Introducing of acyclonucleoside analogues tethered 1,2,4-triazole as anticancer agents with dual epidermal growth factor receptor kinase and microtubule inhibitors.

This study reports an efficient and convenient regioselective synthesis of a novel series of S- and S,N-bis(acyclonucleoside) analogues carrying 5-(2-chlorophenyl)-2,4-dihydro-1,2,4-triazole-3-thione. A facile and straightforward synthesis of thiazolotriazole and triazolothiazines has also been reported. Structures of all newly synthesized compounds were well characterized by infrared IR, 1H and 13C nuclear magnetic resonance (NMR) and mass (MS) spectra analyses. Cytotoxic screening was performed according to (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assay method using staurosporine as a reference drug against three different types: human liver cancer cell line (Hep G2), Michigan cancer foundation-7 (MCF-7) and human colorectal carcinoma cell line (HCT116). These data showed considerable anticancer activity for these newly synthesized compounds. Biological data for most of the S-acyclonucleoside analogues and S,N-bis(acyclonucleoside) analogues showed excellent activity with micromolar (µM) half maximal inhibitory concentration (IC50) values against tumor cells. EGFR assay and tubulin inhibition assay analysis were performed for the most active compounds to get more details about their mechanism of action. In order to assess and explain their binding affinities, molecular docking simulation was studied against EGFR and tubulin binding sites. The results obtained from molecular docking study and those obtained from cytotoxic screening were correlated. Extensive structure activity relationship (SAR) analyses were also carried out.

Towards xeno-free cultures of human limbal stem cells for ocular surface reconstruction.

Isolated limbal epithelial stem cells (LESCs) were cultured with or without a 3T3 murine fibroblast feeder-layer (FL) in 4 different culture media on culture plates or on denuded human amniotic membrane (AM) support and fibrin gel support: (1) control medium supplemented with fetal bovine serum; (2) control medium supplemented with the synthetic serum "XerumFree™ XF205" (XF); (3) CnT-20 medium supplemented with "XerumFree™ XF205" (CnT-XF) and (4) CnT-20 medium supplemented with human AB serum (CnT-AB). The three xenogeneic media were compared to standard condition (control + FL) and parameters assessed included cell morphology, proliferative potential, number of passages, assessment of clonogenic and abortive colonies, life span, ∆Np63α expression and epithelial morphology on AM. During serial cultivation of LESCs, most of the tested xeno-free media supported similar numbers of cell passages, total colony number, cumulative cell doublings (CCD) rates and expression of ∆Np63α compared to control. The conditions cultivated with a FL showed a non-statistically significant higher number of cell passages and CCD rates before senescence when compared to the same conditions cultured without FL. Except for the control medium, only XF medium enabled the growth of cells on AM. The expression of ∆Np63α was comparable in all the cultures grown onto AM, when compared to the controls on fibrin gel. In conclusion, the xeno-free media enabled LESC culture both on plastic and on denuded human AM. Despite the analyses were carried out in a statistically low number of samples and need re-assessment in a larger cohort, our results suggest that the production of a completely xeno-free LESC graft could be beneficial for future clinical applications.

Corneal Epithelial Stem Cells Repopulate the Donor Area within 1 Year from Limbus Removal for Limbal Autograft.

PURPOSE: To determine whether limbal epithelial stem cells (LESCs) repopulate the site harvested for limbal autograft transplantation (LAT), the expression of LESCs markers was evaluated in bioptic specimens obtained from the donor area 12 months or more after surgery. DESIGN: Interventional case series. PARTICIPANTS: Patients who underwent LAT for unilateral acquired limbal stem cell deficiency after chemical burn. METHODS: Corneal limbal explants were obtained from 2 sites, the harvested area and the untouched control area, in the donor eyes of 6 patients who previously underwent LAT for unilateral acquired limbal stem cell deficiency after chemical burn. Limbal epithelial stem cells were isolated, and cellular, immunohistochemistry, and histologic parameters were assessed to compare differences between LESCs isolated from harvested or control sites. MAIN OUTCOME MEASURES: Presence of LESCs 1 year or more after LAT. RESULTS: Specific markers (p63, Ki67, K12), percentage of LESCs, cell doubling, and number of passages in culture did not differ significantly between harvested and control sites. However, the distinctive structure of the palisades of Vogt was found only in 2 of 6 harvested sites. CONCLUSIONS: Limbal epithelial stem cells repopulate the donor site as early as 1 year after limbus removal for LAT. Autologous transplantation of conjunctiva and limbus are safe procedures and can be performed in cases that cannot be treated by simple grafting of LESCs cultured ex vivo.

Amniotic membranes in ophthalmology: long term data on transplantation outcomes.

The use of amniotic membrane (AM) is a widespread clinical practice for eye surgeries and the treatment of an increasing number of ocular surface pathologies. Here we describe the AM collection methods and donor selection criteria adopted by our tissue bank to distribute 5349 amniotic membrane patches over the last 12 years for the treatment of several ocular pathologies. Specific quality control measures are described and the long term results attained using the reported procedure are presented. A case of AM utilized to treat severe ocular ulceration is also described as an example of AM transplantation. Collective data for the total amniotic membrane patches deployed to treat various ocular diseases are discussed and success rates for AM transplantations are reported. An extensive follow-up is illustrated. The results suggest that the procedures and protocols used by the Treviso Tissue Bank Foundation and Veneto Eye Bank Foundation for collection, preservation, distribution and follow-up are of an optimal standard. Accordingly, the authors conclude that the safety and efficiency of the proposed procedure for the therapeutic use of AM to treat various ocular pathologies are reproducible, with additional evidence favoring the use of AM as an alternative to conventional medical treatment for certain ocular conditions.

Surgical Excision of Orbital Progressive Granular Cell Tumour.

Granular cell tumour (GCT) is mostly benign lesion first described by Abrikossoff and named after him. Most cases are reported in the head and neck area, where the tongue is the most common site. Here we review previous cases in the literature for GCT in the orbit and present a new case. A 49-year-old male presented with apparent exophthalmos. Examination of the patient revealed the presence of a mass in the bottom side of the orbit. A substantial progress was noted after two months from the initial examination using computed tomography (CT) scan. An orbital mass was extracted and histological analysis showed signs typical for GCT. Immunohistochemistry was positive for S-100; the biopsy showed no mitotic or necrotic areas. Proptosis was resolved after surgery and a six-year follow-up CT scan was performed. We conclude that rapid progress of the tumour does not necessarily suggest malignancy.

A superfusion apparatus for ex vivo human eye irritation investigations.

A superfusion apparatus (SA) was developed to maintain isolated human corneas ex vivo under conditions which mimic the natural eye environment in vivo, including controlled temperature, tear flow and intraocular pressure. The SA was designed, developed and tested for use in ophthalmic pre-clinical research and to test new pharmaceutical formulations. Corneas undergo an equilibration process in the new physiological environment for one day. The test was then initiated by the application of the test substance, incubation, and temporal assessment of corneal damage using various parameters. The effects of mild and severe irritant concentrations of NaOH (2% and 8%, respectively) on corneal opacity, swelling and epithelial integrity were studied, and the inflammatory status assessed using F4/80 and MPO as macrophages and neutrophils markers, respectively. The SA was then used to test new artificial tear formulations supplemented with silver ions as an active constituent, showing different degrees of inflammatory responses as indicated by the migration of MPO and F4/80 positive cells towards the epithelium. The human cornea superfusion apparatus was proposed as a model for acute eye irritation research.