Limbal stem cells (LSCs) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell transplantations have contributed substantially to ...the treatment of the visually disabling condition known as LSC deficiency. In this protocol, we describe a method of establishing human limbal epithelial cell cultures by a feeder-free explant culture technique using a small limbal biopsy specimen and human amniotic membrane (hAM) as the culture substrate. This protocol is free of animal-derived products and involves the use of human recombinant growth factors. In addition, the recombinant cell dissociation enzyme TrypLE is used to replace trypsin and autologous serum replaces FBS. It takes approximately 2 weeks to establish a confluent monolayer from which approximately 3 x 10(6) cells can be harvested. This procedure can be adopted for both basic research purposes and clinical applications.
Dry eye syndrome is a multifactorial chronic disabling disease mainly caused by the functional disruptions in the lacrimal gland. The treatment involves palliation like ocular surface lubrication and ...rehydration. Cell therapy involving replacement of the gland is a promising alternative for providing long-term relief to patients. This study aimed to establish functionally competent lacrimal gland cultures in-vitro and explore the presence of stem cells in the native gland and the established in-vitro cultures.
Fresh human lacrimal gland from patients undergoing exenteration was harvested for cultures after IRB approval. The freshly isolated cells were evaluated by flow cytometry for expression of stem cell markers ABCG2, high ALDH1 levels and c-kit. Cultures were established on Matrigel, collagen and HAM and the cultured cells evaluated for the presence of stem cell markers and differentiating markers of epithelial (E-cadherin, EpCAM), mesenchymal (Vimentin, CD90) and myofibroblastic (α-SMA, S-100) origin by flow cytometry and immunocytochemistry. The conditioned media was tested for secretory proteins (scIgA, lactoferrin, lysozyme) post carbachol (100 µM) stimulation by ELISA.
Native human lacrimal gland expressed ABCG2 (mean±SEM: 3.1±0.61%), high ALDH1 (3.8±1.26%) and c-kit (6.7±2.0%). Lacrimal gland cultures formed a monolayer, in order of preference on Matrigel, collagen and HAM within 15-20 days, containing a heterogeneous population of stem-like and differentiated cells. The epithelial cells formed 'spherules' with duct like connections, suggestive of ductal origin. The levels of scIgA (47.43 to 61.56 ng/ml), lysozyme (24.36 to 144.74 ng/ml) and lactoferrin (32.45 to 40.31 ng/ml) in the conditioned media were significantly higher than the negative controls (p<0.05 for all comparisons).
The study reports the novel finding of establishing functionally competent human lacrimal gland cultures in-vitro. It also provides preliminary data on the presence of stem cells and duct-like cells in the fresh and in-vitro cultured human lacrimal gland. These significant findings could pave way for cell therapy in future.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ocular burns can damage the corneal epithelial stem cells located at the limbus. This study evaluated the efficacy of xeno-free autologous cell-based treatment of limbal stem cell deficiency.
This ...retrospective study included 200 patients, above 8 years of age, with clinically diagnosed unilateral total limbal stem cell deficiency due to ocular surface burns treated between 2001 and 2010. A small limbal biopsy was obtained from the unaffected eye. The limbal epithelial cells were expanded ex vivo on human amniotic membrane for 10-14 days using a xeno-free explant culture system. The resulting cultured epithelial monolayer and amniotic membrane substrate were transplanted on to the patient's affected eye. Postoperative corneal surface stability, visual improvement and complications were objectively analysed.
A completely epithelised, avascular and clinically stable corneal surface was seen in 142 of 200 (71%) eyes at a mean follow-up of 3 ± 1.6 (range: 1-7.6) years. A two-line improvement in visual acuity, without further surgical intervention, was seen in 60.5% of eyes. All donor eyes remained healthy.
Autologous cultivated limbal epithelial transplantation using a xeno-free explant culture technique was effective in long-term restoration of corneal epithelial stability and improvement of vision in eyes with ocular surface burns.
Purpose
Lacrimal gland (LG) dysfunction leading to dry eye syndrome (DES) is an important cause of ocular morbidity. One of the potential and promising long-term management therapies for restoration ...of LG function could be transplantation of autologous ex vivo expanded stem cells. The present study was aimed at exploring the 2D and 3D cultures of human LG, identifying inherent stem cells and evaluating their secretory potential.
Methods
Fresh human lacrimal gland (HuLG) (
n
= 5) from patients undergoing therapeutic exenteration was harvested after ethical approval and informed consent. The gland was enzymatically digested and the isolated cells plated in Hepato-STIM media supplemented with l-glutamine, epidermal growth factor, fibroblast growth factor, and N-2 supplement. The native HuLG and the cultured spheres (
DIV
14-16) were evaluated for presence of stem cells (CD117 expression, quiescence, BrdU label retention, cell cycle, colony forming efficiency) and differentiation (secretion of tear proteins).
Results
Under the established culture conditions, suspension 3D cultures of human “lacrispheres” could be maintained and propagated for 3–4 weeks. The spheres consist of both acinar as well as ductal cells with evidence of stem cells (0.8 ± 0.05% CD117
+
cells), BrdU label retention (9.31 ± 0.41%), G0/G1 profile similar to native lacrimal cells at isolation (76.9 versus 79.9%) and colony forming units (3.1%). The lacrispheres also secreted quantifiable levels of tear proteins (lysozyme, lactoferrin, scIgA) into the conditioned media.
Conclusion
The study provides promising, first-of-its-kind evidence for the generation of lacrispheres from fresh HuLG, with enriched population of stem cells and secretory competent differentiated cells. The dual properties of these spheres make them a highly suitable source of transplantable cells for restoring the structure and function of damaged lacrimal gland.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The human lacrimal gland is an essential component of the lacrimal functional unit (LFU). Any perturbation of this unit can lead to the debilitating morbid condition called the dry eye syndrome ...(DES). The current line of therapy available for dry eye remains supportive and palliative with the patient being dependent on life long and frequent administration of lubricating eye drops. Even advanced therapies like punctual plugs, cyclosporine B administration, and salivary gland auto-transplantation have led to a limited success. Under these scenarios, the option of cell based therapy needs to be explored to provide better and long term relief to these patients. This review gives an overview of the efforts in lacrimal gland regeneration and examines the past and ongoing research in cell based therapies in animals as well as human lacrimal gland cultures. The authors discuss their first of its kind functionally viable human lacrimal gland in vitro culture system from fresh exenteration specimens. A brief overview of research in near future and the potential implications of lacrimal gland regenerative therapies have been discussed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Despite advances in orbital radiotherapy (XRT), a significant proportion of patients develop ophthalmic complication like dry eye syndrome (DES). The study evaluates the prevalence of aqueous ...deficient DES (ADDE) and lacrimal gland (LG) changes through histologic evaluation and ex-vivo expansion potential postorbital XRT.
With the approval of the institutional review board, medical records of patients who underwent orbital XRT as management protocol were reviewed for evidence of ADDE using DEWS (Dry Eye Workshop) 2007 criteria (
= 51). HuLG was harvested from three of these patients who underwent subsequent orbital exenteration and used for histological studies/ex-vivo culture.
ADDE was noted in 47.07% of the patients, status postorbital XRT, with a prediction of nearly 50% developing it within 0.5 to 2.9 years. ADDE severity was grade 2 (18%), grade 3 (14%), and grade 4 (17%). Other comorbidities were radiation retinopathy (33.4%), radiation-induced cataract (24.9%), and radiation keratopathy (20.8%). Multivariate and univariate analysis showed that fraction of radiation and dose of radiation/fraction were significant risk factors; male gender and young age were protective factors. The post-XRT exenterated HuLG showed near-total effacement of histoarchitecture with intra/periductal and intra/interlobular fibrosis, loss of acini, and reduced secretory activity. The potential of the LG to expand and grow in culture was impaired with loss of stem cells as compared to normal HuLG.
This study documents that orbital-XRT is associated with morphological and functional loss of lacrimal function in nearly 50% of the patients with a prediction of two-third developing ADDE by the end of 5 years.
The study provides objective clinical evidence for DES development due to architectural/functional damage to the LG postorbital XRT. Based on recent findings that the LG can be cultured in-vitro, with preservation of stem cells and secretory potential, it would be logical to harvest a portion of LG before radiation, and expand and transplant it to rescue the damaged gland if indicated.
A Survey on Filtering Techniques for Recommendation System Pereira, Janisa Gosbel; Tiwari, Shubha; Ajoy, Sheetal
2020 IEEE International Symposium on Sustainable Energy, Signal Processing and Cyber Security (iSSSC),
2020-Dec.-16
Conference Proceeding
The advancement of technology in recent years have caused a tremendous development in the ecommerce industry. With the growth in ecommerce, recommendation systems have gained importance in research. ...The recommendation system is based on different filtering techniques to provide appropriate personalized suggestions to the user. In this paper we focus on the analysis of the commonly used filtering techniques of recommendation system and provide a comparison of these techniques.
We aimed to study the impression cytology (IC) of the ocular surface in eyes with vernal keratoconjunctivitis (VKC) and clinical evidence of limbal stem cell deficiency (LSCD).
This is a prospective ...comparative study.
This study included 78 eyes of 40 patients with VKC. Limbal stem cell deficiency was diagnosed clinically based on the presence of corneal findings such as dull irregular epithelial reflex, superficial neovascularization, conjunctivalization, and loss of limbal palisades of Vogt. The study group consisted of 28 eyes of 15 patients with clinically diagnosed LSCD and control group of 50 eyes of 25 patients without LSCD. Conjunctival and corneal IC was done in all eyes. Presence of goblet cells in the corneal samples on IC was considered confirmatory of LSCD.
Compared with controls, patients with LSCD were older and had longer duration of disease. On IC, goblet cells were present on the cornea in 53.6% of eyes with clinically diagnosed LSCD and in none of the control eyes (P < 0.0001). Clinically diagnosed LSCD in study eyes correlated with cytologic findings of greater conjunctival squamous metaplasia, decreased conjunctival goblet cells, greater corneal cell metaplasia, and increased inflammation as compared with control eyes.
Most of the eyes with VKC and clinical evidence of LSCD have cytologic evidence of LSCD with goblet cells on the cornea.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A huge amount of data is available on the web. Several users tend to access the data available on the internet. This makes it difficult to understand the browsing behavior of the user. The browsing ...history of the user is recorded in the web server log files. Such log files need to be analyzed. The approach described in this paper is based on a novel hybrid technique for analyzing the browsing patterns. It involves a novel and hybrid approach based on clustering and pattern mining algorithm. The approach discussed in this paper is being implemented and the results are analyzed and compared with other related system using two different datasets. The results are also compared concerning the execution time, accuracy and number of frequent patterns.
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