Characterisation of limbal stem cells

PhD ThesisThe cornea is the anterior most structure at the front of the eye and its most important function is to transmit light to the retina for a clear vision. The external surface of the cornea is composed of an epithelium which is continuous with the surrounding conjunctiva. The corneal limbus forms the narrow transition zone between the corneal and conjunctival epithelia and is believed to harbour the cornea stem cells. Limbal stem cell (LSC) deficiency (LSCD) is a painful and blinding condition of the eye. The recent technique of ex vivo expansion of healthy autologous limbal epithelia for transplantation is the mainstay of cellular therapy for this condition. Successful outcome of ex vivo expanded LSC transplantation is dependent on the quality of the transplanted tissues. The main aims of this thesis are; i) to characterise human LSC from both primary LEC (LEC) and from a human telomerase-immortalised corneal epithelial cell (HTCEC) line, ii) to enrich for LSC by utilising the side population (SP) discrimination assay. Further, adult stem cells are promising candidates for promoting donor-specific tolerance and adult stem cells have immunosuppressive mechanisms to protect them from immunological reactions which are damaging to the survival of the transplanted tissues. Therefore, another aim of the study was iii) to investigate the immunobiological aspects of LSC including HLA expression and cellular migration which can promote the success of cellular transplantation. LSC markers were characterised in both LEC and HTCEC. Using an optimised protocol, SP were identified in LEC and HTCEC. Isolated limbal mesenchymal stem cells (MSC) fulfil the minimum requirement of a human MSC. These limbal MSC (LMSC) exhibited plasticity, could maintain the expression of limbal markers and demonstrated viable growth on a biological substrate, qualities making them a suitable alternative to cultured limbal explants for clinical transplantation. SP cells in HTCEC and LMSC expressed known common limbal markers, stem cell antigens and the chemokine CXCR4. The presence of CXCR4 and CXCL12-mediated cellular migration were demonstrated in HTCEC. Further, HTCEC constitutively expressed HLA Class I antigens, while HLA Class II expressions were induced by Interferon-γ stimulation. In this study, HTCEC HLA-typing was presented and that HTCEC were in many ways comparable to LEC, therefore suitable as a LSC replacement or as a robust model for further understanding of LSC biology.Ministry of Education, Malaysia and my employe

A study on the capability of frequency doubling perimetry in the detection of visual field abnormalities in primary open angle glaucoma patients.

Frequency Doubling Peri1netry (FDP) has become a widely used technique for both screening and follow up of glaucomatous field loss. It is thought to be sensitive and superior in the detection of early visual field loss Objective: To determine the capability ofFDP in the detection of visual field abnormalities in the primary open angle glaucoma (POAG) patients. Design: Prospective, comparative, cross sectional study. Method: 150 POAG patients underwent both FDP in screening and threshold mode 30- 2 and Humphrey Visual Field (HVF) 30-2 tests in Eye clinic Hospital Universiti Sains Malaysia (HUSM). All patients had a comprehensive ophthalmology assessment. Data analysis including sensitivity, specificity, area under the receiver operating characteristic (ROC) curve and kappa agreement between FDP and HVF was performed using SPSS system version 12.0. Results: Data from 120 POAG patients were analyzed following inclusion, exclusion criteria and reliability of the visual field testing. For FDP in screening mode, when "at least 1 missed point" \Vas used as the definition for abnormal FDP, the sensitivity was high between 92.2% to 96.2% and specificity was between 24.2% to 50.0%. When "2 or more missed points", was used as definition for abnormal visual field, the sensitivity was 89.5% to 95.0% and specificity between 33.3% to 47.0% was achieved. For FDP in threshold n1ode and HVF with various definitions for abnormal visual field, the sensitivity was between 83.5% to 100% and specificity between 0.0% to 47.0%. Kappa value for FDP and HVF was between 0.155 to 0.391 which showed poor to slight agreement. Conclusion: FDP showed high sensitivity but low specificity in the detection of visual field abnormalities among our patients. The new classification for severity of glaucoma \Vith Glaucoma Scoring System 2 (GSS2) and Frequency Doubling Technology Scoring System 2 (FDT SS2) was able to provide immediate and reliable classification for both severity and characteristic of visual field

Preliminary study to compare the prediction error of post operative refraction in pediatric cataract surgery between 2 different intraocular lens power calculation formula

Introduction: The treatment of paediatric cataracts has progressed tremendously in the past 15 to 20 years. There is a growing trend towards intraocular lens implantation in infants and younger children whose eyes are still undergoing rapid growth and refractive changes. Objective: This study is intended to assess the predictability of desired refractive outcomes at 3 month postoperative period in paediatric patients undergoing cataract surgery with primary placement of an intraocular lens. Methodology: This randomized interventional study of 31 eyes (24 patients) that successfully underwent cataract surgery and intraocular lens implantations. All patients were 12 years old and below. Intraocular lens power calculations were made using either SRK II or Modified Formula For Paediatric IOL Calculation. The postoperative refractive outcome was taken as the spherical equivalent of the refraction at 3 month postoperative follow-up. The prediction error was taken as the absolute difference between the predicted and the actual refraction. The data were analysed to compare the mean prediction error between SRK II and Modified Formula and evaluate the predictability. Results: The mean prediction error in the SRK II group was 1.03 D (SD, 0.69 D) while in Modified Formula 1.14 D (SD, 1.19 D). The SRK II group showed lower prediction error of 0.11 D compared to Modified Formula group, but this was not statistically significant (p > 0.05). There were 3 eyes (18.75%) in SRK II group achieved accurate predictability where the refraction postoperatively was within ± 0.5 D from predicted refraction compared to 7 eyes (46.67%) in the Modified Formula group. However the difference of the predictability between the two formulas was also not statistically significant (p = 0.097). Conclusion: The prediction error and the accuracy of predictability of postoperative refraction in paediatric cataract surgery are comparable between SRK II and Modified Formula. The existence of the Modified Formula provides an alternative to the ophthalmologist for intraocular lens calculation in paediatric patients

Human wharton’s jelly-derived mesenchymal stem cells minimally improve the growth kinetics and cardiomyocyte differentiation of aged murine cardiac c-kit cells in in vitro without rejuvenating effect

Cardiac c-kit cells show promise in regenerating an injured heart. While heart disease commonly affects elderly patients, it is unclear if autologous cardiac c-kit cells are functionally competent and applicable to these patients. This study characterised cardiac c-kit cells (CCs) from aged mice and studied the effects of human Wharton’s Jelly-derived mesenchymal stem cells (MSCs) on the growth kinetics and cardiac differentiation of aged CCs in vitro. CCs were isolated from 4-week- and 18-month-old C57/BL6N mice and were directly co-cultured with MSCs or separated by transwell insert. Clonogenically expanded aged CCs showed comparable telomere length to young CCs. However, these cells showed lower Gata4, Nkx2.5, and Sox2 gene expressions, with changes of 2.4, 3767.0, and 4.9 folds, respectively. Direct co-culture of both cells increased aged CC migration, which repopulated 54.6 ± 4.4% of the gap area as compared to aged CCs with MSCs in transwell (42.9 ± 2.6%) and CCs without MSCs (44.7 ± 2.5%). Both direct and transwell co-culture improved proliferation in aged CCs by 15.0% and 16.4%, respectively, as traced using carboxyfluorescein succinimidyl ester (CFSE) for three days. These data suggest that MSCs can improve the growth kinetics of aged CCs. CCs retaining intact telomere are present in old hearts and could be obtained based on their self-renewing capability. Although these aged CCs with reduced growth kinetics are improved by MSCs via cell–cell contact, the effect is minimal

Tualang honey improves human corneal epithelial progenitor cell migration and cellular resistance to oxidative stress in vitro

Stem cells with enhanced resistance to oxidative stress after in vitro expansion have been shown to have improved engraftment and regenerative capacities. Such cells can be generated by preconditioning them with exposure to an antioxidant. In this study we evaluated the effects of Tualang honey (TH), an antioxidant-containing honey, on human corneal epithelial progenitor (HCEP) cells in culture. Cytotoxicity, gene expression, migration, and cellular resistance to oxidative stress were evaluated. Immunofluorescence staining revealed that HCEP cells were holoclonal and expressed epithelial stem cell marker p63 without corneal cytokeratin 3. Cell viability remained unchanged after cells were cultured with 0.004, 0.04, and 0.4% TH in the medium, but it was significantly reduced when the concentration was increased to 3.33%. Cell migration, tested using scratch migration assay, was significantly enhanced when cells were cultured with TH at 0.04% and 0.4%. We also found that TH has hydrogen peroxide (H2O2) scavenging ability, although a trace level of H2O2 was detected in the honey in its native form. Preconditioning HCEP cells with 0.4% TH for 48 h showed better survival following H2O2-induced oxidative stress at 50 µM than untreated group, with a significantly lower number of dead cells (15.3 ± 0.4%) were observed compared to the untreated population (20.5 ± 0.9%, p<0.01). Both TH and ascorbic acid improved HCEP viability following induction of 100 µM H2O2, but the benefit was greater with TH treatment than with ascorbic acid. However, no significant advantage was demonstrated using 5-hydroxymethyl-2-furancarboxaldehyde, a compound that was found abundant in TH using GC/MS analysis. This suggests that the cellular anti-oxidative capacity in HCEP cells was augmented by native TH and was attributed to its antioxidant properties. In conclusion, TH possesses antioxidant properties and can improve cell migration and cellular resistance to oxidative stress in HCEP cells in vitro

Prediction error and accuracy of intraocular lens power calculation in pediatric patient comparing SRK II and Pediatric IOL Calculator

<p>Abstract</p> <p>Background</p> <p>Despite growing number of intraocular lens power calculation formulas, there is no evidence that these formulas have good predictive accuracy in pediatric, whose eyes are still undergoing rapid growth and refractive changes. This study is intended to compare the prediction error and the accuracy of predictability of intraocular lens power calculation in pediatric patients at 3 month post cataract surgery with primary implantation of an intraocular lens using SRK II versus Pediatric IOL Calculator for pediatric intraocular lens calculation. Pediatric IOL Calculator is a modification of SRK II using Holladay algorithm. This program attempts to predict the refraction of a pseudophakic child as he grows, using a Holladay algorithm model. This model is based on refraction measurements of pediatric aphakic eyes. Pediatric IOL Calculator uses computer software for intraocular lens calculation.</p> <p>Methods</p> <p>This comparative study consists of 31 eyes (24 patients) that successfully underwent cataract surgery and intraocular lens implantations. All patients were 12 years old and below (range: 4 months to 12 years old). Patients were randomized into 2 groups; SRK II group and Pediatric IOL Calculator group using envelope technique sampling procedure. Intraocular lens power calculations were made using either SRK II or Pediatric IOL Calculator for pediatric intraocular lens calculation based on the printed technique selected for every patient. Thirteen patients were assigned for SRK II group and another 11 patients for Pediatric IOL Calculator group. For SRK II group, the predicted postoperative refraction is based on the patient's axial length and is aimed for emmetropic at the time of surgery. However for Pediatric IOL Calculator group, the predicted postoperative refraction is aimed for emmetropic spherical equivalent at age 2 years old. The postoperative refractive outcome was taken as the spherical equivalent of the refraction at 3 month postoperative follow-up. The data were analysed to compare the mean prediction error and the accuracy of predictability of intraocular lens power calculation between SRK II and Pediatric IOL Calculator.</p> <p>Results</p> <p>There were 16 eyes in SRK II group and 15 eyes in Pediatric IOL Calculator group. The mean prediction error in the SRK II group was 1.03 D (SD, 0.69 D) while in Pediatric IOL Calculator group was 1.14 D (SD, 1.19 D). The SRK II group showed lower prediction error of 0.11 D compared to Pediatric IOL Calculator group, but this was not statistically significant (p = 0.74). There were 3 eyes (18.75%) in SRK II group achieved acccurate predictability where the refraction postoperatively was within ± 0.5 D from predicted refraction compared to 7 eyes (46.67%) in the Pediatric IOL Calculator group. However the difference of the accuracy of predictability of postoperative refraction between the two formulas was also not statistically significant (p = 0.097).</p> <p>Conclusions</p> <p>The prediction error and the accuracy of predictability of postoperative refraction in pediatric cataract surgery are comparable between SRK II and Pediatric IOL Calculator. The existence of the Pediatric IOL Calculator provides an alternative to the ophthalmologist for intraocular lens calculation in pediatric patients. Relatively small sample size and unequal distribution of patients especially the younger children (less than 3 years) with a short time follow-up (3 months), considering spherical equivalent only.</p

Anti-inflammatory and antioxidant effects of Tualang honey in alkali injury on the eyes of rabbits: Experimental animal study

<p>Abstract</p> <p>Background</p> <p>Alkali injury is one of the most devastating injuries to the eye. It results in permanent unilateral or bilateral visual impairment. Chemical eye injury is accompanied by an increase in the oxidative stress. Anti-inflammatory and antioxidant agents play a major role in the treatment of chemical eye injuries. The purpose of this study is to evaluate the anti-inflammatory (clinical and histopathological) and antioxidant effects of Tualang honey versus conventional treatment in alkali injury on the eyes of rabbits.</p> <p>Methods</p> <p>A preliminary study was carried out prior to the actual study to establish the alkali chemical injury on rabbit's cornea and we found that alkali chemical injury with 2 N NaOH showed severe clinical inflammatory features. In actual study, alkali injury with 2 N NaOH was induced in the right eye of 10 New Zealand White rabbits' cornea. The rabbits were divided into two groups, Group A was given conventional treatment and Group B was treated with both topical and oral Tualang honey. Clinical inflammatory features of the right eye were recorded at 12 hours, 24 hours, 72 hours, 5^thday and 7^thday post induction of alkali burn on the cornea. The histopathological inflammatory features of the right corneas of all rabbits were also evaluated on day-7. The level of total antioxidant status and lipid peroxidation products in the aqueous humour, vitreous humour and serum at day-7 were estimated biochemically. Fisher's Exact, Chi-Square and Mann-Whitney test were used to analyse the data.</p> <p>Results</p> <p>There was no statistically significant difference in clinical inflammatory features (p > 0.05) between honey treated and the conventional treated group at different times of examination. Histopathological examination of the cornea showed the number of polymorphonuclear leucocytes was below 50 for both groups (mild grade). There was also no significant difference in the level of total antioxidant status as well as lipid peroxidation products in aqueous humour (p = 0.117, p = 0.382 respectively), vitreous humour (p = 0.917, p = 0.248 respectively) and serum (p = 0.917, p = 0.332 respectively) between honey treated and the conventional treated group.</p> <p>Conclusion</p> <p>Tualang honey has almost the equal effects when compared with the conventional treatment in treating alkali injury on rabbit's eye. Future research with more number of rabbits and control group is warranted to explore the anti-inflammatory and antioxidant effects of Tualang honey.</p

Intravitreal triamcinolone versus laser photocoagulation as a primary treatment for diabetic macular oedema - a comparative pilot study

Abstract Background Diabetic macular oedema is the leading causes of blindness. Laser photocoagulation reduces the risk of visual loss. However recurrences are common and despite laser treatment, patients with diabetic macular oedema experienced progressive loss of vision. Stabilization of the blood retinal barrier introduces a rationale for intravitreal triamcinolone treatment in diabetic macular oedema. This study is intended to compare the best corrected visual acuity (BCVA) and the macular oedema index (MEI) at 3 month of primary treatment for diabetic macular oedema between intravitreal triamcinolone acetonide (IVTA) and laser photocoagulation. Methods This comparative pilot study consists of 40 diabetic patients with diabetic macular oedema. The patients were randomized into two groups using envelope technique sampling procedure. Treatment for diabetic macular oedema was based on the printed envelope technique selected for every patient. Twenty patients were assigned for IVTA group (one injection of IVTA) and another 20 patients for LASER group (one laser session). Main outcome measures were mean BCVA and mean MEI at three months post treatment. The MEI was quantified using Heidelberg Retinal Tomography II. Results The mean difference for BCVA at baseline [IVTA: 0.935 (0.223), LASER: 0.795 (0.315)] and at three months post treatment [IVTA: 0.405 (0.224), LASER: 0.525 (0.289)] between IVTA and LASER group was not statistically significant (p = 0.113 and p = 0.151 respectively). The mean difference for MEI at baseline [IVTA: 2.539 (0.914), LASER: 2.139 (0.577)] and at three months post treatment [IVTA: 1.753 (0.614), LASER: 1.711 (0.472)] between IVTA and LASER group was also not statistically significant (p = 0.106 and p = 0.811 respectively). Conclusions IVTA demonstrates good outcome comparable to laser photocoagulation as a primary treatment for diabetic macular oedema at three months post treatment. Trial Registration ISRCTN05040192 (http://www.controlled-trial.com)</p

A lean case study on managing interlibrary loan and document delivery service in an academic library

Introduction: Interlibrary loan and document delivery (ILL/DD) is an essential service provided by academic libraries to support research activities, publication and academic programmes. ILL/DD service capitalises on resource sharing or library networking by providing library materials that are not available at the host library. The Lean Six Sigma method was applied to identify the root problems. A construction of a web-based system branded as “Request Book and Article System (RBAS)” soon followed as an intervention. A retrospective study was conducted from March 2020 to August 2021 (16 months) to evaluate the key indicators of the service. Customer feedback on service quality was also surveyed. The majority of request was for articles, 649 (87.5%), followed by book request 84 (11.3%) and ebook, 9 (1.2%). Turnaround time fell within 24 hours for the majority of requests, 494 (68.4%). The highest number was from article request category, 460 (71.8%). A high proportion of users (78%) deemed the service as “high quality”. The use of a lean approach and the development of a homegrown system have improved the ILL/DD service at our library. Here we share lean practices in ILL/DD management, focusing on transforming a manual method to a digital homegrown system development and its advantages