CAOMECS grafting successfully replaced the D-CEC with oral mucosal epithelial cells with higher levels of CPR. The increase in constitutive proteasome expression is possibly responsible for the recovery and improvement in CAOMECS-grafted corneas.

Purpose: The purpose of the present study is to investigate the expression of aldehyde dehydrogenases (ALDHs) in rabbit corneas with limbal stem cell deficiency (LSCD) and corneas treated with cultured autologous oral mucosa epithelial cell sheet CAOMECS designed to reconstruct the ocular surface with LSCD. Methods: New Zealand white rabbit autologous oral mucosal epithelial cells were isolated from a buccal biopsy and cultured to be grafted back onto corneas of rabbit model of LSCD. Immunofluorescent staining and Western blot analysis were used to compare the expression of ALDH1A1 and ALDH1A3 in healthy, LSCD-diseased, CAOMECS treated corneas. Human oral mucosal and corneal epithelial cells (OMECS and CECs) were cultured and treated with retinoic acid (RA) to further investigate the expression of ALDHs. Results: In healthy corneas, ALDH1A1 and ALDH1A3 were markedly expressed in basal cells of corneal epithelium. In LSCD diseased corneas, ALDH1A1 and ALDH1A3 were markedly expressed in the conjunctivalized apical epithelial cells, the goblet cells, and the stroma. CAOMECS grafted corneas showed a decreased expression of ALDHs as compared to LSCD diseased corneas. Western blot analysis confirmed the up regulation of ALDH1A1 and ALDH1A3 expression in LSCD-diseased corneal epithelial cells. CAOMECS expressed low levels of ALDH1A1 and ALDH1A3, as compared to diseased CECs (D-CEC). When ALDH1A3 was up regulated by retinoic acid treatment in OMECS, Pax-6 expression was down regulated, suggesting a decrease in regenerative capacity when ALDH enzymes are up regulated. Conclusions: These findings report for the first time the up regulation of ALDH1A1 and ALDH1A3 in rabbit corneas with LSCD and document that CAOMECS grafting used to reconstruct corneal epithelium may reduce the expression levels of ALDH enzymes.

Purpose: Ubiquitin proteasome pathway (UPP) failure has been reported in case of rabbit Limbal Stem Cell Deficiency (LSCD). In the present study, we examined the effect of LSCD on autophagy in comparison to LSCD's effect on UPP. Methods: Rabbits with surgically induced LSCD were used. Corneal epithelial cells (CEC) were collected to measure the levels of autophagy and UPP biomarkers. Rabbit oral epithelial cells were isolated, cultured and treated with autophagy and proteasome inhibitors. Results: Immunofluorescent staining showed that both pathways were positive in normal corneal epithelium. However, while proteasome subunits were decreased in LSCD-CEC, autophagy biomarker MAPLC3B and ATG12-ATG5 complex were significantly increased in LSCD-CEC, compared to healthy CEC. These results indicate that LSCD-induced impairment of UPP may cause a compensatory stimulation of autophagy. However, despite autophagy up regulation, damaged and unwanted proteins, such as modified keratin K4 and K13 aggregates, still deposited and accumulated in LSCD-CEC without clearance, possibly contributing to CEC haziness. Proteasome inhibition in cultured oral epithelial cell sheet caused an increase in the expression of MAPLC3B and ATG12-ATG5 complex, supporting the observation that when UPP is failing, autophagy is stimulated. When autophagy was inhibited with chloroquine, proteasome activity was significantly increased compared to untreated cells. In addition, there a slight decrease in unmodified K4 and K13 expression levels with no keratin high molecular weight deposition when autophagy was inhibited. Conclusions: We report that inhibition of autophagy leads to an activation in proteasome activity, which may stimulate protein degradation to alleviate the symptoms of LSCD.

Fine-needle aspiration (FNA) is one of the most accurate modes of obtaining thyroid nodule biopsies, however, up to 25% of biopsies still yield indeterminate results. There is an increasing number of thyroidectomies due to indeterminate nodules by FNA alone. Therefore, more accurate and time efficient diagnostic approaches for analyzing indeterminate thyroid nodules is required. Recent studies showed that Enigma is associated with different cancer types, including thyroid cancer progression and calcification through its interaction with bone morphogenic protein-1 (BMP-1) and tyrosine kinases linked to mitogen-activated protein kinase (MAPK) signaling pathway. Our published data on Enigma protein analysis with immunohistochemistry showed promising findings to discriminate malignant versus benign nodules. We also showed a thyroid cancer stage-dependent enhancement of Enigma protein expression. In this study, we are investigating Enigma at a gene expression level by quantitative reverse transcription polymerase chain reaction (RT-qPCR), which is more time-efficient, quantitative, and requires less tissue than immunohistochemistry. We extracted mRNA/DNA/proteins from fresh malignant and benign thyroid nodules using a Qiagen AllPrep DNA/RNA/Protein Mini Kit. After verification of the quantity and purity by NanoDrop, isolated mRNA was then run through Enigma-RT-qPCR. MAPK assay was done by western blotting using MAPK-antibody. Our initial results found that Enigma-mRNA expression level was 3-fold higher in malignant compared to benign thyroid tissues. This finding supports our previous protein expression data with a relative quantitative difference in Enigma-mRNA expression level between malignant and benign thyroid nodules. MAPK expression was upregulated in thyroid cancer compared to benign nodules. We conclude that Enigma-RT-qPCR can be used effectively in FNA samples derived from thyroid nodules, which could potentially enhance the diagnostic accuracy of indeterminate nodules and decrease unnecessary thyroidectomies. Furthermore, both Enigma and MAPK were highly expressed in advanced tumor in the same tissues. Future study is needed to establish the functional interaction of Enigma-MAPK activity in thyroid cancer cells.