Corneal Densitometry Changes after Epithelium-Off Corneal Collagen Crosslinking

Background Keratoconus is a progressive non-inflammatory, degenerative disease that affects the integrity of the collagen matrix within the corneal stroma. Corneal cross-linking (CXL) has been described as the only modality in halting the disease progression over the past decade. Aim of the Work To compare the change in corneal densitometry before and 6 months after CXL using our unique protocol. Patients and Methods A prospective observational study was conducted on 30 eyes of 30 patients with progressive keratoconus. Epitheliumoff CXL was done with application of Riboflavin (VibeX RapidTM) for 6 minutes at a rate of 1 drop/ minute. UVA was delivered using the Avedro KXL machine (Avedro, Waltham, MA, USA) using the following parameters: 7.2 joules, 12 milli watt/ minute and 10 minutes of accelerated continuous UV delivery. Results Within the anterior (120 um) stromal layer, the concentric zones (0 to 2 mm) and (2 to 6 mm) showed a significant elevation of mean densitometry 6 months post-surgery (P = 0.020) and (P = 0.023) respectively compared to the baseline, However the concentric zones (6 to 10 mm) and (10 to 12 mm) showed a non-significant elevation of mean densitometry 6 months post-surgery (P = 0.167) and (P = 0.234) respectively compared to the baseline. Conclusion There is a significant increase in corneal densitometry 6 months postsurgery of the anterior stromal layer within the central 6 mm zone.

Treatment with Regenerating Agent Cacicol�

The importance of extracellular matrix (ECM) integrity in maintaining normal tissue function is demonstrated by numerous pathologies of acute or chronic injury associated with destruction or disruption of its components. Regenerating agent therapy (RGTA) showed a strong anti-oxidative and protective effect resulting in reduced inflammation in chronic periodontitis, corneal lesions, oral and skin ulcers [1-5].Two cases of corneal healing with Cacicol�. The first one was a chemical corneal burn with persistent lesions which resulted in restitutio ad integrum after treatment with Cacicol�. The second case was a contact lens wearer whose cornea had an ulcer with profuse lesions within the stromal layer and the evolution under conventional treatment for 2 weeks was insignificant and after the use of Cacicol� the lesions improved. Cacicol� is an innovative treatment that can be used in non-healing corneal lesions, eg: unresponsive corneal burns with conventional treatment.

Corneal sterile infiltration induced by topical use of ocular hypotensive agent

Purpose: To report two cases with corneal sterile infiltration presumably due to topical ocular hypotensive agent. Method: Case report. Results: Case 1: A 65-year-old man presented with corneal opacity and neovascularization in his left eye. A diagnosis of glaucoma was made 2 years previously, and anti-glaucoma agents were prescribed (brimonidine tartrate, ripasudil hydrochloride hydrate, and brinzolamide) for both eyes. Case 2: A 75-year-old woman noticed corneal opacity in the left eye. A diagnosis of glaucoma was made 35 years previously, and anti-glaucoma agents were prescribed (brimonidine tartrate, 1% dorzolamide, and bimatoprost) for both eyes. In both cases, ocular examination revealed follicular conjunctivitis and blepharitis in both eyes, and corneal sterile infiltration with neovascularization in the left eyes. The three topical drugs were discontinued and replaced with 0.1% fluorometholone. Both the blepharitis and corneal sterile infiltration improved thereafter, although corneal opacity remained across the stromal layer. Conclusion: We encountered two cases of corneal and conjunctival complications that were suspected as side effects after brimonidine eye drop use. Special care should be taken to observe the condition of ocular surface when topical brimonidine is administered.

Thymic Precursor Cells Generate Acute Myeloid Leukemia in NUP98-PHF23/NUP98-HOXD13 Double Transgenic Mice

Introduction: Oncogenic fusion genes have been identified in a large number of hematologic malignancies. Using Vav regulatory elements, we previously developed transgenic mice that expressed either a NUP98-PHF23 (NP23) or NUP98-HOXD13 (NHD13) fusion in the hematopoietic compartment. Both NP23 and NHD13 mice develop a wide variety of leukemias, including myeloid, erythroid, megakaryocytic and lymphoid, with an age of onset typically between 9 and 14 months, but rarely before 6 months of age. Results: NP23-NHD13 double transgenic mice were generated by interbreeding NP23 and NHD13 single transgenic mice. Surprisingly, 100% of the NP23-NHD13 double transgenic mice showed rapid onset of acute myeloid leukemia (AML) within three months. In contrast, none of the single transgenic mice had developed leukemia by 3 months of age. The leukemias were characterized by extraordinarily high WBC (>500,000/uL) and almost complete replacement of the thymus with Mac1+/Gr1+ myeloblasts, with the percent of malignant myeloid cells in the thymus often higher than the percent in the bone marrow or spleen. These findings led to the intriguing hypothesis that the AML generated in NP23-NHD13 mice arose in the thymus, as opposed to the bone marrow. We harvested cells from the thymus of a NP23-NHD13 mouse that had been invaded by Mac1+/Gr1+ AML cells, and transplanted unfractionated cells (10E06 per recipient) as well as residual CD4-/CD8- double negative (DN) thymocytes (10E05 per recipient; these thymocytes were negative for Mac1 and Gr1 as well as CD4 and CD8) into sub-lethally(600 rds) irradiated recipients. All mice (transplanted with unfractionated thymocytes or DN cells from same leukemic mouse) developed Mac1+/Gr1+ AML within 26 days, indicating that the AML was aggressive and transplantable, and could be transmitted by DN thymocytes. The observation that there was no difference in onset of AML transmitted by the DN thymocytes or Mac1+/Gr1+ AML suggested that the DN thymocytes may be the cell of origin for this leukemia. To rule out the possibility that the leukemia in this experiment was transmitted by rare, contaminant Mac1+/Gr1+ cells, we repeated the experiment, twice, using DN thymocytes from 4-5 wk old mice with no signs of leukemia. In these experiments, the DN thymocytes again transmitted a Mac1+/Gr1+ AML. Finally, we further fractioned DN thymocytes into DN1, DN2, DN3 and DN4 populations; the recipients of DN1 and DN2 populations developed a Mac1+/Gr1+ AML, whereas the DN3 and DN4 recipients were healthy with no signs of engraftment in the peripheral blood. To gain further insight into this phenomenon, DN thymocytes from non-leukemic NP23-NHD13 mice were co-cultured on an OP9 stromal layer, which has been shown to support myeloid, B, and T lymphocyte (when supported with Notch1 ligands) differentiation in vitro. DN thymocytes from non-leukemic NP23-NHD13 mice co-cultured on a OP9 stromal layer showed a markedly enhanced ability to differentiate into Mac1+/Gr1+ myeloid lineage cells compared to WT (56% vs 1.4 % at day 10). However, this "wave" of myeloid differentiation was extinguished by 26 days. The NP23-NHD13 cells lost expression of Mac1 and Gr1; an expanded immunophenotype was CD4-, CD8-, CD25-, CD44+, Thy-1.2+ and cKit+, consistent with a self-renewing DN1 thymocyte. These cells were transplanted; all recipients were anemic, and demonstrated engraftment of NP23-NHD13 myeloid cells as well as a less prominent (7.85%-38.5%) population of CD71+/Ter119+ erythroid cells in the BM and spleen. There was no evidence of T-cell differentiation of the transplanted NP23-NHD13 cells. Further thymocyte fractionation experiments using the OP9 co-culture system demonstrated that myeloid differentiation potential resided in the DN1 population. Conclusions: Taken together, these results indicate that NP23-NHD13 thymic progenitors are potently leukemogenic, and retain myeloid and erythroid differentiation potential. Disclosures No relevant conflicts of interest to declare.

327 PORCINE AMNION: A SOURCE OF EPITHELIAL STEM CELLS

The use of pig models for preclinical testing is well established, and the availability of stem cells from this species would open the way to preclinical studies for application of cell therapy. According to the developmental stage from which they are obtained, stem cells are classified as being embryonic, fetal, or adult. Embryonic stem cells have unlimited self-renewing capacity and multilineage differentiation potential, but their clinical application seems to be hindered by the high tumorigenic rate after transplantation. Mesenchymal stem cells (MSC) derived from adult tissues are considered to be more limited in their potential and the risk of the immunological rejection of the transplanted stem cells by the recipient is an important limiting factor. The MSC derived from extra-fetal tissues could overcome many of these restrictions. Indeed, in veterinary medicine, MSC isolated from equine term placenta were the ideal candidates for tendon disease treatment, specifically for their plasticity and their reduced immunogenicity compared to bone marrow-derived cells. Extra-fetal derived MSC in porcine have been isolated from the umbilical cord matrix and amniotic fluid. The aim of this work was to provide, for the first time, an isolation protocol and the characterisation of stem cells from porcine amniotic membrane, which could hold potential uses in regenerative medicine. The amnion is a thin, avascular membrane made of an epithelial layer and an outer layer of connective tissue. From 3 samples of allanto-amnion retrieved at delivery, each amniotic membrane was stripped from the overlying allantois and, for isolation of the epithelial cells, it was digested with trypsin. After removal of epithelial cells, the stromal layer was digested with collagenase to obtain amniotic mesenchymal cells. The cellular yield from term amnion resulted only in epithelial cells (AEC) at a concentration of 10 × 106 for 1 g of digested tissue while no MSC were obtained. Histology, indeed, revealed very few cells in the stromal layer. The AEC readily attached to plastic culture dishes. Culture was established in DMEM-HG medium, supplemented with 10% serum and 10 ng mL–1 of EGF where the cells proliferated robustly. The AEC displayed typical cuboidal morphology. These cells showed a mean of 31 ± 0.24 cell population doublings after 31 days. The mean frequency of colony-forming unit fibroblasts was 1 for each of the 75 plated cells. The AEC expressed MSC mRNA markers (CD29, CD166, CD90, CD73, CD117) and pluripotent markers (Nanog and Oct4), while were negative for CD34 and MHC-II. Osteogenic, adipogenic, and neurogenic differentiations were confirmed by von Kossa, Red Oil, and Nissle stains, respectively, and by expression of specific markers (osteocalcin and osteopontin for osteogenic differentiation, adiponectin and leptin for adipogenic differentiation, and glial fibrillary acid protein and nestin for neurogenic differentiation). We conclude that porcine amnion contain unique and primitive cells whose potential is as yet undefined. Ease of collection and propagation of AEC make this tissue an attractive candidate as a resource for stem cell biotechnology and biomedical research.