Prospective Observational Study Evaluating Systemic Hormones and Corneal Crosslinking Effects in Keratoconus.

Purpose: To evaluate associations between hormone levels and corneal parameters in patients with keratoconus (KC), before and after photooxidative corneal collagen crosslinking (CXL). Design: Prospective, observational cohort study. Participants: Twenty-eight patients with KC who were scheduled for CXL at Aarhus University Hospital in Denmark. Methods: Androgen (dehydroepiandrosterone sulfate [DHEA-S]) and estrogen (estrone and estriol) plasma levels were measured and clinical assessments were performed before CXL and 2 to 3 months post-CXL, comparing the CXL eye with the control eye from the same participant. Main Outcome Measures: Associations between hormone levels and maximum corneal curvature (Kmax) and minimum central corneal thickness (CCtmin) before and after CXL. Results: Corneal collagen crosslinking was associated with a 2% reduction in Kmax values in the CXL eye, post-CXL, from baseline (median, 56.8 diopters [D]; 95% confidence interval [CI], 50.4-60.3) to the second visit (55.7 D; 95% CI, 50.4-58.8; P < 0.001). Systemic DHEA-S levels were 5 to 6 orders of magnitude higher than estriol or estrone concentrations in plasma. Importantly, estriol levels, rather than DHEA-S or estrone levels, were more closely correlated with Kmax before CXL (Spearman's r = 0.55, P = 0.01). Post-CXL Kmax and CCtmin were not associated with DHEA-S, estrone, or estriol plasma levels at the same timepoint. Conclusions: This study provides supporting evidence based on a KC clinical population that systemic estrogen levels may influence corneal parameters (curvature and thickness) pre-CXL. Further studies evaluating the interplay between the therapeutic benefits of CXL and systemic hormone distributions are needed to determine if perturbation of the local corneal microenvironment influences endocrine function. Financial Disclosures: The authors have no proprietary or commercial interest in any materials discussed in this article.

Cell sheet-based approach to study the diabetic corneal stroma.

Prolonged hyperglycemia during diabetes mellitus (DM) is associated with severe complications that may affect both the anterior and posterior ocular segments, leading to impaired vision or blindness. The cornea is a vital part of the eye that has a dual role as a protective transparent barrier and as a major refractive structure and is likewise negatively affected by hyperglycemia in DM. Understanding the cellular and molecular mechanisms underlying the phenotypic changes associated with DM is critical to developing targeted therapies to promote tissue integrity. In this proof-of-concept study, we applied a cell sheet-based approach to generate stacked constructs of physiological corneal thickness using primary human corneal fibroblasts isolated from cadaveric control (healthy), Type 1 DM and Type 2 DM corneal tissues. Self-assembled corneal stromal sheets were generated after 2 weeks in culture, isolated, and subsequently assembled to create stacked constructs, which were evaluated using transmission electron microscopy. Analysis of gene expression patterns revealed significant downregulation of fibrotic markers, α-smooth muscle actin, and collagen type 3, with stacking in Type 2 DM constructs when compared to controls. IGF1 expression was significantly upregulated in Type 2 DM constructs compared to controls with a significant reduction induced by stacking. This study describes the development of a thicker, self-assembled corneal stromal construct as a platform to evaluate phenotypic differences associated with DM-derived corneal fibroblasts and enable the development of targeted therapeutics to promote corneal integrity.

Temperature during pupal development affects hoverfly developmental time, adult life span, and wing length.

Hoverflies (Diptera, Syrphidae) are cosmopolitan, generalist flower visitors and among the most important pollinators after bees and bumblebees. The dronefly Eristalis tenax can be found in temperate and continental climates across the globe, often synanthropically. Eristalis tenax pupae of different generations and different climate zones are thus exposed to vastly different temperatures. In many insects, the ambient temperature during the pupal stage affects development, adult size, and survival; however, the effect of developmental temperature on these traits in hoverflies is comparatively poorly understood. We here reared E. tenax pupae at different temperatures, from 10°C to 25°C, and quantified the effect on adult hoverflies. We found that pupal rearing at 17°C appeared to be optimal, with high eclosion rates, longer wings, and increased adult longevity. Rearing temperatures above or below this optimum led to decreased eclosion rates, wing size, and adult survival. Similar thermal dependence has been observed in other insects. We found that rearing temperature had no significant effect on locomotor activity, coloration or weight, despite evidence of strong sexual dimorphism for each of these traits. Our findings are important as hoverflies are key pollinators, and understanding the effects of developmental temperature could potentially be useful for horticulture.

Novel Correlation between TGF-ß1/-ß3 and Hormone Receptors in the Human Corneal Stroma.

This study investigated the interplay between transforming growth factor beta (TGF-ß1/T1 and TGF-ß3/T3), and sex hormone receptors using our 3D in vitro cornea stroma model. Primary human corneal fibroblasts (HCFs) from healthy donors were plated in transwells at 106 cells/well and cultured for four weeks. HCFs were supplemented with stable vitamin C (VitC) and stimulated with T1 or T3. 3D construct proteins were analyzed for the androgen receptor (AR), progesterone receptor (PR), estrogen receptor alpha (ERα) and beta (ERß), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin-releasing hormone receptor (GnRHR), KiSS1-derived peptide receptor (KiSS1R/GPR54), and follicle-stimulating hormone subunit beta (FSH-B). In female constructs, T1 significantly upregulated AR, PR, ERα, FSHR, GnRHR, and KiSS1R. In male constructs, T1 significantly downregulated FSHR and FSH-B and significantly upregulated ERα, ERß, and GnRHR. T3 caused significant upregulation in expressions PR, ERα, ERß, LHR, FSHR, and GNRHR in female constructs, and significant downregulation of AR, ERα, and FSHR in male constructs. Semi-quantitative Western blot findings present the interplay between sex hormone receptors and TGF-ß isoforms in the corneal stroma, which is influenced by sex as a biological variable (SABV). Additional studies are warranted to fully delineate their interactions and signaling mechanisms.

Descending neurons of the hoverfly respond to pursuits of artificial targets.

Many animals use motion vision information to control dynamic behaviors. Predatory animals, for example, show an exquisite ability to detect rapidly moving prey, followed by pursuit and capture. Such target detection is not only used by predators but is also important in conspecific interactions, such as for male hoverflies defending their territories against conspecific intruders. Visual target detection is believed to be subserved by specialized target-tuned neurons found in a range of species, including vertebrates and arthropods. However, how these target-tuned neurons respond to actual pursuit trajectories is currently not well understood. To redress this, we recorded extracellularly from target-selective descending neurons (TSDNs) in male Eristalis tenax hoverflies. We show that they have dorso-frontal receptive fields with a preferred direction up and away from the visual midline. We reconstructed visual flow fields as experienced during pursuits of artificial targets (black beads). We recorded TSDN responses to six reconstructed pursuits and found that each neuron responded consistently at remarkably specific time points but that these time points differed between neurons. We found that the observed spike probability was correlated with the spike probability predicted from each neuron's receptive field and size tuning. Interestingly, however, the overall response rate was low, with individual neurons responding to only a small part of each reconstructed pursuit. In contrast, the TSDN population responded to substantially larger proportions of the pursuits but with lower probability. This large variation between neurons could be useful if different neurons control different parts of the behavioral output.

Pediatric Acute Liver Failure Secondary to Autoimmune Hepatitis in an Infant With Thrombocytopenia-Absent Radius (TAR) Syndrome: A Case Report.

Thrombocytopenia absent radius (TAR) syndrome is a rare genetic disorder that has been associated with food protein-induced allergic proctocolitis and transient leukemoid reactions, among other manifestations. There has been no prior reports of its association with autoimmune disease, more specifically, autoimmune hepatitis (AIH) or the development of pediatric acute liver failure (PALF). We present a case of an 8-month-old infant with TAR syndrome who presented with PALF, secondary to AIH with elevated liver-kidney microsomal antibody (>1:2560). She received a liver transplant and had a very complicated postoperative course including severe T-cell-mediated rejection, infection, biliary stricture, persistently elevated liver-kidney microsomal antibodies, and antibody-mediated rejection. Ultimately, these complications led to graft failure, severe sepsis, and death. This case highlights a new association of TAR syndrome with AIH and PALF and a potentially aggressive nature of AIH both pre- and post-transplant.

Dual Receptive Fields Underlying Target and Wide-Field Motion Sensitivity in Looming-Sensitive Descending Neurons.

Responding rapidly to visual stimuli is fundamental for many animals. For example, predatory birds and insects alike have amazing target detection abilities, with incredibly short neural and behavioral delays, enabling efficient prey capture. Similarly, looming objects need to be rapidly avoided to ensure immediate survival, as these could represent approaching predators. Male Eristalis tenax hoverflies are nonpredatory, highly territorial insects that perform high-speed pursuits of conspecifics and other territorial intruders. During the initial stages of the pursuit, the retinal projection of the target is very small, but this grows to a larger object before physical interaction. Supporting such behaviors, E. tenax and other insects have both target-tuned and loom-sensitive neurons in the optic lobes and the descending pathways. We here show that these visual stimuli are not necessarily encoded in parallel. Indeed, we describe a class of descending neurons that respond to small targets, to looming and to wide-field stimuli. We show that these descending neurons have two distinct receptive fields where the dorsal receptive field is sensitive to the motion of small targets and the ventral receptive field responds to larger objects or wide-field stimuli. Our data suggest that the two receptive fields have different presynaptic input, where the inputs are not linearly summed. This novel and unique arrangement could support different behaviors, including obstacle avoidance, flower landing, and target pursuit or capture.

Atypical infiltrates on endomyocardial biopsy are associated with adverse outcomes in pediatric heart transplantation.

BACKGROUND: The significance of atypical infiltrates (eosinophils or plasma cells) on endomyocardial biopsy (EMB) after pediatric heart transplant (HTx) is not known. We hypothesized that atypical infiltrates are associated with worse post-HTx outcomes. METHODS: We performed a retrospective cohort study of consecutive patients <21 years old who underwent primary HTx between 2013 and 2017. Multiorgan transplants were excluded. The presence of atypical infiltrates and burden of atypical infiltrates (rare vs predominant) on EMB were recorded. Primary outcome was a composite of cardiac allograft vasculopathy, graft failure (relisting or retransplant), or death. Presence of atypical infiltrates was evaluated: (1) overall using Cox regression with time-dependent covariates and (2) if present by 1 year post-HTx using Kaplan-Meier analysis. RESULTS: Atypical infiltrates were present in 24 out of 95 patients (25%) and were associated with a higher likelihood of reaching the composite outcome (hazard ratio (HR) 6.22, 95% confidence interval (CI) 2.60-14.89, p < 0.0001). This persisted when controlling for rejection in multivariable analysis. There was also a greater risk of the composite outcome if ≥2 nonconsecutive EMBs had atypical infiltrates (HR 11.80, 95%CI 3.17-43.84, p = 0.0002) or if atypical infiltrates were the predominant feature on EMB (HR 30.58, 95%CI 9.34-100.06, p < 0.0001). Patients with atypical infiltrates by 1-year post-HTx had a 5-year freedom from the composite outcome of 48%, compared to 90% if no atypical infiltrates had been present by this timepoint (log rank p = 0.002). CONCLUSIONS: The presence of atypical infiltrates on EMB is associated with significantly worse outcomes in children following HTx. These patients require closer follow-up to assess for developing graft dysfunction.

Dominant negative variants in IKZF2 cause ICHAD syndrome, a new disorder characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay.

BACKGROUND: Helios (encoded by IKZF2), a member of the Ikaros family of transcription factors, is a zinc finger protein involved in embryogenesis and immune function. Although predominantly recognised for its role in the development and function of T lymphocytes, particularly the CD4+ regulatory T cells (Tregs), the expression and function of Helios extends beyond the immune system. During embryogenesis, Helios is expressed in a wide range of tissues, making genetic variants that disrupt the function of Helios strong candidates for causing widespread immune-related and developmental abnormalities in humans. METHODS: We performed detailed phenotypic, genomic and functional investigations on two unrelated individuals with a phenotype of immune dysregulation combined with syndromic features including craniofacial differences, sensorineural hearing loss and congenital abnormalities. RESULTS: Genome sequencing revealed de novo heterozygous variants that alter the critical DNA-binding zinc fingers (ZFs) of Helios. Proband 1 had a tandem duplication of ZFs 2 and 3 in the DNA-binding domain of Helios (p.Gly136_Ser191dup) and Proband 2 had a missense variant impacting one of the key residues for specific base recognition and DNA interaction in ZF2 of Helios (p.Gly153Arg). Functional studies confirmed that both these variant proteins are expressed and that they interfere with the ability of the wild-type Helios protein to perform its canonical function-repressing IL2 transcription activity-in a dominant negative manner. CONCLUSION: This study is the first to describe dominant negative IKZF2 variants. These variants cause a novel genetic syndrome characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay.

Collagen Crosslinking for Keratoconus: Cellular Signaling Mechanisms.

Collagen crosslinking (CXL) is a widely used treatment to halt the progression of keratoconus (KC). Unfortunately, a significant number of patients with progressive KC will not qualify for CXL, including those with corneas thinner than 400 µm. The present study aimed to investigate the molecular effects of CXL using in vitro models, mirroring the normal, as well as thinner corneal stroma seen in KCs. Primary human corneal stromal cells were isolated from healthy (HCFs) and keratoconus (HKCs) donors. Cells were cultured and stimulated with stable Vitamin C resulting in 3D self-assembled extracellular matrix (ECM), cell-embedded, constructs. CXL was performed on (a) thin ECM with CXL performed at week 2 and (b) normal ECM with CXL performed at week 4. Constructs without CXL served as controls. All constructs were processed for protein analysis. The results showed modulation of Wnt signaling, following CXL treatment, as measured by the protein levels of Wnt7b and Wnt10a, correlated to the expression of α-smooth muscle actin (SMA). Further, the expression of a recently identified KC biomarker candidate, prolactin-induced protein (PIP), was positively impacted by CXL in HKCs. CXL-driven upregulation of PGC-1 and the downregulation of SRC and Cyclin D1 in HKCs were also noted. Although the cellular/molecular impacts of CXL are largely understudied, our studies provide an approximation to the complex mechanisms of KC and CXL. Further studies are warranted to determine factors influencing CXL outcomes.

Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus-A Transcriptomic Profiling Study.

Biomechanical and molecular stresses may contribute to the pathogenesis of keratoconus (KC). We aimed to profile the transcriptomic changes in healthy primary human corneal (HCF) and KC-derived cells (HKC) combined with TGFß1 treatment and cyclic mechanical stretch (CMS), mimicking the pathophysiological condition in KC. HCFs (n = 4) and HKCs (n = 4) were cultured in flexible-bottom collagen-coated 6-well plates treated with 0, 5, and 10 ng/mL of TGFß1 with or without 15% CMS (1 cycle/s, 24 h) using a computer-controlled Flexcell FX-6000T Tension system. We used stranded total RNA-Seq to profile expression changes in 48 HCF/HKC samples (100 bp PE, 70-90 million reads per sample), followed by bioinformatics analysis using an established pipeline with Partek Flow software. A multi-factor ANOVA model, including KC, TGFß1 treatment, and CMS, was used to identify differentially expressed genes (DEGs, |fold change| ≥ 1.5, FDR ≤ 0.1, CPM ≥ 10 in ≥1 sample) in HKCs (n = 24) vs. HCFs (n = 24) and those responsive to TGFß1 and/or CMS. PANTHER classification system and the DAVID bioinformatics resources were used to identify significantly enriched pathways (FDR ≤ 0.05). Using multi-factorial ANOVA analyses, 479 DEGs were identified in HKCs vs. HCFs including TGFß1 treatment and CMS as cofactors. Among these DEGs, 199 KC-altered genes were responsive to TGFß1, thirteen were responsive to CMS, and six were responsive to TGFß1 and CMS. Pathway analyses using PANTHER and DAVID indicated the enrichment of genes involved in numerous KC-relevant functions, including but not limited to degradation of extracellular matrix, inflammatory response, apoptotic processes, WNT signaling, collagen fibril organization, and cytoskeletal structure organization. TGFß1-responsive KC DEGs were also enriched in these. CMS-responsive KC-altered genes such as OBSCN, CLU, HDAC5, AK4, ITGA10, and F2RL1 were identified. Some KC-altered genes, such as CLU and F2RL1, were identified to be responsive to both TGFß1 and CMS. For the first time, our multi-factorial RNA-Seq study has identified many KC-relevant genes and pathways in HKCs with TGFß1 treatment under CMS, suggesting a potential role of TGFß1 and biomechanical stretch in KC development.

Salivary Exosomes in Health and Disease: Future Prospects in the Eye.

Exosomes are a group of vesicles that package and transport DNA, RNA, proteins, and lipids to recipient cells. They can be derived from blood, saliva, urine, and/or other biological tissues. Their impact on several diseases, such as neurodegenerative, autoimmune, and ocular diseases, have been reported, but not fully unraveled. The exosomes that are derived from saliva are less studied, but offer significant advantages over exosomes from other sources, due to their accessibility and ease of collection. Thus, their role in the pathophysiology of diseases is largely unknown. In the context of ocular diseases, salivary exosomes have been under-utilized, thus creating an enormous gap in the literature. The current review discusses the state of exosomes research on systemic and ocular diseases and highlights the role and potential of salivary exosomes as future ocular therapeutic vehicles.

Potentiation of Sphingolipids and TGF-ß in the human corneal stroma reveals intricate signaling pathway crosstalks.

Corneal haze brought on by fibrosis due to insult can lead to partial or complete vision loss. Currently, corneal transplantation is the gold standard for treating severe corneal fibrosis, which comes with the risk of rejection and the issue of donor tissue shortages. Sphingolipids (SPLs) are known to be associated with fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to Transforming Growth Factor ß (TGF-ß) signaling and corneal fibrogenesis. This study aimed to elucidate the interplay of SPLs, specifically sphingosine-1-phosphate (S1P) signaling, and its' interactions with TGF-ß signaling through detailed analyses of the corresponding downstream signaling targets in the context of corneal fibrosis, in vitro. Healthy human corneal fibroblasts (HCFs) were isolated, plated on polycarbonate membranes, and stimulated with a stable Vitamin C derivative. The 3D constructs were treated with either 5 µM sphingosine-1-phosphate (S1P), 5 µM SPHK I2 (I2; inhibitor of sphingosine kinase 1, one of the two enzymes responsible for generating S1P in mammalian cells), 0.1 ng/mL TGF-ß1, or 0.1 ng/mL TGF-ß3. Cultures with control medium-only served as controls. All 3D constructs were examined for protein expression of fibrotic markers, SPLs, TGF-ßs, and relevant downstream signaling pathways. This data revealed no significant changes in any LTBP (latent TGF-ß binding proteins) expression when stimulated with S1P or I2. However, LTBP1 was significantly upregulated via stimulation of TGF-ß1 and TGF-ß3, whereas LTBP2 was significantly upregulated only with TGF-ß3 stimulation. Significant downregulation of TGF-ß receptor II (TGF-ßRII) following S1P stimulation but significant upregulation following I2 stimulation was observed. Following TGF-ß1, S1P, and I2 stimulation, phospho-SMAD2 (pSMAD2) was significantly downregulated. Furthermore, I2 stimulation led to significant downregulation of SMAD4. Adhesion/proliferation/transcription regulation targets, SRC, FAK, and pERK 1/2 were all significantly downregulated by exogenous S1P, whereas I2 only significantly downregulated FAK. Exogenous TGF-ß3 caused significant upregulation of AKT. Interestingly, both I2 and TGF-ß3 caused significant downregulation of JNK expression. Lastly, TGF-ß1 led to significant upregulation of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1PR3), whereas TGF-ß3 caused significant upregulation of only SphK1. Together with previously published work from our group and others, S1P inhibition exhibits great potential as an efficacious anti-fibrotic modality in human corneal stromal ECM. The current findings shed further light on a very complex and rather incompletely investigated mechanism, and cement the intricate crosstalk between SPLs and TGF-ß in corneal fibrogenesis. Future studies will dictate the potential of utilizing SPLs/TGF-ß signaling modulators as novel therapeutics in corneal fibrosis.

Selective Modulation of the Keratoconic Stromal Microenvironment by FSH and LH.

Keratoconus (KC) affects the corneal structure, with thinning and bulging outward into a conelike shape. Irregular astigmatism and decreased visual acuity appear during puberty and progress into the mid-30s, with unpredictable disease severity. The cause of KC is recognized as multifactorial, but remains poorly understood. Hormone imbalances are a significant modulator of the onset of KC. This study sought to investigate the role of gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in KC, using a three-dimensional, self-assembled matrix in vitro model. Healthy corneal fibroblasts and human KC cells in the corneal stroma were isolated, cultured, and stimulated with stable vitamin C to promote extracellular matrix assembly. Cultures were further stimulated with 2.5 or 10 mIU/mL FSH and 5 or 35 mIU/mL LH. Samples were evaluated for cell proliferation and morphology via BrdU assay and imaging; protein expression was assessed via Western blot analysis. Proliferation was significantly greater in human KC cells compared to healthy corneal fibroblasts with LH stimulation, but no changes were found with FSH stimulation. Additionally, in sex hormone receptors, fibrotic markers, proteoglycans, and members of the gonadotropin signaling pathway were significantly changed, largely driven by exogenous LH. The impact of exogenous FSH/LH in the KC stromal microenvironment was demonstrated. These results highlight the need to further examine the role of FSH/LH in KC and in human corneal homeostasis.

Cord blood transplantation for nonmalignant disorders: early functional immunity and high survival.

There is no consensus on the best donor for children with nonmalignant disorders and immune deficiencies in the absence of a matched related donor (MRD). We evaluated the 2-year overall survival (OS) after umbilical cord blood transplantation (UCBT) in patients with nonmalignant disorders from 2009 to 2020 enrolled in a prospective clinical trial using either 5/6 or 6/6 UCB as the cell source. Patients receive a fully ablative busulfan, cyclophosphamide, and fludarabine without serotherapy. Fifty-five children were enrolled, median age 5 months (range, 1-111 months); primary immune deficiency (45), metabolic (5), hemophagocytic lymphohistiocytosis (1), and hematologic disorders (4). Twenty-six patients had persistent infections before transplant. Nineteen of them (34%) were 6/6 matched, and 36 (66%) were 5/6 human leukocyte antigen-matched. The OS at 2 years was 91% (95% cumulative incidence, 79-96), with a median follow-up of 4.3 years. The median time to neutrophil and platelet recovery were 17 days (range, 5-39 days) and 37 days (range, 20-92 days), respectively. All but one evaluable patient achieved full donor chimerism. The cumulative incidence of acute GVHD grades 2-4 on day 100 was 16% (n = 9). All patients with viral infections at the time of transplant cleared the infection at a median time of 54 days (range, 44-91 days). All evaluable patients underwent correction of their immune or metabolic defects. We conclude that in the absence of MRD, UCBT following myeloablative conditioning without serotherapy is an excellent curative option in young children with nonmalignant disorders. This trial has been registered at www.clinicaltrials.gov as NCT00950846.

Monocarboxylate Transporters: Role and Regulation in Corneal Diabetes.

Diabetes mellitus (DM) is a group of metabolic diseases that is known to cause structural and functional ocular complications. In the human cornea, DM-related complications affect the epithelium, stroma, and nerves. Monocarboxylate transporters (MCTs) are a family of proton-linked plasma membrane transporters that carry monocarboxylates across plasma membranes. In the context of corneal health and disease, their role, presence, and function are largely undetermined and solely focused on the most common MCT isoforms, 1 through 4. In this study, we investigated the regulation of MCT1, 2, 4, 5, 8, and 10, in corneal DM, using established 3D self-assembled extracellular matrix (ECM) in vitro models. Primary stromal corneal fibroblasts were isolated from healthy (HCFs), type I (T1DMs), and type II (T2DMs) DM donors. Monoculture 3D constructs were created by stimulating stromal cells on transwells with stable vitamin C for two or four weeks. Coculture 3D constructs were created by adding SH-SY5Y neurons at two different densities, 12 k and 500 k, on top of the monocultures. Our data showed significant upregulation of MCT1 at 4 weeks for HCF, T1DM, and T2DM monocultures, as well as the 500 k nerve cocultures. MCT8 was significantly upregulated in HCF and T1DM monocultures and all of the 500 k nerve cocultures. Further, MCT10 was only expressed at 4 weeks for all cocultures and was limited to HCFs and T1DMs in monocultures. Immunofluorescence analysis showed cytoplasmic MCT expression for all cell types and significant downregulation of both MCT2 and MCT4 in HCFs, when compared to T1DMs and T2DMs. Herein, we reveal the existence and modulation of MCTs in the human diabetic cornea in vitro. Changes appeared dependent on neuronal density, suggesting that MCTs are very likely critical to the neuronal defects observed in diabetic keratopathy/neuropathy. Further studies are warranted in order to fully delineate the role of MCTs in corneal diabetes.

Ablation of Sphingosine Kinase 1 Protects Cornea from Neovascularization in a Mouse Corneal Injury Model.

The purpose of this study was to investigate the role of sphingosine kinase 1 (SphK1), which generates sphingosine-1-phosphate (S1P), in corneal neovascularization (NV). Wild-type (WT) and Sphk1 knockout (Sphk1-/-) mice received corneal alkali-burn treatment to induce corneal NV by placing a 2 mm round piece of Whatman No. 1 filter paper soaked in 1N NaOH on the center of the cornea for 20 s. Corneal sphingolipid species were extracted and identified using liquid chromatography/mass spectrometry (LC/MS). The total number of tip cells and those positive for ethynyl deoxy uridine (EdU) were quantified. Immunocytochemistry was done to examine whether pericytes were present on newly forming blood vessels. Cytokine signaling and angiogenic markers were compared between the two groups using multiplex assays. Data were analyzed using appropriate statistical tests. Here, we show that ablation of SphK1 can significantly reduce NV invasion in the cornea following injury. Corneal sphingolipid analysis showed that total levels of ceramides, monohexosyl ceramides (HexCer), and sphingomyelin were significantly elevated in Sphk-/- corneas compared to WT corneas, with a comparable level of sphingosine among the two genotypes. The numbers of total and proliferating endothelial tip cells were also lower in the Sphk1-/- corneas following injury. This study underscores the role of S1P in post-injury corneal NV and raises further questions about the roles played by ceramide, HexCer, and sphingomyelin in regulating corneal NV. Further studies are needed to unravel the role played by bioactive sphingolipids in maintenance of corneal transparency and clear vision.

Quercetin Decreases Corneal Haze In Vivo and Influences Gene Expression of TGF-Beta Mediators In Vitro.

We have previously reported the flavonoid, quercetin, as a metabolic regulator and inhibitor of myofibroblast differentiation in vitro. Our current study evaluated the effects of topical application of quercetin on corneal scar development using two different animal models followed by RNA analysis in vitro. Wild-type C57BL/6J mice were anesthetized and the corneal epithelium and stroma were manually debrided, followed by quercetin (0.5, 1, 5, or 50 mM) or vehicle application. Corneal scarring was assessed for 3 weeks by slit lamp imaging and clinically scored. In a separate animal study, six New Zealand White rabbits underwent lamellar keratectomy surgery, followed by treatment with 5 mM quercetin or vehicle twice daily for three days. Stromal backscattering was assessed at week 3 by in vivo confocal microscopy. In mice, a single dose of 5 mM quercetin reduced corneal scar formation. In rabbits, stromal backscattering was substantially lower in two out of three animals in the quercetin-treated group. In vitro studies of human corneal fibroblasts showed that quercetin modulated select factors of the transforming growth factor-ß (TGF-ß) signaling pathway. These results provide evidence that quercetin may inhibit corneal scarring. Further studies in a larger cohort are required to validate the efficacy and safety of quercetin for clinical applications.

A low-input strategy for chromium removal from industrial stormwater using peat sorbent.

Low-cost and low-input water treatment systems are important for industrial stormwater remediation. Here we examine a flow-through reactor treatment installation where water exceeds the allowable maximum concentration for drinking water in multiple metals (e.g., chromium [Cr], cadmium [Cd], zinc [Zn]) prior to treatment. Specifically, we seek to understand why Cr attenuated in the reactors is not leachable by identifying the specific chemical form of Cr and dominant mechanisms promoting sequestration in the reactors. Total solid-phase Cr concentration in the peat media ranged from 50 to 150 mg/kg after 1 yr of exposure to stormwater to 300 to 900 mg/kg after 3.5 yr. X-ray fluorescence mapping images show Cr, iron (Fe), and Zn spatially correlated over a scale of 10 µm to 5 mm. Chromium rinds form on the edges of peat particles as Cr accumulates. Chromium and Fe K-edge X-ray absorption near edge structure spectroscopy reveal chromium predominately in the 3+ oxidation state with lesser amounts of elemental Cr. We propose the primary means of chromium attenuation in the reactors is precipitation as Cr-Fe hydroxides combined with trivalent Cr adsorption onto peat surfaces.

Splice factor polypyrimidine tract-binding protein 1 (Ptbp1) primes endothelial inflammation in atherogenic disturbed flow conditions.

NF-κB-mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through adhesion molecules Icam1 and Vcam1. The endothelium is primed for cytokine activation of NF-κB by exposure to low and disturbed blood flow (LDF)but the molecular underpinnings are not fully understood. In an experimental in vivo model of LDF, platelets were required for the increased expression of several RNA-binding splice factors, including polypyrimidine tract binding protein (Ptbp1). This was coordinated with changes in RNA splicing in the NF-κB pathway in primed cells, leading us to examine splice factors as mediators of priming. Using Icam1 and Vcam1 induction by tumor necrosis factor (TNF)-α stimulation as a readout, we performed a CRISPR Cas9 knockout screen and identified a requirement for Ptbp1 in priming. Deletion of Ptbp1 had no effect on cell growth or response to apoptotic stimuli, but reversed LDF splicing patterns and inhibited NF-κB nuclear translocation and transcriptional activation of downstream targets, including Icam1 and Vcam1. In human coronary arteries, elevated PTBP1 correlates with expression of TNF pathway genes and plaque. In vivo, endothelial-specific deletion of Ptbp1 reduced Icam1 expression and myeloid cell infiltration at regions of LDF in atherosclerotic mice, limiting atherosclerosis. This may be mediated, in part, by allowing inclusion of a conserved alternative exon in Ripk1 leading to a reduction in Ripk1 protein. Our data show that Ptbp1, which is induced in a subset of the endothelium by platelet recruitment at regions of LDF, is required for priming of the endothelium for subsequent NF-κB activation, myeloid cell recruitment and atherosclerosis.