ABSTRACT.Purpose: Failure of retinal pigment epithelial (RPE) cells and macrophages to engulf different dying cells in the retina may result in accumulation of debris and development of age-related macular degeneration (AMD). The dynamics and influence of different treatments on this clearance process can be studied in vitro using human ARPE-19 cells and macrophages as phagocytes modelling dry and wet type of AMD, respectively.Methods: Death through extracellular matrix detachment using polyHEMA-coated surfaces (anoikis) and UV irradiation (apoptosis) was induced in ARPE-19 cells. Twocoloured phagocytic assays were performed to quantify the amount of dying cells phagocytes engulfed (flow cytometry) and for visualization (fluorescent and scanning electron microscopy). The effect of phosphatidylserine inhibition with recombinant annexin-V and glucocorticoid (triamcinolone) treatment on the phagocytic process was tested.Results: The clearance of anoikic and apoptotic cells by nondying ARPE-19 cells over 8 hr of co-incubation increased over time (at 8 hr, over 53% and 35% of the phagocytes contained engulfed dying cells, respectively). The human macrophages engulfed the anoikic and apoptotic ARPE-19 cells with seven and four times lower capacity, respectively. Phosphatidylserine appearance on the dying cells did not affect, but triamcinolone treatment enhanced the phagocytosis of the dying cells by macrophages.Conclusions: ARPE-19 cells are more efficient in clearing anoikic than UV-induced apoptotic cells. Macrophages are less efficient in the clearance process than ARPE-19 cells. The present model can be used for studying both dry and wet type of AMD in vitro and for testing different pharmacological aspects affecting this disease.

ABSTRACT.Purpose: The identification of cells with properties of retinal progenitor cells (RPCs) in the adult human ciliary margin (CM) prompted a number of studies of their proliferative and differentiation potential. One of the remaining challenges is to find a feasible method of isolating RPCs from the patient's eye. In the human CM, only the iris pigment epithelium (IPE) is easily obtained by a minimally invasive procedure. In the light of recent studies questioning the existence of RPCs in the adult mammalian CM, we wanted to assess the potential of the adult human IPE as source of RPCs. Methods: The IPE were isolated from peripheral iridectomies during glaucoma surgery, and IPE and ciliary body (CB) epithelium were also isolated from post-mortem tissue. Cells were cultivated in sphere-promoting conditions or as monolayers. Whole-tissue samples, undifferentiated and differentiated cells were studied by immunocytochemistry, RT-PCR and transmission electron microscopy. Results: The adult human IPE, like the CB, expressed markers of RPCs such as Pax6, Sox2 and Nestin in vivo. Both sphere-promoting and monolayer cultures preserved this phenotype. However, both IPE ⁄ CB cultures expressed markers of differentiated epithelial cells such as Claudin, microphtalmia-associated transcription factor (MITF) and Cytokeratin-19. Ultrastructurally, IPE spheres displayed epithelial-like junctions and contained mature melanosomes. After induced differentiation, IPE-derived cells showed only partial neuronal differentiation expressing b-III-tubulin, Map-2 and Rhodopsin, whereas no mature glial markers were found. Conclusion: Proliferative cells with some properties of RPCs can be isolated from the adult human IPE by peripheral iridectomies. Yet, many cells retain properties of differentiated epithelial cells and lack central properties of somatic stem cells.

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Uveal melanoma, more than other solid tumors, is resistant to most of the chemotherapeutic protocols as indicated by the high mortality rate of metastatic disease. The authors showed that s4UMP, a naturally occurring nucleotide, could induce apoptosis in uveal melanoma cells, suggesting a potential supplementary therapeutic application of the compound.

In this short communication, it is shown that 4-thio-uridylate (s(4)UMP, designated as UD29) inhibits glyceraldehyde 3-phosphate dehydrogenase (GAPDH), suggesting that the enol-form of the thiolated nucleotide may interfere with the function of the essential -SH group in the active center of the enzyme. Since HIV entry requires thiol/disulfide exchange processes, this activity prompted us to study the anti-HIV activity of the nucleotide. Indeed, UD29 inhibited the replication of HIV-1(IIIB) in the MT-4 cell line and HIV-1(Ada-M) in peripheral blood mononuclear cells (PBMC). Furthermore, UD29 was not toxic in PBMCs in vitro or in mice when the compound was administered intravenously.