a-non-invasive-way-to-isolate-and-phenotype-cells-from-the-conjunctiva

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

a-chronic-autoimmune-dry-eye-rat-model-with-increase-effector-memory

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

Singapore Eye Research Institute

Singapore Eye Research Insitute, A Member of SingHealth

DUKE-National University of Singapore Medical School

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders resulting from deficiency of imprinted gene expression from paternal or maternal chromosome 15q11-15q13, respectively. In humans, expression of the imprinted genes is under control of a bipartite cis-acting imprinting center (IC). Families with deletions causing PWS imprinting defects localize the PWS-IC to 4.3 kb overlapping with SNRPN exon 1. Families with deletions causing AS imprinting defects localize the AS-IC to 880 bp 35 kb upstream of the PWS-IC. We report two mouse mutations resulting in defects similar to that seen in AS patients with deletion of the AS-IC. An insertion/duplication mutation 13 kb upstream of Snrpn exon 1 resulted in lack of methylation at the maternal Snrpn promoter, activation of maternally repressed genes, and decreased expression of paternally repressed genes. The acquisition of a paternal epigenotype on the maternal chromosome in the mutant mice was demonstrated by the ability to rescue the lethality and growth retardation in a mouse model of a PWS imprinting defect. A second mutation, an 80-kb deletion extending upstream of the first mutation, caused a similar imprinting defect with variable penetrance. These results suggest that there is a mouse functional equivalent to the human AS-IC.

Mouse imprinting defect mutations that model Angelman syndrome.

Prader-Willi syndrome (PWS) is a neurogenetic disorder associated with recurrent genomic recombination involving low copy repeats (LCRs) located in the human chromosome 15q11-q13. Previous studies of PWS patients from Asia suggested that there is a higher incidence of deletion and lower incidence of maternal uniparental disomy (mUPD) compared to that of Western populations. In this report, we present genetic etiology of 28 PWS patients from Taiwan. Consistent with the genetic etiology findings from Western populations, the type II deletion appears to be the most common deletion subtype. Furthermore, the ratio of the two most common deletion subtypes and the ratio of the maternal heterodisomy to isodisomy cases observed from this study are in agreement with previous findings from Western populations. In addition, we identified and further mapped the deletion breakpoints in two patients with atypical deletions using array CGH (comparative genomic hybridization). Despite the relatively small numbers of patients in each subgroup, our findings suggest that the genomic architecture responsible for the recurrent recombination in PWS is conserved in Taiwanese of the Han Chinese heritage and Western populations, thereby predisposing chromosome 15q11-q13 to a similar risk of rearrangements.

Genetic studies of Prader-Willi patients provide evidence for conservation of genomic architecture in proximal chromosome 15q.

Migration of cells in the ocular surface underpins physiological wound healing as well as many human diseases. Transglutaminase (TG)-2 is a multifunctional cross-linking enzyme involved in the migration of skin fibroblasts and wound healing, however, its functional role in epithelial migration has not been evaluated. This study investigated the importance of TG-2 in a murine corneal wound healing model as well as the mechanistic role of TG-2 in the regulation of related biological processes such as cell adhesion and migration of cultured human corneal epithelial (HCE-T) cells. Corneal wound closure was delayed in homozygous TG-2 deleted mice compared to wild type mice. HCE-T cells that were knocked-down for TG-2 expression through stable expression of a short-hairpin (sh) RNA targeting TG-2, were delayed in closure of scratch wounds (48 compared to 12h in control cells expressing scrambled shRNA). TG-2 knockdown did not influence epithelial cell cycle progression or proliferation, rather, it led to reduced epithelial cell adhesion, spreading and velocity of migration. At the molecular level, TG-2 knockdown reduced phosphorylation of β-3 integrin at Tyr747, paxillin at Ser178, vinculin at Tyr822 and focal adhesion kinase at Tyr925 simultaneous with reduced activation of Rac and CDC42. Phosphorylation of paxillin at Ser178A has been shown to be indispensable for the migration of corneal epithelial cells (Kimura et al., 2008) [18]. TG-2 dependent β-3 integrin activation, serine-phosphorylation of paxillin, and Rac and CDC42 activation may thus play a key functional role in enhancing corneal epithelial cell adhesion and migration during wound healing.

Molecular mechanism of transglutaminase-2 in corneal epithelial migration and adhesion.

Pterygium is a fibrovascular growth on the ocular surface with corneal tissue destruction, matrix degradation and varying extents of chronic inflammation. To facilitate investigation of pterygium etiology, we immortalized pterygium fibroblast cells and profiled their global transcript levels compared to primary cultured cells.

Comparison of gene expression profiles in primary and immortalized human pterygium fibroblast cells.

Pterygium is a fibrovascular proliferative condition of the ocular surface with no known pathological mechanism. This condition affects vision due to dry eyes, astigmatism or physical occlusion of the visual axis for severe cases. The only definitive treatment for this condition is surgical excision. Interestingly, it is a lesion that may be related to UV radiation and elaboration of proteases. Conjunctivochalasis is a dry eye related condition that is exemplified by excessive conjunctiva or the mucous membrane of the front of the eye around the cornea. Both pterygium and conjunctivochalasis are associated with elaboration of matrix metalloproteinases as well as inflammatory cytokines. We propose that under specific conditions, conjunctivochalasis in the nasal part of the conjunctiva can progress to pterygium. The progression of conjunctivochalasis to pterygium may be related to special kinds of oxidative or inflammatory damage that affects only the part of the loose conjunctival tissue adjacent to the cornea. Protease expressed may then breakdown the conjunctival and corneal epithelium causing the head of pterygium to be very adherent to the cornea. This explains the fact that surgically excised pterygium tissue has stromal tissue enclosed by epithelia on both surfaces. In addition, it explains the existence of a surgical plane when an instrument is passed under the neck of the pterygium tissue but not at the apex. The implications of this hypothesis are first, treatment should be directed to the protection of conjunctivochalasis before it transforms to pterygium. This may be achieved by anti-inflammatory measures, anti-protease treatment, or preventing the triggering of the changes at the head of pterygium, such as avoidance of sunlight. Second, during resection of pterygium, it may not be necessary to resect the pterygium too extensively away from the cornea, since this effectively removes relatively normal conjunctiva.

Conjunctivochalasis is the precursor to pterygium.

Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration.

Rho GTPases and regulation of cell migration and polarization in human corneal epithelial cells.

Pterygium is a wing shaped fibrovascular growth on the ocular surface, characterized by fibrosis, angiogenesis, extracellular matrix remodeling, and inflammatory infiltrates. Epidemiologic studies have linked pterygium formation to various chronic inflammatory conditions, such as ultraviolet radiation, sawdust exposure, and dry eye disease. The purpose of this study is to identify proteins that are differentially expressed in primary pterygium by using a combination of gene microarray and proteomic platforms.

Evaluation of global differential gene and protein expression in primary Pterygium: S100A8 and S100A9 as possible drivers of a signaling network.

Pterygium is a triangular shaped ocular fibrous surface lesion growing from conjunctiva towards central cornea, causing ocular irritation, astigmatism, and visual disturbance. The condition is characterized by epithelial proliferation, fibrovascular growth, chronic inflammation, and prominent extracellular matrix remodeling. Studies have suggested that aberrant extracellular proteins secreted by fibroblasts lead to abnormal matrix production and tissue invasion contributing to the development of the disease. In this study, secreted proteins collected from paired pterygium and conjunctival fibroblasts in vitro were identified and quantified by LC-MS iTRAQ-based analysis, in which 433 proteins common to all samples were identified. Among these proteins, 48.0% (208) were classified as classically secreted proteins, 17.1% (74) were exported out of the cells via non-classical secretion pathways, and 31.2% (135) were exosome proteins. A minority (3.7%) was not previously known to be secreted, or might be contaminants. 31 and 27 proteins were found up- or down-regulated in the conditioned media of pterygium fibroblasts relative to the media of control cells, respectively. Molecular function analysis showed that these proteins either belonged to catalytic proteins, structural molecules or were involved with receptor activities and protein binding. Further pathway analysis revealed that these proteins were involved in ECM-receptor interaction, focal adhesion, cancer-related, p53 signaling, complement and coagulation, and TGF-beta signaling pathways. These molecules identified may serve as extracellular ligands to activate intracellular pathways, possibly serving as potential therapeutic targets.

In vitro secretomics study of pterygium-derived fibroblasts by iTRAQ-based quantitative proteomics strategy.

Non-recirculating resident memory (TRM) and recirculating T cells mount vigorous immune responses to both self and foreign antigens in barrier tissues like the skin, lung and gastrointestinal tract. Using impression cytology followed by flow cytometry we identified two TRM subsets and four recirculating T-subsets in the healthy human ocular surface. In dry eye disease, principal component analysis (PCA) revealed two clusters of patients with distinct T-cell signatures. Increased conjunctival central memory and naïve T cells characterized Cluster-1 patients, and increased CD8(+) TRMs and CD4(+) recirculating memory T cells characterized Cluster-2 patients. Interestingly these T-cell signatures are associated with different clinical features: the first signature correlated with increased ocular redness, and the second with reduced tear break up times. These findings open the door to immune-based characterization of dry eye disease and T-subset specific immunotherapies to suppress T-subsets involved in disease. They may also help with patient stratification during clinical trials of immunomodulators.

Tissue resident memory T cells in the human conjunctiva and immune signatures in human dry eye disease.

The aim of this study was to evaluate effects of Shen-Cao granules for the prevention of thrombocytopenia caused by anticancer chemotherapy.

Shen-Cao granules formulated based on traditional Chinese medicine alleviates bone marrow suppression caused by platinum-based anticancer reagents.

To study the effects of Qingzao Runfei Huazhuo Xingxue decoction (QRHXD) on inflammatory reaction and histopathology in mice with PM2.5-induced pulmonary injury, and to approach the possible mechanism of prevention and treatment of traditional Chinese medicine on lung injury induced by haze.

[Intervention effect of Qingzao Runfei Huazhuo Xingxue decoction on PM2.5-induced pulmonary injury in mice].

To evaluate the basal and possibly stimulated expression of interleukin (IL)-17 in the context of the ocular surface and potential downstream effects.

Expression, Regulation, and Effects of Interleukin-17f in the Human Ocular Surface.

This study aimed at finding the relationship between the level of expression of the PRR11 protein in pancreatic carcinoma, and the clinical characteristics of the tumor. PCR technique was used to analyze the expression levels of the PRR11 gene in 38 samples from pancreatic cancer patients and 10 samples from normal pancreatic tissues. Western blot analysis and immunohistochemistry were used to measure the expression of the PRR11. Additionally, the migration ability of cancerous cells expressing PRR11 and those with inhibited expression were compared using a wound healing assay. Finally, the relationships between the expression level of PRR11 protein and variables such as tumor size, tumor invasion, TNM stages, and the overall survival time of patients with pancreatic cancer were calculated. Our results showed the expression level of the PRR11 gene in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues. The detection of PRR11 protein in cancer tissues versus normal tissues was 78.9 (30/38) vs. 0 (0/10), respectively. The western blot results confirmed this by showing a significantly higher level of expression of the PRR11 protein in pancreatic cancer tissues than in normal tissues (P<0.05). Inhibiting the expression of PRR11 in cancer cells reduced the migration ability of the cells. Finally, the expression of PRR11 was positively correlated with the invasion, disease and tissue differentiation stages of the pancreatic cancer. By comparing clinical data and expression patterns in patients, we found the survival rate in those expressing the PRR11 protein by immunohistochemistry to be lower than in those with tissues negative to the PRR11 protein. Our results show, the expression of PRR11 protein in pancreatic cancer is closely related to the development of the cancer and a poor prognosis. These findings provide a theoretical and experimental basis for approaching the diagnosis and treatment of pancreatic cancer using PRR11 as a molecular target.

ArticleTotal : 2	Year
Publication title Cited by 5	2017
Publication title Cited by 9	2017

Article	Year
Mouse imprinting defect mutations that model Angelman syndrome. Genesis (New York, N.Y. : 2000)\| PubMed ID: 16397868	2006
Genetic studies of Prader-Willi patients provide evidence for conservation of genomic architecture in proximal chromosome 15q. Annals of human genetics\| PubMed ID: 21198515	2011
Molecular mechanism of transglutaminase-2 in corneal epithelial migration and adhesion. Biochimica et biophysica acta\| PubMed ID: 23466867	2013
Comparison of gene expression profiles in primary and immortalized human pterygium fibroblast cells. Experimental cell research\| PubMed ID: 24012806	2013
Conjunctivochalasis is the precursor to pterygium. Medical hypotheses\| PubMed ID: 24053977	2013
Rho GTPases and regulation of cell migration and polarization in human corneal epithelial cells. PloS one\| PubMed ID: 24130842	2013
Evaluation of global differential gene and protein expression in primary Pterygium: S100A8 and S100A9 as possible drivers of a signaling network. PloS one\| PubMed ID: 24825356	2014
In vitro secretomics study of pterygium-derived fibroblasts by iTRAQ-based quantitative proteomics strategy. Experimental eye research\| PubMed ID: 27725197	2016
Tissue resident memory T cells in the human conjunctiva and immune signatures in human dry eye disease. Scientific reports\| PubMed ID: 28345628	2017
Shen-Cao granules formulated based on traditional Chinese medicine alleviates bone marrow suppression caused by platinum-based anticancer reagents. Medicine\| PubMed ID: 28489759	2017
[Intervention effect of Qingzao Runfei Huazhuo Xingxue decoction on PM2.5-induced pulmonary injury in mice]. Zhonghua wei zhong bing ji jiu yi xue\| PubMed ID: 28524038	2017
Expression, Regulation, and Effects of Interleukin-17f in the Human Ocular Surface. Ocular immunology and inflammation\| PubMed ID: 28537453	2017
Expression of PRR11 protein and its correlation with pancreatic cancer and effect on survival. Oncology letters\| PubMed ID: 28599413	2017

Aihua Hou

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