds for biomedical applications.

In situ investigation of acute exposure of graphene oxide on activated sludge: Biofilm characteristics, microbial activity and cytotoxicity.

Graphene Oxide (GO) has wide applications in many fields which has caused a large expected quantity of the graphene-based wastes. It is necessary to understand the toxic effects of the GO on the activated sludge (AS) considering its inevitable discharge to the wastewater treatment plants as the ultimate repositories for these wastes. In this study, the acute exposures of the multilayer Nano-graphene oxide (MNGO) at different dosages were conducted in order to investigate its integrated effects on the formation of the biofilm, mature biofilm and the microbial activity of the activated sludge. Raman spectroscopy and laser scanning confocal microscopy (LSCM) were adopted for the in-situ characterization of the biofilm with the exposure of the MNGO. The results showed that the activated sludge was tolerable to the acute exposure of the less than 100 mg/L of the MNGO, especially for the mature biofilm, and only a subtle decrease was found in the size and thickness during the formation of the biofilm, while the amount of 300 mg/L of the MNGO caused the sever deterioration on the activated sludge system. The microbial metabolic activity, viability, and the biological removal of the nutrients were significantly affected with the more than 100 mg/L of the MNGO. It was also demonstrated by the microbial cytotoxicity tests that the increase in the exposure of the MNGO was related to the increase in the reactive oxygen species (ROS) and the damaging degree of the cell membrane.

Tissue-Clearing Techniques Enable Three-Dimensional Visualization of Aerosolized Model Compound and Lung Structure at the Alveolar Scale.

In this study, we examined the usefulness of a tissue-clearing technique for the evaluation of the lung distribution of aerosolized drugs. An aerosol formulation of TexasRed dextran (70 kDa), a model compound of drug carrier for aerosolized drugs, was administered intrapulmonarily to mice using a MicroSprayer, and then DyLight 488-conjugated tomato lectin was administered intravenously to visualize general lung structure via the fluorescent labeling of alveolar and bronchial epithelial cells. Tissue clearing followed by laser scanning confocal microscopy enabled the three-dimensional visualization of intrapulmonary TexasRed dextran and the evaluation of its distribution at the alveolar scale without the preparation of thin tissue sections. These findings suggest that tissue-clearing techniques are useful for the evaluation of intrapulmonary distribution and development of pulmonary drug delivery systems.

Evaluation of the corneal epithelium in non-Sjögren's and Sjögren's dry eyes: an in vivo confocal microscopy study using HRT III RCM.

BACKGROUND: The corneal epithelium is directly affected in dry eye syndrome. Thus, we attempted to describe the morphological features and evaluate the cellular density within the corneal epithelial layers in patients with non-Sjögren's (NSDE) and Sjögren's syndrome dry eyes (SSDE) by in vivo confocal microscopy (IVCM). METHODS: Central cornea was prospectively imaged by IVCM in 68 clinically diagnosed aqueous tear-deficient dry eyes and 10 healthy age-matched control eyes. Morphological characteristics of corneal epithelial layers and cellular densities were evaluated by four trained graders from the Doheny Eye Institute. RESULTS: Corneal epithelium in dry eyes presents morphological changes such as areas of enlarged and irregular shaped cells. In comparison with controls, the density of superficial epithelial cells was decreased in both the NSDE (P < 0.05) and SSDE groups (P < 0.01); the density of the outer layer of wing cells was smaller but not significantly different in NSDE (P > 0.05), but was lower in the SSDE group (P < 0.01); the density of the inner layer of wing cells was decreased in both the NSDE (P < 0.05) and SSDE groups (P < 0.01) and the density of basal epithelial cells was lower in both the NSDE (P < 0.01) and SSDE groups (P = 0.01). For all cell counts, the interclass correlation coefficient showed good agreement between graders (ICC =0.75 to 0.93). CONCLUSIONS: IVCM represents a reliable technique for examining the corneal epithelial microstructural changes associated with dry eyes, as well as for objectively and reproducibly quantifying cell densities within all corneal epithelial layers.

Effects of fluoride and epigallocatechin gallate on soft-drink-induced dental erosion of enamel and root dentin.

BACKGROUND/PURPOSE: Fluoride and epigallocatechin gallate (EGCG) have been proven to prevent dental caries. The purpose of this study was to evaluate the effects of fluoride and EGCG on soft-drink-induced dental erosion in vitro. METHODS: Forty enamel and dentin specimens were prepared from extracted human teeth. The specimens were divided into 4 groups and treated separately with distilled water (as control), 0.5 M sodium fluoride (NF), 400 µM EGCG (EG), and a solution containing 0.5 M NaF and 400 µM EGCG (FG). Cyclic erosive treatment was performed according to the experimental procedures. The specimens were analyzed using laser scanning confocal microscopy, scanning electron microscopy, and a microhardness tester. The data were analyzed using ANOVA and Bonferroni's post hoc test. The significance level was set at 5%. RESULTS: The amount of substance loss was lower in the NF and EG groups than in the control group (p < 0.05). The erosion-caused substance loss was more pronounced in the dentin than in the enamel specimens. Surface microhardness loss was lower in the NF and EG groups than in the control group (p < 0.05). The diameter of the dentinal tubule was wider in the control group than in the NF and EG groups (p < 0.05). No combined effects were observed in the FG group. CONCLUSION: Both fluoride and EGCG are effective in preventing soft-drink-induced erosion compared with the control group. Fluoride and EGCG may interfere with each other. The mechanisms of the anti-erosive effect need to be explored in the future.

Effect of Dibutyl Phthalate on the Tolerance and Lipid Accumulation in the Green Microalgae Chlorella vulgaris.

In the present study, Chlorella vulgaris were cultured in the presence of the common plasticizer dibutyl phthalate (DBP) with different concentrations for 10 days. The cell density, DBP concentrations, neutral lipid concentrations, and lipid morphology in C. vulgaris were studied using optical microscopy, gas chromatography (GC), fluorescence spectrophotometry, and laser scanning confocal microscopy (LSCM). We observed that the neutral lipid contents and cell density of C. vulgaris were negatively influenced by DBP of high concentrations (50 and 100 mg/L), but significantly stimulated by DBP of low concentrations (5, 10, and 20 mg/L). Lipid bodies were destroyed into pieces by DBP of high concentrations (50 and 100 mg/L), but were slightly suppressed by DBP at low concentrations (5, 10, and 20 mg/L). Chlorella vulgaris treated with DBP (50 mg/L) for 2 days showed the highest removal efficiency (31.69%). The results suggested that C. vulgaris could be used in practice to remove DBP and has the potential of being oleaginous microalgae in DBP contaminated water.

Vegetating erosive cutaneous lesions and pyogenic granuloma in the course of mucous membrane pemphigoid: a case report and review of literature.

Mucous membrane pemphigoid (MMP) is a group of chronic autoimmune sub-epithelial blistering disorders, which mostly affect the oral mucosa and the conjunctiva. MMP is very diverse in terms of both the clinical and immunological features (IgG and IgA autoantibodies may react with different antigens). MMP can be induced by infections and medication, including ophthalmologic medication, which may lead to the development of eye lesions. In contrast, a vegetating variant of MMP is extremely rare. Here, we report an MMP case that demonstrated unusual clinical features, that is, pyogenic granulomas on the conjunctivae and extensive vegetating erosions on the skin of intertriginous regions. All these lesions were considered to be induced by unconventional medication containing arsenic.

[Involutional blepharitis: modern approaches to diagnostic and treatment]. / Involiutsionnyi blefarit: sovremennye podkhody k diagnostike i lecheniiu.

PURPOSE: to identify the role of laser scanning confocal microscopy (LSCM) in diagnostic and to assess the effectiveness of involutional blepharitis treatment. MATERIAL AND METHODS: The study involved 32 patients (64 eyes) with chronic blepharitis aged in average 75.21±3.56 years. All patients completed questionnaire (SPEED), underwent Schirmer I, Norn tear break-up time and compression tests, had the number of functioning meibomian glands (NFMG) calculated. The patients' eyelids were examined with LSCM, and the condition of conjunctiva was evaluated by microbiological study. The patients were also tested for Demodex spp. Based on the results of LSCM the patients were divided into two groups: atrophic and obstructive. The first group (13 patients, 26 eyes) was prescribed treatment of eyelid margins with 'Blephaclean' tissues 3 times a day and artificial tears 'Thealoz' 4 times a day. The second group (19 patients, 38 eyes) was recommended therapy with warm compress 2 times a day, application of eyelid gel 'Theagel' 3 times a day and instillations of 'Thealoz' 4 times a day. The follow-up began 2 months after therapy start. RESULTS: Clinical-functional, morphological parameters and laboratory findings had a tendency for improvements after the treatment. The application of tissues, eyelid gel and artificial tears contributed to increase of NFMG, improvement of tear film stability and tear production. The treatment resulted in decreased secretion reflectivity of the lacrimal canal orifice and lower degree of heterogeneity of acinar and interstitial walls according to LSCM readings. CONCLUSION: The data acquired on the discriminate application of eyelid hygiene products based on LSCM and clinical-functional examination allows the recommendation of such therapy for the treatment of various forms of involutional blepharitis.

[Modern methods of evaluating the morphological and functional state of the eyelids in chronic blepharitis]. / Sovremennye metody issledovaniia morfofunktsional'nogo sostoianiia vek pri disfunktsii meibomievykh zhelez.

The article reviews the literature on methods of evaluating the morphological and functional state of the eyelids in chronic blepharitis. Development of methods continues together with further research on the etiology and pathogenesis of the disease.

Assessment of statistical agreement of three techniques for the study of cut marks: 3D digital microscope, laser scanning confocal microscopy and micro-photogrammetry.

In the last few years, the study of cut marks on bone surfaces has become fundamental for the interpretation of prehistoric butchery practices. Due to the difficulties in the correct identification of cut marks, many criteria for their description and classification have been suggested. Different techniques, such as three-dimensional digital microscope (3D DM), laser scanning confocal microscopy (LSCM) and micro-photogrammetry (M-PG) have been recently applied to the study of cut marks. Although the 3D DM and LSCM microscopic techniques are the most commonly used for the 3D identification of cut marks, M-PG has also proved to be very efficient and a low-cost method. M-PG is a noninvasive technique that allows the study of the cortical surface without any previous preparation of the samples, and that generates high-resolution models. Despite the current application of microscopic and micro-photogrammetric techniques to taphonomy, their reliability has never been tested. In this paper, we compare 3D DM, LSCM and M-PG in order to assess their resolution and results. In this study, we analyse 26 experimental cut marks generated with a metal knife. The quantitative and qualitative information registered is analysed by means of standard multivariate statistics and geometric morphometrics to assess the similarities and differences obtained with the different methodologies.

Correlation between corneal innervation and inflammation evaluated with confocal microscopy and symptomatology in patients with dry eye syndromes: a preliminary study.

PURPOSE: To evaluate corneal innervation and inflammatory cell infiltration using in vivo confocal microscopy (IVCM) and to correlate these findings with subjective symptoms of dry eye, as measured by the Ocular Surface Disease Index (OSDI) in patients with non-Sjögren's (NSDE) and Sjögren's syndrome dry eyes (SSDE). METHODS: Central corneal images were prospectively captured from 10 age-matched healthy control eyes, 24 eyes with clinically diagnosed NSDE and 44 eyes with clinically diagnosed SSDE, using IVCM (HRT III RCM). Density, tortuosity and reflectivity of corneal nerves, presence of inflammatory dendritic cells (DCs) and OSDI scores were evaluated. RESULTS: Images obtained by IVCM from 78 eyes were analyzed. The density of nerve fibers was 1562 ± 996 µm/frame in the SSDE group, 2150 ± 1015 µm/frame in the NSDE group and 2725 ± 687 µm/frame in the control group (P < 0.05, ANOVA). In comparison to the control group, the density of nerve fibers was decreased in the SSDE (P < 0.001) and the NSDE groups (P = 0.06), with increased nerve tortuosity and decreased reflectivity in both groups (both P < 0.05). The density of DCs was 71.65 ± 72.54 cells/mm2 in the SSDE group, 40.33 ± 31.63 cells/mm2 in the NSDE group and 27.53 ± 5.58 cells/mm2 in the control group (P < 0.05, ANOVA). In comparison to the control group, the density of DCs was increased in the SSDE (P < 0.001) and the NSDE groups (P = 0.07). Significant correlations were found between the nerve density and DC density (r = -0.57, P < 0.001), between the nerve density and OSDI scores (r = -0.91, P < 0.001) and between the nerve reflectivity and OSDI scores (r = -0.75, P < 0.001). CONCLUSIONS: The corneas of eyes affected with NSDE and SSDE are characterized by alterations in corneal innervation and infiltration of inflammatory DCs. Corneal nerve density and reflectivity are correlated with severity of subjective dry eye symptoms, as measured by OSDI score.

Corneal features in ocular graft-versus-host disease by in vivo confocal microscopy.

PURPOSE: The purpose of our study was to determine the morphological features of the corneal epithelial layers, sub-basal nerve plexus and anterior stroma in patients with ocular graft-versus-host disease (oGVHD) compared to non-GVHD dry eyes and normal controls, using in vivo confocal microscopy (IVCM). METHODS: IVCM was used to capture central cornea images from eight volunteers with normal healthy eyes, ten patients with non-GVHD dry eye syndrome (DES) and 15 patients with clinically diagnosed oGVHD, in a cross-sectional study. Morphological changes of the corneal epithelial layers and anterior stroma, characteristics of corneal nerves and presence of dendritic cells (DCs) were then evaluated. RESULTS: IVCM images obtained from 66 eyes were analyzed. The density of superficial epithelial cells was 636.07 ± 101.05 cells/mm2 in the oGVHD group, 827 ± 99.62 cells/mm2 in the DES group and 1277.2 ± 121.42 cells/mm2 in the control group (P < 0.001). The density of wing cells was 4499.79 ± 976.36 cells/mm2 in the oGVHD group, 4662.85 ± 319.72 cells/mm2 in DES group and 6556.38 ± 503.99 cells/mm2 in the control group (p < 0.001). The density of basal cells was 7850.93 ± 723.51 cells/mm2 in the oGVHD group, 8570 ± 913.32 cells/mm2 in DES group and 9759.8 ± 251.99 cells/mm2 in the control group (p < 0.01). The density of nerve fibers was 11.22 ± 5.46 mm/mm2 in the oGVHD group, 14.50 ± 4.27 mm/mm2 in DES group and 19.56 ± 4.75 mm/mm2 in the control group (p < 0.01). The DC density was 67.88 ± 71.82 cells/mm2 in the oGVHD group, 40.06 ± 31.95 cells/mm2 in the DES group and 29.45 ± 8.1 cells/mm2 in the control group (P > 0.05). Visible networks of activated keratocytes were seen in the anterior stroma of eyes with oGVHD and DES, but not in normal controls. CONCLUSIONS: IVCM revealed distinct microstructural changes in the corneas of patients with oGVHD and DES, similar between the two groups. Our findings suggest implications for use of IVCM to evaluate and monitor patients with dry eyes associated or not with GVHD.

[Diagnostic capabilities of optical coherence tomography and confocal laser scanning microscopy in studying manifestations of aniridia-associated keratopathy]. / Diagnosticheskie vozmozhnosti opticheskoi kogerentnoi tomografii i lazernoi skaniruiushchei konfokal'noi mikroskopii v izuchenii proiavlenii aniridiinoi keratopatii.

AIM: to investigate the possible use of anterior segment optical coherence tomography (AS-OCT) and laser scanning confocal microscopy (LSCM) for visualization of limbal progenitor structures and epithelial changes at different stages of aniridia-associated keratopathy (AAK) and to analyze genotype-phenotype correlations of corneal damage. MATERIAL AND METHODS: Thirty-four patients (63 eyes) with congenital aniridia (CA) were subjected to epithelial cell density measurement in the central cornea as well as epithelial surface assessment with limbal palisades of Vogt (POV) detection in the corresponding sites of the two corneas. For that, LSCM (HRT3) and AS-OCT (RTVue XR Avanti) were performed. Central corneal and epithelial thicknesses were measured using the Pachymetry protocol. RESULTS: There has been found an increase in the central corneal thickness (CCT) of CA patients, which correlated with the stage of AAK, and a decrease in the central epithelial thickness as compared with healthy subjects (p<0.05). The difference between the basal and wing epithelial cells density in eyes with stages I and II AAK and normal cells density at stage 0 AAK was statistically significant (p<0.05). Intact or disturbed POV were detected in all patients with PAX6 3' deletion. At that, AS-OCT findings highly agreed with LSCM images for both the inferii (rS=0.85, p<0.05) and superior limbi (rS=0.53, p<0.05). A negative correlation was established between the stage of AAK and in vivo morphology of POV (rS=-0.5, p<0.05). However, no correlation was found between the stage of AAK and patient's age (rS=0.169, p=0.174). CONCLUSION: AS-OCT and LSCM are both important diagnostic tools for corneal surface monitoring in patients with limbal stem cells deficiency.

Characterization of the cell penetrating properties of a human salivary proline-rich peptide.

Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins. Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents.

Morphological, ultrastructural and functional imaging of frozen/thawed and vitrified/warmed human ovarian tissue retrieved from oncological patients.

STUDY QUESTION: Which is the best method for human ovarian tissue cryopreservation: slow freezing/rapid thawing (SF/RT) or vitrification/warming (V/W)? SUMMARY ANSWER: The conventional SF/RT protocol used in this study seems to better preserve the morpho-functional status of human cryopreserved ovarian tissue than the used open carrier V/W protocol. WHAT IS KNOWN ALREADY: Cryopreservation of human ovarian tissue is generally performed using the SF/RT method. However, reduction in the follicular pool and stroma damage are often observed. An emerging alternative procedure is represented by V/W which seems to allow the maintenance of the morphological integrity of the stroma. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study including six patients affected by oncological diseases and enrolled from January to December 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissue was laparoscopically harvested from the right and left ovaries and was cryopreserved using a routinary SF/RT protocol or a V/W method, involving tissue incubation in two solutions (containing propylene glycol, ethylene glycol and sucrose at different concentrations) and vitrification in an open system. For each patient, three pieces from each ovary were collected at the time of laparoscopy (fresh tissue) and after storage (SF/RT or V/W) and processed for light microscopy (LM) and transmission electron microscopy (TEM), to assess the morphological and ultrastructural features of follicles and stroma, and for laser scanning confocal microscopy (LSCM), to determine the functional energetic/redox stroma status. The preservation status of SF/RT and V/W ovarian tissues was compared with that of fresh ones, as well as between them. MAIN RESULTS AND THE ROLE OF CHANCE: By LM and TEM, SF/RT and V/W samples showed cryodamage of small entity. Interstitial oedema and increased stromal cell vacuolization and chromatin clumping were observed in SF/RT samples; in contrast, V/W samples showed oocyte nuclei with slightly thickened chromatin and irregular shapes. The functional imaging analysis by LSCM revealed that the mitochondrial activity and intracellular reactive oxygen species levels were reduced both in SF/RT and in V/W samples compared with fresh samples. The study also showed progressive dysfunction of the mitochondrial activity going from the outer to the inner serial section of the ovarian cortex. The reduction of mitochondrial activity of V/W samples compared with fresh samples was significantly higher in the inner section than in the outer section. LIMITATIONS, REASONS FOR CAUTION: The results report the bioenergetic and oxidative status assessment of fresh and cryopreserved human ovarian tissue by LSCM, a technique recently applied to tissue samples. The use of LSCM on human ovarian tissues after SF/RT or V/W is a new application that requires validation. The procedures for mitochondrial staining with functional probes and fixing are not yet standardized. Xenografting of the cryopreserved ovarian tissue in severe combined immunodeficient mice and in vitro culture have not yet been performed. WIDER IMPLICATIONS OF THE FINDINGS: The identification of a cryopreservation method able to maintain the morpho-functional integrity of the ovarian tissue and a number of follicles comparable with those observed in fresh tissue might optimize results in clinical practice, in terms of recovery, duration of ovarian function and increased delivery outcomes after replanting. The SF/RT protocol allowed better morpho-functional tissue integrity than the V/W procedure. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Fondazione del Monte di Bologna e Ravenna, Italy. Dr N.A.M. was granted by the project ONEV MIUR PONa3 00134-n.254/R&C 18 5 2011 and the project GR-2011-02351396 (Ministry of Health, Young Researchers Grant 2011/2012). There are no competing interests. TRIAL REGISTRATION NUMBER: Clinical trial 74/2001/0 (approved:13 2 2002): 'Pilot study on cryopreservation of human ovarian tissue: morphological and immunohistochemical analysis before and after cryopreservation'.