Enrichment, Characterization, and Proteomic Profiling of Small Extracellular Vesicles Derived from Human Limbal Mesenchymal Stromal Cells and Melanocytes.

Limbal epithelial progenitor cells (LEPC) rely on their niche environment for proper functionality and self-renewal. While extracellular vesicles (EV), specifically small EVs (sEV), have been proposed to support LEPC homeostasis, data on sEV derived from limbal niche cells like limbal mesenchymal stromal cells (LMSC) remain limited, and there are no studies on sEVs from limbal melanocytes (LM). In this study, we isolated sEV from conditioned media of LMSC and LM using a combination of tangential flow filtration and size exclusion chromatography and characterized them by nanoparticle tracking analysis, transmission electron microscopy, Western blot, multiplex bead arrays, and quantitative mass spectrometry. The internalization of sEV by LEPC was studied using flow cytometry and confocal microscopy. The isolated sEVs exhibited typical EV characteristics, including cell-specific markers such as CD90 for LMSC-sEV and Melan-A for LM-sEV. Bioinformatics analysis of the proteomic data suggested a significant role of sEVs in extracellular matrix deposition, with LMSC-derived sEV containing proteins involved in collagen remodeling and cell matrix adhesion, whereas LM-sEV proteins were implicated in other cellular bioprocesses such as cellular pigmentation and development. Moreover, fluorescently labeled LMSC-sEV and LM-sEV were taken up by LEPC and localized to their perinuclear compartment. These findings provide valuable insights into the complex role of sEV from niche cells in regulating the human limbal stem cell niche.

Severe ulcerative keratopathy following implantation of an acellular porcine corneal stromal lenticule in a patient with keratoconus.

PURPOSE: To report a case of ulcerative keratopathy following implantation of acellular porcine corneal stroma (APCS) in a patient with keratoconus (KC). METHODS: A 58 year-old patient initially presented with an ulcerative keratopathy in the left eye. Previously, several corneal procedures (including radial keratotomy, laser-in-situ keratomileusis, crosslinking) were performed for KC. Eight months ago, an APCS lenticule (Xenia corneal implant, Gebauer Medizintechnik GmbH, Neuhausen, Germany) was implanted into a stromal pocket because of progressive keratectasia. Visual acuity was hand movement. Anterior segment optical coherence tomography showed a space between the APCS lenticule and the host stroma. Excimer laser-assisted penetrating keratoplasty (PKP, 8.0/8.1 mm) was performed in the left eye. The corneal explant was investigated by light and transmission electron microscopy. RESULTS: Best-corrected visual acuity was 20/40 six weeks after PKP. Light microscopy demonstrated a stromal ulceration down to the APCS lenticule. No stromal cells could be found within the APCS lenticule eight months after implantation. The APCS lenticule did not show a green stain of the collagens with Masson-Goldner staining and exhibited a strong Periodic acid-Schiff positive reaction. Electron microscopy of the APCS lenticule revealed cross-linked collagen lamellae without cellular components. Close to the interface, corneal collagen lamellae of the host cornea were disorganized. Few vital keratocytes were present on the surface of the lenticule and appeared to cause mechanical disruption of the host stroma along the lenticule-stroma interface. CONCLUSION: APCS implantation may lead to severe complications such as ulcerative keratopathy in otherwise uncomplicated KC corneas. In such cases, excimer laser-assisted PKP or Deep Anterior Lamellar Keratoplasty are the methods of choice to restore visual acuity.

Differences in Guttae Ultramorphology in Relation to Visual Function in Fuchs Endothelial Corneal Dystrophy.

PURPOSE: The purpose of this study was to investigate the differences in guttae ultramorphology and their relation to visual function in eyes with Fuchs endothelial corneal dystrophy (FECD). METHODS: Thirty FECD eyes without ocular comorbidities were included. Visual functional parameters (best-corrected visual acuity with high-contrast and low-contrast letters and contrast sensitivity/LogCS) and corneal morphology measured with Scheimpflug tomography (Pentacam) were assessed. The surgically removed Descemet membranes were examined by light and transmission electron microscopy. RESULTS: Preoperative mean best-corrected visual acuity (logarithm of the minimum angle of resolution) was 0.52 ± 0.18, LogCS 0.96 ± 0.21 and central corneal thickness 640 ± 55 µm. All eyes had signs of subclinical corneal edema in Scheimpflug tomography; clinically visible corneal edema was present in 40% of eyes. Histological findings included a posterior fibrillar zone (PFZ) in 10 specimens (33%) and abnormal collagen depositions in Descemet membranes in 14 specimens (47%). Guttae buried within the PFZ were present only in eyes with clinically visible edema (n = 4, 13%). There was no difference in visual function results and tomography parameters between eyes with and without PFZ or between protruding guttae and guttae embedded in a PFZ, respectively. CONCLUSIONS: Guttae morphology and density were not correlated with visual functional parameters. Guttae buried in a PFZ occurred only in eyes with clinically manifest edema, and thereby, they are an ultramorphological sign for advanced FECD. Subclinical edema was present in all eyes and might be more relevant for quality of vision than guttae ultramorphology.

L-arginine metabolism inhibits arthritis and inflammatory bone loss.

OBJECTIVES: To investigate the effect of the L-arginine metabolism on arthritis and inflammation-mediated bone loss. METHODS: L-arginine was applied to three arthritis models (collagen-induced arthritis, serum-induced arthritis and human TNF transgenic mice). Inflammation was assessed clinically and histologically, while bone changes were quantified by µCT and histomorphometry. In vitro, effects of L-arginine on osteoclast differentiation were analysed by RNA-seq and mass spectrometry (MS). Seahorse, Single Cell ENergetIc metabolism by profilIng Translation inHibition and transmission electron microscopy were used for detecting metabolic changes in osteoclasts. Moreover, arginine-associated metabolites were measured in the serum of rheumatoid arthritis (RA) and pre-RA patients. RESULTS: L-arginine inhibited arthritis and bone loss in all three models and directly blocked TNFα-induced murine and human osteoclastogenesis. RNA-seq and MS analyses indicated that L-arginine switched glycolysis to oxidative phosphorylation in inflammatory osteoclasts leading to increased ATP production, purine metabolism and elevated inosine and hypoxanthine levels. Adenosine deaminase inhibitors blocking inosine and hypoxanthine production abolished the inhibition of L-arginine on osteoclastogenesis in vitro and in vivo. Altered arginine levels were also found in RA and pre-RA patients. CONCLUSION: Our study demonstrated that L-arginine ameliorates arthritis and bone erosion through metabolic reprogramming and perturbation of purine metabolism in osteoclasts.

Influence of Organ Culture on the Characteristics of the Human Limbal Stem Cell Niche.

Organ culture storage techniques for corneoscleral limbal (CSL) tissue have improved the quality of corneas for transplantation and allow for longer storage times. Cultured limbal tissue has been used for stem cell transplantation to treat limbal stem cell deficiency (LSCD) as well as for research purposes to assess homeostasis mechanisms in the limbal stem cell niche. However, the effects of organ culture storage conditions on the quality of limbal niche components are less well described. Therefore, in this study, the morphological and immunohistochemical characteristics of organ-cultured limbal tissue are investigated and compared to fresh limbal tissues by means of light and electron microscopy. Organ-cultured limbal tissues showed signs of deterioration, such as edema, less pronounced basement membranes, and loss of the most superficial layers of the epithelium. In comparison to the fresh limbal epithelium, organ-cultured limbal epithelium showed signs of ongoing proliferative activity (more Ki-67+ cells) and exhibited an altered limbal epithelial phenotype with a loss of N-cadherin and desmoglein expression as well as a lack of precise staining patterns for cytokeratin ((CK)14, CK17/19, CK15). The analyzed extracellular matrix composition was mainly intact (collagen IV, fibronectin, laminin chains) except for Tenascin-C, whose expression was increased in organ-cultured limbal tissue. Nonetheless, the expression patterns of cell-matrix adhesion proteins varied in organ-cultured limbal tissue compared to fresh limbal tissue. A decrease in the number of melanocytes (Melan-A+ cells) and Langerhans cells (HLA-DR+, CD1a+, CD18+) was observed in the organ-cultured limbal tissue. The organ culture-induced alterations of the limbal epithelial stem cell niche might hamper its use in the treatment of LSCD as well as in research studies. In contrast, reduced numbers of donor-derived Langerhans cells seem associated with better clinical outcomes. However, there is a need to consider the preferential use of fresh CSL for limbal transplants and to look at ways of improving the limbal stem cell properties of stored CSL tissue.

Tracking cell turnover in human brain using 15N-thymidine imaging mass spectrometry.

Microcephaly is often caused by an impairment of the generation of neurons in the brain, a process referred to as neurogenesis. While most neurogenesis in mammals occurs during brain development, it thought to continue to take place through adulthood in selected regions of the mammalian brain, notably the hippocampus. However, the generality of neurogenesis in the adult brain has been controversial. While studies in mice and rats have provided compelling evidence for neurogenesis occurring in the adult rodent hippocampus, the lack of applicability in humans of key methods to demonstrate neurogenesis has led to an intense debate about the existence and, in particular, the magnitude of neurogenesis in the adult human brain. Here, we demonstrate the applicability of a powerful method to address this debate, that is, the in vivo labeling of adult human patients with 15N-thymidine, a non-hazardous form of thymidine, an approach without any clinical harm or ethical concerns. 15N-thymidine incorporation into newly synthesized DNA of specific cells was quantified at the single-cell level with subcellular resolution by Multiple-isotype imaging mass spectrometry (MIMS) of brain tissue resected for medical reasons. Two adult human patients, a glioblastoma patient and a patient with drug-refractory right temporal lobe epilepsy, were infused for 24 h with 15N-thymidine. Detection of 15N-positive leukocyte nuclei in blood samples from these patients confirmed previous findings by others and demonstrated the appropriateness of this approach to search for the generation of new cells in the adult human brain. 15N-positive neural cells were easily identified in the glioblastoma tissue sample, and the range of the 15N signal suggested that cells that underwent S-phase fully or partially during the 24 h in vivo labeling period, as well as cells generated therefrom, were detected. In contrast, within the hippocampus tissue resected from the epilepsy patient, none of the 2,000 dentate gyrus neurons analyzed was positive for 15N-thymidine uptake, consistent with the notion that the rate of neurogenesis in the adult human hippocampus is rather low. Of note, the likelihood of detecting neurogenesis was reduced because of (i) the low number of cells analyzed, (ii) the fact that hippocampal tissue was explored that may have had reduced neurogenesis due to epilepsy, and (iii) the labeling period of 24 h which may have been too short to capture quiescent neural stem cells. Yet, overall, our approach to enrich NeuN-labeled neuronal nuclei by FACS prior to MIMS analysis provides a promising strategy to quantify even low rates of neurogenesis in the adult human hippocampus after in vivo15N-thymidine infusion. From a general point of view and regarding future perspectives, the in vivo labeling of humans with 15N-thymidine followed by MIMS analysis of brain tissue constitutes a novel approach to study mitotically active cells and their progeny in the brain, and thus allows a broad spectrum of studies of brain physiology and pathology, including microcephaly.

Type II Diabetes Mellitus Causes Extracellular Matrix Alterations in the Posterior Cornea That Increase Graft Thickness and Rigidity.

Purpose: There is a pressing need to investigate the impact of type II diabetes mellitus on the posterior cornea in donor tissues given its increasing prevalence and potential impact on endothelial keratoplasty surgical outcomes. Methods: Immortalized human cultured corneal endothelial cells (CECs; HCEC-B4G12) were grown in hyperglycemic media for 2 weeks. Extracellular matrix (ECM) adhesive glycoprotein expression and advanced glycation end products (AGEs) in cultured cells and corneoscleral donor tissues, as well as the elastic modulus for the Descemet membrane (DMs) and CECs of diabetic and nondiabetic donor corneas, were measured. Results: In CEC cultures, increasing hyperglycemia resulted in increased transforming growth factor beta-induced (TGFBI) protein expression and colocalization with AGEs in the ECM. In donor corneas, the thicknesses of the DM and the interfacial matrix (IFM) between the DM and stroma both increased from 8.42 ± 1.35 µm and 0.504 ± 0.13 µm in normal corneas, respectively, to 11.13 ± 2.91 µm (DM) and 0.681 ± 0.24 µm (IFM) in non-advanced diabetes (P = 0.013 and P = 0.075, respectively) and 11.31 ± 1.76 µm (DM) and 0.744 ± 0.18 µm (IFM) in advanced diabetes (AD; P = 0.0002 and P = 0.003, respectively). Immunofluorescence in AD tissues versus controls showed increased AGEs (P < 0.001) and markedly increased labeling intensity for adhesive glycoproteins, including TGFBI, that colocalized with AGEs. The elastic modulus significantly increased between AD and control tissues for the DMs (P < 0.0001) and CECs (P < 0.0001). Conclusions: Diabetes and hyperglycemia alter human CEC ECM structure and composition, likely contributing to previously documented complications of endothelial keratoplasty using diabetic donor tissue, including tearing during graft preparation and reduced graft survival. AGE accumulation in the DM and IFM may be a useful biomarker for determining diabetic impact on posterior corneal tissue.

The Blood-ocular Barriers and their Dysfunction: Anatomy, Physiology, Pathology. / Die Blut-Augen-Schranken und ihre Störungen: Anatomie, Physiologie, Pathologie.

Complex barriers comprise the blood-aqueous (BAB) and the blood-retinal barrier (BRB), and separate anterior and posterior eye chambers, vitreous body, and sensory retina from the circulation. They prevent pathogens and toxins from entering the eye, control movement of fluid, proteins, and metabolites, and contribute to the maintenance of the ocular immune status. Morphological correlates of blood-ocular barriers are tight junctions between neighboring endothelial and epithelial cells, which function as gatekeepers of the paracellular transport of molecules, thereby limiting their uncontrolled access to ocular chambers and tissues. The BAB is composed of tight junctions between endothelial cells of the iris vasculature, endothelial cells of Schlemm's canal inner wall, and cells of the nonpigmented ciliary epithelium. The BRB consists of tight junctions between endothelial cells of the retinal vessels (inner BRB) and epithelial cells of the retinal pigment epithelium (outer BRB). These junctional complexes respond rapidly to pathophysiological changes, thus enabling vascular leakage of blood-derived molecules and inflammatory cells into ocular tissues and chambers. Blood-ocular barrier function, which can be clinically measured by laser flare photometry or fluorophotometry, is compromised in traumatic, inflammatory, or infectious processes, but also frequently contributes to the pathophysiology of chronic diseases of the anterior eye segment and the retina, as exemplified by diabetic retinopathy and age-related macular degeneration.

RNA-Seq-based transcriptome analysis of corneal endothelial cells derived from patients with Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is the most common inherited corneal disease. Fibrillar focal excrescences called guttae and corneal edema due to corneal endothelial cell death result in progressive vision loss. Multiple genetic variants have been reported, but the pathogenesis of FECD is not fully understood. In this study, we used RNA-Seq to analyze differential gene expression in the corneal endothelium obtained from patients with FECD. Differential expression analysis of transcriptomic profiles revealed that expression of 2366 genes (1092 upregulated and 1274 downregulated genes) was significantly altered in the corneal endothelium of patients with FECD compared to healthy subjects. Gene ontology analysis demonstrated an enrichment of genes involved in extracellular matrix (ECM) organization, response to oxidative stress, and apoptotic signaling. Several pathway analyses consistently indicated the dysregulation of ECM-associated pathways. Our differential gene expression findings support the previously proposed underlying mechanisms, including oxidative stress and apoptosis of endothelial cells, as well as the phenotypic clinical FECD hallmark of ECM deposits. Further investigation focusing on differentially expressed genes related to these pathways might be beneficial for elucidating mechanisms and developing novel therapies.

Characterization of Porcine Ocular Surface Epithelial Microenvironment.

The porcine ocular surface is used as a model of the human ocular surface; however, a detailed characterization of the porcine ocular surface has not been documented. This is due, in part, to the scarcity of antibodies produced specifically against the porcine ocular surface cell types or structures. We performed a histological and immunohistochemical investigation on frozen and formalin-fixed, paraffin-embedded ocular surface tissue from domestic pigs using a panel of 41 different antibodies related to epithelial progenitor/differentiation phenotypes, extracellular matrix and associated molecules, and various niche cell types. Our observations suggested that the Bowman's layer is not evident in the cornea; the deep invaginations of the limbal epithelium in the limbal zone are analogous to the limbal interpalisade crypts of human limbal tissue; and the presence of goblet cells in the bulbar conjunctiva. Immunohistochemistry analysis revealed that the epithelial progenitor markers cytokeratin (CK)15, CK14, p63α, and P-cadherin were expressed in both the limbal and conjunctival basal epithelium, whereas the basal cells of the limbal and conjunctival epithelium did not stain for CK3, CK12, E-cadherin, and CK13. Antibodies detecting marker proteins related to the extracellular matrix (collagen IV, Tenascin-C), cell-matrix adhesion (ß-dystroglycan, integrin α3 and α6), mesenchymal cells (vimentin, CD90, CD44), neurons (neurofilament), immune cells (HLA-ABC; HLA-DR, CD1, CD4, CD14), vasculature (von Willebrand factor), and melanocytes (SRY-homeobox-10, human melanoma black-45, Tyrosinase) on the normal human ocular surface demonstrated similar immunoreactivity on the normal porcine ocular surface. Only a few antibodies (directed against N-cadherin, fibronectin, agrin, laminin α3 and α5, melan-A) appeared unreactive on porcine tissues. Our findings characterize the main immunohistochemical properties of the porcine ocular surface and provide a morphological and immunohistochemical basis useful to research using porcine models. Furthermore, the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

Accumulation of T-cell-suppressive PD-L1high extracellular vesicles is associated with GvHD and might impact GvL efficacy.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents the only curative treatment option for a number of hemato-oncological disorders. In fact, allo-HSCT is considered as one of the most successful immunotherapies as its clinical efficacy is based on the donor T-cells' capacity to control residual disease. This process is known as the graft-versus-leukemia (GvL) reaction. However, alloreactive T-cells can also recognize the host as foreign and trigger a systemic potentially life-threatening inflammatory disorder termed graft-versus-host disease (GvHD). A better understanding of the underlying mechanisms that lead to GvHD or disease relapse could help us to improve efficacy and safety of allo-HSCT. In recent years, extracellular vesicles (EVs) have emerged as critical components of intercellular crosstalk. Cancer-associated EVs that express the immune checkpoint molecule programmed death-ligand 1 (PD-L1) can suppress T-cell responses and thus contribute to immune escape. At the same time, it has been observed that inflammation triggers PD-L1 expression as part of a negative feedback network.Here, we investigated whether circulating EVs following allo-HSCT express PD-L1 and tested their efficacy to suppress the ability of (autologous) T-cells to effectively target AML blasts. Finally, we assessed the link between PD-L1 levels on EVs to (T-)cell reconstitution, GvHD, and disease relapse.We were able to detect PD-L1+ EVs that reached a peak PD-L1 expression at 6 weeks post allo-HSCT. Development of acute GvHD was linked to the emergence of PD-L1high EVs following allo-HSCT. Moreover, PD-L1 levels correlated positively with GvHD grade and declined (only) on successful therapeutic intervention. T-cell-inhibitory capacity was higher in PD-L1high EVs as compared with their PD-L1low counterparts and could be antagonized using PD-L1/PD-1 blocking antibodies. Abundance of T-cell-suppressive PD-L1high EVs appears to also impact GvL efficacy as patients were at higher risk for relapse. Finally, patients of PD-L1high cohort displayed a reduced overall survival.Taken together, we show that PD-L1-expressing EVs are present following allo-HSCT. PD-L1 levels on EVs correlate with their ability to suppress T-cells and the occurrence of GvHD. The latter observation may indicate a negative feedback mechanism to control inflammatory (GvHD) activity. This intrinsic immunosuppression could subsequently promote disease relapse.

Light and electron microscopic features of preclinical pseudoexfoliation syndrome.

PURPOSE: This study sought to explore the features of the anterior lens capsule in patients with preclinical pseudoexfoliation syndrome (pPEX) via light microscopy (LM) and transmission electron microscopy (TEM). DESIGN: Cross-sectional, prospective, and observational case series. METHODS: We recruited consecutive patients with and without pPEX who underwent routine cataract surgery at Ramathibodi Hospital, between April 2018 and November 2020. pPEX can be characterized by pigmented spoke-wheel deposition (P) on the anterior lens capsule, midperiphery cleft/lacunae (C), faint central disc present within the photopic pupil (D), white-spoke pattern (W) noted at the midperiphery, and a combination of at least two signs (Co). LM and TEM were used to examine anterior lens capsule specimens for the presence of characteristic pseudoexfoliation material (PXM). The features of the anterior lens capsule in pPEX explored via LM and TEM were recorded. RESULTS: This study included a total of 96 patients (101 excised anterior lens capsules); among them, 34 (35 excised anterior lens capsules) exhibited pPEX signs (pPEX group) but 62 (66 excised anterior lens capsules) did not (control group). The patients had a mean age of 74 ± 7 (range, 58-89) years. LM and TEM revealed no definite PXM in any patient. In the pPEX group, LM analysis identified two capsule specimens with suspected PXM; PXM precursors were observed in 1 of the 34 excised capsule specimens analyzed via TEM. Furthermore, 39 eyes (59.09%) exhibited signs of true exfoliation syndrome (TEX) in LM analysis (12.82%, 25.64%, 10.26%, 10.26%, and 41.03% for patients exhibiting P, D, C, W, and Co, respectively). However, no TEX signs were observed in the control group. We found that the anterior lens capsules exhibiting C and D were significantly associated with TEX (odds ratio = 5.4 and 7.9; P = 0.007 and 0.004, respectively). CONCLUSIONS: LM analysis revealed no definite PXMs were detected in the excised anterior lens capsules, whereas TEM analysis showed PXM precursors in one specimen (2.94%). Notably, a significant association was observed between C and D signs and TEX.

Analysis of genetically determined gene expression suggests role of inflammatory processes in exfoliation syndrome.

BACKGROUND: Exfoliation syndrome (XFS) is an age-related systemic disorder characterized by excessive production and progressive accumulation of abnormal extracellular material, with pathognomonic ocular manifestations. It is the most common cause of secondary glaucoma, resulting in widespread global blindness. The largest global meta-analysis of XFS in 123,457 multi-ethnic individuals from 24 countries identified seven loci with the strongest association signal in chr15q22-25 region near LOXL1. Expression analysis have so far correlated coding and a few non-coding variants in the region with LOXL1 expression levels, but functional effects of these variants is unclear. We hypothesize that analysis of the contribution of the genetically determined component of gene expression to XFS risk can provide a powerful method to elucidate potential roles of additional genes and clarify biology that underlie XFS. RESULTS: Transcriptomic Wide Association Studies (TWAS) using PrediXcan models trained in 48 GTEx tissues leveraging on results from the multi-ethnic and European ancestry GWAS were performed. To eliminate the possibility of false-positive results due to Linkage Disequilibrium (LD) contamination, we i) performed PrediXcan analysis in reduced models removing variants in LD with LOXL1 missense variants associated with XFS, and variants in LOXL1 models in both multiethnic and European ancestry individuals, ii) conducted conditional analysis of the significant signals in European ancestry individuals, and iii) filtered signals based on correlated gene expression, LD and shared eQTLs, iv) conducted expression validation analysis in human iris tissues. We observed twenty-eight genes in chr15q22-25 region that showed statistically significant associations, which were whittled down to ten genes after statistical validations. In experimental analysis, mRNA transcript levels for ARID3B, CD276, LOXL1, NEO1, SCAMP2, and UBL7 were significantly decreased in iris tissues from XFS patients compared to control samples. TWAS genes for XFS were significantly enriched for genes associated with inflammatory conditions. We also observed a higher incidence of XFS comorbidity with inflammatory and connective tissue diseases. CONCLUSION: Our results implicate a role for connective tissues and inflammation pathways in the etiology of XFS. Targeting the inflammatory pathway may be a potential therapeutic option to reduce progression in XFS.

Ultrastructural Examination of the Corneal Interface after Predescemetic Deep Anterior Lamellar Keratoplasty (DALK) - A Case Report with Light and Transmission Electron Microscopy.

PURPOSE: To examine corneal buttons with light and transmission electron microscopy (TEM) to visualize the interface area and highlight the ultrastructural corneal changes after deep anterior lamellar keratoplasty (DALK). METHODS: Two patients underwent excimer laser-assisted penetrating repeat keratoplasty after predescemetic DALK. The corneal buttons were examined by light microscopy and TEM. RESULTS: The light microscopic examination of the corneal buttons revealed fragments of a second Descemet's membrane in the central and midperipheral areas (Case 1). In both cases, visualization of the interface area was not possible by light microscopy. The donor and host stroma were tightly attached without dehiscence. TEM identified the interface area by irregularities in the collagen distribution between the donor and host stroma. The thickness of the remaining recipient corneal stroma measured approximately 30 µm (Case 1) and 100 µm (Case 2), respectively. In the host stroma, TEM revealed the absence or degeneration of keratocytes, accumulation of amorphous material between the collagen lamellae, and vacuolar inclusions dispersed in the stroma, forming a band-like zone anterior to Descemet's membrane. CONCLUSION: The interface area after DALK has been mainly investigated by in vivo confocal microscopy. Light microscopy and TEM findings indicate remodeling processes after DALK that are associated with increased keratocyte degeneration and structural alterations of the extracellular matrix in the host stroma. The choice of surgical method may influence the postoperative morphological and functional outcome since these findings were primarily apparent in the remaining host stroma. Therefore, complete exposure of Descemet's membrane is an important prognostic factor for the postoperative visual outcome.

Retinal regions shape human and murine Müller cell proteome profile and functionality.

The human macula is a highly specialized retinal region with pit-like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone- and rod-rich retinae from human and mice and identified different expression profiles of cone- and rod-associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell-derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region-specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo.

Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy.

Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).

Transcriptomic Landscape and Functional Characterization of Human Induced Pluripotent Stem Cell-Derived Limbal Epithelial Progenitor Cells.

Limbal stem cell deficiency (LSCD) is a complex, multifactorial disease affecting limbal epithelial progenitor cells (LEPC), which are essential for maintaining corneal stability and transparency. Human induced pluripotent stem cell-derived (hiPSC-) LEPC are a promising cell source for the treatment of LSCD. However, their similarity to native tissue-derived (T-) LEPC and their functional characterization has not been studied in detail. Here, we show that hiPSC-LEPC and T-LEPC have rather similar gene expression patterns, colony-forming ability, wound-healing capacity, and melanosome uptake. In addition, hiPSC-LEPC exhibited lower immunogenicity and reduced the proliferation of peripheral blood mononuclear cells compared with T-LEPC. Similarly, the hiPSC-LEPC secretome reduced the proliferation of vascular endothelial cells more than the T-LEPC secretome. Moreover, hiPSC-LEPC successfully repopulated decellularized human corneolimbal (DHC/L) scaffolds with multilayered epithelium, while basal deposition of fibrillary material was observed. These findings suggest that hiPSC-LEPC exhibited functional properties close to native LEPC and that hiPSC-LEPC-DHC/L scaffolds might be feasible for transplantation in patients suffering from LSCD in the future. Although hiPSC-LEPC-based stem cell therapy is promising, the current study also revealed new challenges, such as abnormal extracellular matrix deposition, that need to be overcome before hiPSC-LEPC-based stem cell therapies are viable.

Infectious Crystalline Keratopathy after Penetrating Keratoplasty with Light and Electron Microscopic Examination.

PURPOSE: To highlight the typical histological and ultrastructural features of severe infectious crystalline keratopathy (ICK) in a corneal graft, which required excimer laser-assisted repeat penetrating keratoplasty (PKP) and to present the challenging treatment conditions associated with ICK. METHODS: An 85-year-old female patient underwent PKP for secondary graft failure after Descemet membrane endothelial keratoplasty (DMEK) for Fuchs' endothelial corneal dystrophy in the left eye. One year later, white branched opacities were observed in the superficial corneal stroma of the graft without surrounding inflammation in the left eye. The patient underwent excimer laser-assisted repeat PKP (8.0/8.1 mm) in the left eye after prolonged refractory topical anti-infectious treatment for 1 month. The corneal explant was further examined by light and transmission electron microscopy (TEM). RESULTS: The light microscopic examination of the corneal explant demonstrated aggregates of coccoid bacteria in the superficial and mid-stromal region that were positive for periodic acid-Schiff (PAS) and Gram stain. The bacterial aggregates extended into the interlamellar spaces, showed a spindle-shaped appearance, and were not surrounded by an inflammatory cellular reaction. TEM demonstrated lamellae separation within the anterior corneal stroma with spindle-shaped aggregates of bacteria, which were embedded in an extracellular amorphous matrix with incipient calcification, being consistent with a biofilm. No inflammatory cellular reaction was evident by TEM. At discharge from hospital, the corrected visual acuity was 20/80 in the left eye. CONCLUSION: ICK is often challenging due to the difficult diagnosis and treatment conditions. The refractory courses are mainly attributed to a biofilm formation, which inhibits effective topical anti-infectious treatment. In such cases, (repeat) PKP may be necessary to completely remove the pathology, prevent recurrences, and improve vision.

Corneal epithelial ingrowth after perforating corneal injury: a case report.

BACKGROUND: Epithelial ingrowth is a rare complication after ocular perforation and can become manifest many years after the primary trauma. CASE PRESENTATION: A 49-year-old patient presented with a positive Seidel test of unclear origin at her left eye, as well as a sharply defined anterior-stromal corneal scar at both eyes. Prior operations included a bilateral laser-assisted blepharoplasty 3 months earlier. The patient indicated to have been on holiday to France 5 months earlier, during an ongoing oak processionary moth caterpillars infestation. The examination using confocal microscopy confirmed a corneal perforation at the left eye and revealed corneal epithelial ingrowth capped with scarred stroma in both eyes. We performed a penetrating keratoplasty at the left eye. The scarred and perforated host cornea was divided into 4 pieces for further investigation: microbiology (negative), virology (negative), histology and transmission electron microscopy (TEM). Histology revealed differently structured epithelium, centrally inverted into the stroma through defects in Bowman's layer. TEM revealed full thickness corneal perforation with an epithelial plug extending to the lower third of the cornea, but without evidence of epithelial cell migration into the anterior chamber. Our differential diagnosis of the unclear positive Seidel test with epithelial ingrowth was as follows: (1) corneal perforation by hairs of the oak processionary moth caterpillar, although no hairs could be found histologically; (2) corneal perforation during laser-assisted blepharoplasty, which may be supported by the presence of pigmented cells on the posterior surface of Descemet´s membrane, pointing to a possible iris injury. CONCLUSION: Consequently, we highlighted that contact lenses can be useful, safe and inexpensive protective devices in upper eyelid procedures to protect the cornea against mechanical iatrogenic trauma.

Desmin Knock-Out Cardiomyopathy: A Heart on the Verge of Metabolic Crisis.

Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.