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In a phase 2 study, pimavanserin demonstrated efficacy as adjunctive treatment for major depressive disorder (MDD). Subsequently, two phase 3 studies (NCT03968159 in the US; NCT03999918 in Europe) were initiated to examine the efficacy and safety of adjunctive pimavanserin in subjects with MDD and inadequate response to antidepressant treatment. Studies were combined with a prespecified statistical analysis plan owing to recruitment challenges related to the COVID-19 pandemic.The randomized, double-blind studies enrolled 298 patients with MDD and inadequate response to current antidepressants. Patients were randomly assigned 1:1 to pimavanserin or placebo added to current antidepressant for 6 weeks. Primary endpoint was change from baseline to week 5 in the Hamilton Rating Scale for Depression, 17-item version (HAM-D-17).There was no effect of pimavanserin in change from baseline to week 5 in the HAM-D-17 (pimavanserin [n = 138]: least-squares mean [LSM] [standard error {SE}], -9.0 [0.58]; placebo [n = 135]: -8.1 [0.58]; mixed-effects model for repeated measures LSM [SE] difference, -0.9 [0.82],Adjunctive pimavanserin did not significantly improve depressive symptoms, although pimavanserin was well tolerated.

BACKGROUND AND OBJECTIVES All US Food and Drug Administration-approved medications for Tourette syndrome are antipsychotics, and their use is limited by the risk of weight gain, metabolic changes, and drug-induced movement disorders. Several small trials suggest that ecopipam, a first-in-class, selective dopamine 1 receptor antagonist, reduces tics with a low risk for these adverse events. This trial sought to further evaluate the efficacy, safety, and tolerability of ecopipam in children and adolescents with moderate to severe Tourette syndrome. METHODS This was a multicenter, randomized, double-blind, placebo-controlled, phase 2b trial. Subjects aged ≥6 to RESULTS Total tic scores were significantly reduced from baseline to 12 weeks in the ecopipam group compared with placebo (least squares mean differences −3.44, 95% confidence interval −6.09 to −0.79, P = .01). Improvement in Clinical Global Impression of Tourette Syndrome Severity was also greater in the ecopipam group (P = .03). More weight gain was seen in subjects assigned to placebo. No metabolic or electrocardiogram changes were identified. Headache (15.8%), insomnia (14.5%), fatigue (7.9%), and somnolence (7.9%) were the most common adverse events. CONCLUSIONS Among children and adolescents with TS, ecopipam reduces tics to a greater extent than placebo, without observable evidence of common antipsychotic-associated side effects.

TWEAK (tumor necrosis factor-like weak inducer of apoptosis) is a member of the TNF superfamily that controls a multitude of cellular events including proliferation, migration, differentiation, apoptosis, angiogenesis, and inflammation. TWEAK control of these events is via an expanding list of intracellular signalling pathways which include NF-κB, ERK/MAPK, Notch, EGFR and AP-1. Two receptors have been identified for TWEAK – Fn14, which targets the membrane bound form of TWEAK, and CD163, which scavenges the soluble form of TWEAK. TWEAK appears to elicit specific events based on the receptor to which it binds, tissue type in which it is expressed, specific extrinsic conditions, and the presence of other cytokines. TWEAK signalling is protective in healthy tissues, but in chronic inflammatory states become detrimental to the tissue. Consistent data show a role for the TWEAK/FN14/CD163 axis in metabolic disease, chronic autoimmune diseases, and acute ischaemic stroke. Low circulating concentrations of soluble TWEAK are predictive of poor cardiovascular outcomes in those with and without diabetes. This review details the current understanding of the TWEAK/Fn14/CD163 axis as one of the chief regulators of immune signalling and its cell-specific role in metabolic disease development and progression.

TWEAK (tumor necrosis factor-like weak inducer of apoptosis) is a member of the TNF superfamily that controls a multitude of cellular events including proliferation, migration, differentiation, apoptosis, angiogenesis, and inflammation. TWEAK control of these events is via an expanding list of intracellular signalling pathways which include NF-κB, ERK/MAPK, Notch, EGFR and AP-1. Two receptors have been identified for TWEAK - Fn14, which targets the membrane bound form of TWEAK, and CD163, which scavenges the soluble form of TWEAK. TWEAK appears to elicit specific events based on the receptor to which it binds, tissue type in which it is expressed, specific extrinsic conditions, and the presence of other cytokines. TWEAK signalling is protective in healthy tissues, but in chronic inflammatory states become detrimental to the tissue. Consistent data show a role for the TWEAK/FN14/CD163 axis in metabolic disease, chronic autoimmune diseases, and acute ischaemic stroke. Low circulating concentrations of soluble TWEAK are predictive of poor cardiovascular outcomes in those with and without diabetes. This review details the current understanding of the TWEAK/Fn14/CD163 axis as one of the chief regulators of immune signalling and its cell-specific role in metabolic disease development and progression.

Pseudoexfoliation glaucoma (XFG) is an aggressive form of secondary open angle glaucoma, characterised by the production of exfoliation material and is estimated to affect 30 million people worldwide. Activation of the TGF-β pathway by TGF-β1 has been implicated in the pathogenesis of pseudoexfoliation glaucoma. To further investigate the role of TGF-β1 in glaucomatous changes in the trabecular meshwork (TM), we used RNA-Seq to determine TGF-β1 induced changes in the transcriptome of normal human trabecular meshwork (HTM) cells. The main purpose of this study was to perform a hypothesis-independent RNA sequencing analysis to investigate genome-wide alterations in the transcriptome of normal HTMs stimulated with TGF-β1 and investigate possible pathophysiological mechanisms driving XFG. Our results identified multiple differentially expressed genes including several genes known to be present in exfoliation material. Significantly altered pathways, biological processes and molecular functions included extracellular matrix remodelling, Hippo and Wnt pathways, the unfolded protein response, oxidative stress, and the antioxidant system. This cellular model of pseudoexfoliation glaucoma can provide insight into disease pathogenesis and support the development of novel therapeutic interventions.

TNFAIP3 encodes a zinc finger protein called A20, which has potent anti-inflammatory and anti-apoptotic properties. A20 promotes beta-cell survival and protects against islet graft rejection in experimental models. The current study sought to investigate the mechanisms underlying the protective role of A20 in the pancreatic beta-cell. Two islet cell types were used for experiments: the insulin-secreting BRIN-BD11 cell line and human islet cells. A20 was silenced using siRNA against TNFAIP3, and knockdown was confirmed by qPCR and immunostaining of cells. Cell viability, cytotoxicity and apoptosis were assessed using the ApotoxGlo assay. Glucose-stimulated insulin secretion and production of inflammatory cytokines (TNFa, IL1b and IFNg) were measured by ELISA. Expression of beta-cell regulatory genes (Abcc8, Kcnj11, Kcnq1, Gck, Scl2a2) and transcription factors (Hnf1a, Pdx1, Nkx6.1, Ngn3) was determined by qPCR. A20 deficiency increased apoptosis, impaired glucose-induced insulin secretion, and reduced expression of beta-cell regulatory genes and transcription factors. Addition of recombinant A20 normalized gene expression profiles. TNFa, IL1b and IFNg were elevated in A20 deficient cells and found to independently elicit changes in gene expression. Analysis of PCR array data suggests that A20 action in the beta cell is largely, although not exclusively, driven by the P65 subunit of NF-kB. The current report demonstrates a role for A20 in controlling beta-cell integrity and survival, which likely results from the regulation of inflammatory signalling. Of particular note is the impact that A20 deficiency has on the expression of transcription factors regulating the maturation and normal function of beta cells.

GWAS studies have identified a number of SNPs connected to the development of type 1 diabetes (T1D). It is unclear how these variants confer risk for T1D development and whether this is based on direct effects on the beta cell, or whether the systemic inflammatory environment is detrimental to the beta cell. Using CRISPR mediated homology directed repair (Life Technologies), T1D risk variants rs10517086 and rs1534422 were introduced into the BRIN-BD11 beta cell line and the Jurkat T-cell line. Experiments were conducted in BRIN-BD11 cells expressing each variant and in native BRIN-BD11 cells treated with conditioned media from Jurkat cells expressing each variant. ApoTox GloTM (Promega) was used to assess cell viability, cytotoxicity and apoptosis. In all instances, cell viability and cytotoxicity were unchanged following the introduction of the two variants. However, rs1534422 significantly (P Disclosure W. Ratajczak: None. S. Atkinson: None. C. Kelly: None. Funding European Union Regional Development Fund; European Union Sustainable Competitiveness Programme for Northern Ireland; Northern Ireland Public Health Agency; Ulster University; Northern Ireland Department of Employment and Learning

Introduction: Management of co- and multi-morbidity continues to represent a major clinical challenge. There remains a lack of understanding of how multiple pathologies or conditions co-evolve or i...

Glaucoma is a group of optic neuropathies characterised by the degeneration of retinal ganglion cells, resulting in damage to the optic nerve head (ONH) and loss of vision in one or both eyes. Increased intraocular pressure (IOP) is one of the major aetiological risk factors in glaucoma, and is currently the only modifiable risk factor. However, 30–40% of glaucoma patients do not present with elevated IOP and still proceed to lose vision. The pathophysiology of glaucoma is therefore not completely understood, and there is a need for the development of IOP-independent neuroprotective therapies to preserve vision. Neuroinflammation has been shown to play a key role in glaucoma and, specifically, the NLRP3 inflammasome, a key driver of inflammation, has recently been implicated. The NLRP3 inflammasome is expressed in the eye and its activation is reported in pre-clinical studies of glaucoma. Activation of the NLRP3 inflammasome results in IL-1β processing. This pro inflammatory cytokine is elevated in the blood of glaucoma patients and is believed to drive neurotoxic inflammation, resulting in axon degeneration and the death of retinal ganglion cells (RGCs). This review discusses glaucoma as an inflammatory disease and evaluates targeting the NLRP3 inflammasome as a therapeutic strategy. A hypothetical mechanism for the action of the NLRP3 inflammasome in glaucoma is presented.

Autosomal dominantly inherited genetic disorders such as corneal dystrophies are amenable to allele-specific gene silencing with small interfering RNA (siRNA). siRNA delivered to the cornea by injection, although effective, is not suitable for a frequent long-term treatment regimen, whereas topical delivery of siRNA to the cornea is hampered by the eye surface's protective mechanisms. Herein we describe an attractive and innovative alternative for topical application using cell-penetrating peptide derivatives capable of complexing siRNA non-covalently and delivering them into the cornea. Through a rational design approach, we modified derivatives of a cell-penetrating peptide, peptide for ocular delivery (POD), already proved to diffuse into the corneal layers. These POD derivatives were able to form siRNA-peptide complexes (polyplexes) of size and ζ-potential similar to those reported able to undergo cellular internalization. Successful cytoplasmic release and gene silencing in vitro was obtained when an endosomal disruptor, chloroquine, was added. A palmitoylated-POD, displaying the best delivery properties, was covalently functionalized with trifluoromethylquinoline, an analog of chloroquine. This modified POD, named trifluoromethylquinoline-palmitoyl-POD (QN-Palm-POD), when complexed with siRNA and topically applied to the eye in vivo, resulted in up to 30% knockdown of luciferase reporter gene expression in the corneal epithelium. The methods developed within represent a valid standardized approach that is ideal for screening of a range of delivery formulations. © 2019 The Authors, This work was supported by the United Kingdom Fight for Sight grant (to C.B.T.M.), The Belfast Association for the Blind (S.D.A., M.A.N., and T.M.), and Northern Ireland Clinical Research Network Vision Research Translation Research Group (M.A.N. and T.M.). Partial financial support from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and the European Regional Development Fund (grant CTQ2015-63919-R to I.H.) is also gratefully acknowledged.

Pterygium is a pathological proliferative condition of the ocular surface, characterised by formation of a highly vascularised, fibrous tissue arising from the limbus that invades the central cornea leading to visual disturbance and, if untreated, blindness. Whilst chronic ultraviolet (UV) light exposure plays a major role in its pathogenesis, higher susceptibility to pterygium is observed in some families, suggesting a genetic component. In this study, a Northern Irish family affected by pterygium but reporting little direct exposure to UV was identified carrying a missense variant in CRIM1 NM_016441.2: c.1235 A > C (H412P) through whole-exome sequencing and subsequent analysis. CRIM1 is expressed in the developing eye, adult cornea and conjunctiva, having a role in cell differentiation and migration but also in angiogenesis, all processes involved in pterygium formation. We demonstrate elevated CRIM1 expression in pterygium tissue from additional individual Northern Irish patients compared to unaffected conjunctival controls. UV irradiation of HCE-S cells resulted in an increase in ERK phosphorylation and CRIM1 expression, the latter further elevated by the addition of the MEK1/2 inhibitor, U0126. Conversely, siRNA knockdown of CRIM1 led to decreased UV-induced ERK phosphorylation and increased BCL2 expression. Transient expression of the mutant H412P CRIM1 in corneal epithelial HCE-S cells showed that, unlike wild-type CRIM1, it was unable to reduce the cell proliferation, increased ERK phosphorylation and apoptosis induced through a decrease of BCL2 expression levels. We propose here a series of intracellular events where CRIM1 regulation of the ERK pathway prevents UV-induced cell proliferation and may play an important role in the in the pathogenesis of pterygium.

CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

The purpose of this study was to evaluate the efficacy and safety of duloxetine flexible dose in children (7-11 years) and adolescents (12-17 years) with major depressive disorder (MDD).Patients (n=337) in this 36 week study (10 week acute and 26 week extension treatment) received duloxetine (60-120 mg once daily [QD], n=117), fluoxetine (20-40 mg QD, n=117), or placebo (n=103). Measures included: Children's Depression Rating Scale-Revised (CDRS-R), treatment-emergent adverse events (TEAEs), and Columbia-Suicide Severity Rating Scale (C-SSRS).Neither active drug (duloxetine or fluoxetine) separated significantly (p0.05) from placebo on mean change from baseline to end-point (10 weeks) on the CDRS-R total score. There were no significant differences between the duloxetine or fluoxetine groups compared with placebo on serious AEs (SAEs), total TEAEs, or discontinuation for AE during acute treatment. There were no completed suicides or deaths, and no clinically significant electrocardiogram (ECG) abnormalities observed during the study. One fluoxetine and one duloxetine patient experienced alanine aminotransferase (ALT) three or more times the upper limit of normal, which resolved during the study. A total of 8 (7.1%) duloxetine patients, 7 (6.8%) placebo patients, and 9 (8.0%) fluoxetine patients had worsening of suicidal ideation from baseline during acute treatment. Of the patients with suicidal ideation at baseline, 15/19 (79%) duloxetine, 19/19 (100%) placebo, and 16/19 (84%) fluoxetine had improvement in suicidal ideation at end-point during acute treatment. One duloxetine and two fluoxetine patients had treatment-emergent suicidal behavior during the 36 week study.Trial results were inconclusive, as neither the investigational drug (duloxetine) nor the active control (fluoxetine) separated from placebo on the CDRS-R at 10 weeks. No new duloxetine safety signals were identified relative to those seen in adults. Clinical Trial Registry Number: NCT00849901.

Purpose: To compare the clinical and molecular diagnoses of Herpes Simplex Keratitis (HSK). Materials and Methods: Conjunctival swabs (after fluorescein and anaesthetic wash out) and detailed questionnaire data were obtained from 146 participants. Corneal rims and conjunctival epithelial cells were infected with Herpes Simplex Virus (HSV) type 1 or HSV2 and supernatant collected. HSV1; HSV2; Varicella Zoster Virus (VZV) and Adenovirus (ADV) DNA was assessed using two real time Polymerase Chain Reaction (PCR) methods. Results: Of the 146 participants recruited, 54 were clinically diagnosed with typical epithelial lesions and 38 with atypical epithelial lesions, 17 with old inactive HSK and 37 healthy volunteers. HSV1 DNA was detected in 28 (30%) of the 92 participants with clinically suspect HSK. Patients who presented with typical epithelial lesions had a higher positive rate (46%) than those who presented with atypical type lesions (8%), when using primers against the Glycoprotein (Gp) G region of the virus. When the same samples were retested with primers against the GpB region, the positive rate for the typical and atypical cases increased to 52% and 11% respectively. Antiviral use at the time of sampling reduced the rate of PCR positivity by 20% (p in vitro control investigations of HSV1 and 2 infected corneal rims and conjunctival epithelial cells were 100% positive for infected and 100% negative for uninfected samples when assessed using both PCR methods. Conclusions: Clinical diagnosis of typical HSK is not always confirmed by PCR. Concomitant use of an antiviral reduces levels of PCR positivity. Given this and the findings that other ocular surface pathogens may mimic HSK pathology, and that choice of gene amplification region can also affect accurate detection of HSV1 by PCR, we propose the use of a multiplex assay. This would perform PCR using primers spanning a number of different regions within one gene and would also target a number of different viral genes to ensure potentially different HSV1 viral strains or other viruses do not affect the test and lead to disagreements between the clinical and molecular diagnosis of HSK. From these findings, this paper proposes a clinical supportive algorithmic guide to manage such disagreements.

Background/aims Cross-linking of the cornea is usually carried out at a young age as a treatment to manage ectasia. The corneal limbal region contains delicate long-lived stem cells, which could potentially be deleteriously affected by Ultraviolet A (UV-A) radiation. Damage to these stem cells may not demonstrate as a clinical problem for many years subsequent to cross-linking treatment. UV-A radiation is known to have potential mutagenic effects upon mammalian DNA and can result in cancer. Methods Cultured corneal epithelial cells and ex vivo corneal tissue were treated with the standard clinical cross-linking protocol for UV-A irradiation. 8-hydroxydeoxyguansoine (8-OHdG) and cyclin-dependent kinase inhibitor genes (CDKN1A and CDKN2A) were assayed as markers of DNA damage using immunohistochemistry, ELISA and quantitative real time PCR. Results Staining of treated limbal tissue demonstrated the presence of 8-OHdG within p63 positive basal limbal cells. Levels of 8-OHdG and CDKN1A mRNA were found to be significantly increased in cultured corneal epithelial cells and limbal epithelial cells but no increase was demonstrated with the use of a polymethyl methylacrylate protective cover. Conclusions This study provides evidence that oxidative nuclear DNA damage can occur through cross-linking in layers of corneal epithelial cells at the limbus and that this can be easily prevented by covering the limbus.

Epidermolysis bullosa simplex (EBS) is an incurable, inherited skin-blistering disorder predominantly caused by dominant-negative mutations in the genes encoding keratins K5 or K14. RNA interference, particularly in the form of small interfering RNA (siRNA), offers a potential therapy route for EBS and related keratin disorders by selectively silencing the mutant allele. Here, using a systemic screening system based on a luciferase reporter gene assay, we have developed mutant-specific siRNAs for two independent EBS-causing missense mutations in the K5 gene (p.Ser181Pro and p.Asn193Lys). The specificity of the allele-specific inhibitors identified in the screen was subsequently confirmed at the protein level, where the lead inhibitors were shown to strongly knock down the expression of mutant proteins with negligible effect on wild-type K5 expression. In a cell-based model system, the lead inhibitors were able to significantly reverse the cytoskeletal aggregation phenotype. Overall, this approach shows promise for the treatment of EBS and paves the way for future clinical trials.

PURPOSE: This study aimed to investigate the potency and specificity of short-interfering RNA (siRNA) treatment for TGFBI-Arg124Cys lattice corneal dystrophy type I (LCDI) using exogenous expression constructs in model systems and endogenous gene targeting in an ex vivo model using corneal epithelial cell cultures. METHODS: A panel of 19 TGFBI-Arg124Cys-specific siRNAs were assessed by a dual-luciferase reporter assay. Further assessment using pyrosequencing and qPCR was used to identify the lead siRNA; suppression of mutant TGFBIp expression was confirmed by Western blot and Congo red aggregation assays. An ex vivo model of LCDI was established using limbal biopsies from corneal dystrophy patients harboring the Arg124Cys mutation. Treatment efficiency of the siRNA was assessed for the inhibition of the mutant allele in the primary patient's corneal epithelial cells using pyrosequencing, quantitative PCR (qPCR), and an ELISA. RESULTS: A lead siRNA was identified, and demonstrated to be potent and specific in inhibiting the TGFBI-Arg124Cys mutant allele at the mRNA and protein levels. Besides high allele specificity, siRNA treatment achieved a 44% reduction of the endogenous Arg124Cys allele in an ex vivo model of LCDI. CONCLUSIONS: We have identified a lead siRNA specific to the TGFBI-Arg124Cys mutant allele associated with LCDI. Silencing of exogenous TGFBI was observed at mRNA and protein levels, and in an ex vivo model of LCDI with an efficient suppression of the endogenous mutant allele. This result indicates the potential of siRNA treatment as a personalized medicine approach for the management of heritable TGFBI-associated corneal dystrophies.

Purpose The aim of this study is to further assess our previously reported keratin 12 (K12)-Leu132Pro specific siRNA in silencing the mutant allele in Meesmann's Epithelial Corneal Dystrophy (MECD) in experimental systems more akin to the in vivo situation through simultaneous expression of both wild-type and mutant alleles. Methods Using KRT12 exogenous expression constructs transfected into cells, mutant allele specific knockdown was quantified using pyrosequencing and infrared Western blot analysis, while the silencing mechanism was assessed by a modified rapid amplification of cDNA ends (5'RACE) method. Corneal limbal biopsies taken from patients suffering from MECD were used to establish cultures of MECD corneal limbal epithelial stem cells and the ability of the siRNA to silence the endogenous mutant KRT12 allele was assessed by a combination of pyrosequencing, qPCR, ELISA, and quantitative-fluorescent immunohistochemistry (Q-FIHC). Results The siRNA displayed a potent and specific knockdown of K12-Leu132Pro at both the mRNA and protein levels with exogenous expression constructs. Analysis by the 5'RACE method confirmed siRNA-mediated cleavage. In the MECD cells, an allele-specific knockdown of 63% of the endogenous mutant allele was observed without effect on wild-type allele expression. Conclusions Combined with an effective delivery vehicle this siRNA approach represents a viable treatment option for prevention of the MECD pathology observed in K12-Leu132Pro heterozygous individuals.

During placentation, the concentration of fibrinous deposits on the surfaces of maternal vasculature plays a role in villous development and has been strongly implicated in the pathophysiology of human fetal growth restriction (FGR). Fibrinous deposits are conspicuous sites of platelet aggregation where there is local activation of the hemostatic cascade. During activation of the hemostatic cascade, a number of pro- and antiangiogenic agents may be generated at the cell surface, and an imbalance in these factors may contribute to the placental pathology characteristic of FGR. We tested the hypothesis that angiostatin(4.5) (AS(4.5)), a cleavage fragment of plasminogen liberated at the cell surface, is capable of causing FGR in mice. Increased maternal levels of AS(4.5) in vivo result in reproducible placental pathology, including an altered vascular compartment (both in decidual and labyrinthine layers) and increased apoptosis throughout the placenta. In addition, there is significant skeletal growth delay and conspicuous edema in fetuses from mothers that received AS(4.5). Maternally generated AS(4.5), therefore, can access maternal placental vasculature and have a severe effect on placental architecture and inhibit fetal development in vivo. These findings strongly support the hypothesis that maternal AS(4.5) levels can influence placental development, possibly by directly influencing trophoblast turnover in the placenta, and contribute to fetal growth delay in mice.

Compare expression of p63 in pterygium and laryngo-onycho-cutaneous (LOC) syndrome with normal conjunctiva.P63 immunohistochemical detection was carried out in normal, pterygium, and LOC conjunctival tissue. In vitro, growth of normal conjunctival biopsy specimens, pterygium, and LOC in growth tissue was compared.In normal conjunctiva, p63 was poorly expressed in the infranasal quadrant, with 36% of cells stained vs. 55 to 59% in other quadrants (p0.05). In pterygium, p63 was overexpressed (59% cells stained) compared to normal supranasal (55%) and normal infranasal conjunctiva (36%, p0.05). In LOC, p63 was only expressed in 39% of cells vs. 58% in normal supratemporal conjunctiva (p0.05). Cytokeratin 19 was expressed by all cells cultured from normal conjunctival tissue.This study emphasizes the importance of using control tissue explanted from the correspondent conjunctival quadrant when studying proliferative disorders. Different pathogenesis may account for the differences in p63 expression between pterygium and LOC.

PURPOSE: Transforming growth factor beta-induced (TGFBI)-related dystrophies constitute the most common heritable forms of corneal dystrophy worldwide. However, other than the underlying genotypes of these conditions, a limited knowledge exists of the exact pathomechanisms of these disorders. This study expands on our previous research investigating dystrophic stromal aggregates, with the aim of better elucidating the pathomechanism of two conditions arising from the most common TGFBI mutations: granular corneal dystrophy type 1 (GCD1; R555W) and lattice corneal dystrophy type 1 (LCD1; R124C).METHODS: Patient corneas with GCD1 and LCD1 were stained with hematoxylin and eosin and Congo red to visualize stromal nonamyloid and amyloid deposits, respectively. Laser capture microdissection was used to isolate aggregates and extracted protein was analyzed by mass spectrometry. Proteins were identified and their approximate abundances were determined. Spectra of TGFBIp peptides were also recorded and quantified.RESULTS: In total, three proteins were found within GCD1 aggregates that were absent in the healthy control corneal tissue. In comparison, an additional 18 and 24 proteins within stromal LCD1 and Bowman's LCD1 deposits, respectively, were identified. Variances surrounding the endogenous cleavage sites of TGFBIp were also noted. An increase in the number of residues experiencing cleavage was observed in both GCD1 aggregates and LCD1 deposits.CONCLUSIONS: The study reveals previously unknown differences between the protein composition of GCD1 and LCD1 aggregates, and confirms the presence of the HtrA1 protease in LCD1-amyloid aggregates. In addition, we find mutation-specific differences in the processing of mutant TGFBIp species, which may contribute to the variable phenotypes noted in TGFBI-related dystrophies.

The purpose of this study was to compare grief among parents who had an adult child with schizophrenia and parents who had "lost" an adult child through death or a head injury that resulted in an organic personality disorder.Twenty-five parents from each group were studied. Self-report scales that assessed grief, depression, anxiety, and substance abuse were administered.There were significant differences with regard to grieving reactions and substance abuse. The parents with schizophrenic children had more ongoing grieving, and the parents of children with head injuries had more substance abuse.Parental loss of a child through schizophrenia leads to a pattern of chronic grief.

Purpose To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment. Methods siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5' rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting. Results The lead siRNA, with an IC(50) of thirty picomolar, showed no keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein. Conclusions A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms.

PURPOSE To investigate the effect of tear hyperosmolarity and signs of clinical ocular surface pathology on conjunctival goblet cell population. METHODS 111 participants were assessed using tear osmolarity (TO) measurements and a comprehensive selection of clinical ophthalmic tests. The resultant clinical database was assessed for evidence of patterns of composite increasing pathology. The total, filled, and empty goblet cell numbers were measured: total number of goblet cells as per cytokeratin 7 (CK7) immunofluorescence and number of filled goblet cells as per periodic acid Schiff's reagent (PAS) or lectin helix pomatia agglutinin (HPA). Goblet cell profile was correlated with composite clinical pathologic grades. RESULTS No significant correlation was found between TO and goblet cell number or function (as indicated by number of filled or unfilled goblet cells). Distinct composite clinical pathologic groups 0-IV with increasing pathology were created based on the frequency of positive pathologic signs, which adhered to the Dry Eye Workshop purported mechanism. Only in group IV was there significantly increased mean tear osmolarity of 344 mOsm/L (P < 0.000) along with significantly decreased empty goblet cell number (CK7+ and HPA-) compared to filled (CK7+ and HPA+, P = 0.000). When the number of filled goblet cells (PAS+) was analyzed there was significant increase in tear osmolarity for the two most severe grades; 3 and 4. CONCLUSIONS The goblet cell population does not appear to be affected by isolated tear hyperosmolarity. Hyperosmolarity when combined with other ocular surface pathology or inflammation alters the goblet cell population.

Numerous risk factors for necrotizing enterocolitis (NEC) including prematurity, bowel ischemia, pathogenic bacteria, and hyperosmolar feedings have been proposed. Recent studies have demonstrated feeding intolerance and bowel dysfunction in children with hypoalbuminemia. No association between hypoalbuminemia and NEC has been suggested. The records of 45 patients with NEC and complete documentation of prenatal and birth histories were reviewed. A control (CONT) group of 90 children matched for maternal age (+/- 1 year), parity, gestational age (+/- 1 week), birth weight (+/- 20 g), type of delivery, sex, race, type of initial feeding, and perinatal stress was compiled. While all other measured parameters were similar in the two groups, premorbid albumin was significantly lower in the patients who subsequently developed NEC (P less than .001). These data suggest that newborns with hypoalbuminemia may have an increased risk of developing NEC.

Accidental ingestion of hydrocarbons is an important cause of childhood poisoning. Due to the number of hydrocarbon products available as solvents, fuels, and cleaning agents, increased awareness is necessary on the part of health caretakers. The scope of complications involving the respiratory system in petroleum products ingestion is frequently overlooked. Physicians may thus apply standard therapeutic modalities used in treating common poisonings to the child who drank a petroleum distillate. Prompt recognition of presenting symptoms and understanding of pathophysiology are important to planning and providing treatment. The two cases of hydrocarbon ingestion reported in this paper illustrate the wide spectrum of problems associated with this condition. The pathophysiology, current management, and a review of the literature of hydrocarbon ingestion are presented.