A comparison of three methods of decellularization of pig corneas to reduce immunogenicity.

AIM: To investigate whether decellularization using different techniques can reduce immunogenicity of the cornea, and to explore the decellularized cornea as a scaffold for cultured corneal endothelial cells (CECs). Transplantation of decellularized porcine corneas increases graft transparency and survival for longer periods compared with fresh grafts. METHODS: Six-month-old wild-type pig corneas were cut into 100-200 µm thickness, and then decellularized by three different methods: 1) 0.1% sodium dodecyl sulfate (SDS); 2) hypoxic nitrogen (N2); and 3) hypertonic NaCl. Thickness and transparency were assessed visually. Fresh and decellularized corneas were stained with hematoxylin/eosin (H&E), and for the presence of galactose-α1,3-galactose (Gal) and N-glycolylneuraminic acid (NeuGc, a nonGal antigen). Also, a human IgM/IgG binding assay was performed. Cultured porcine CECs were seeded on the surface of the decellularized cornea and examined after H&E staining. RESULTS: All three methods of decellularization reduced the number of keratocytes in the stromal tissue by >80% while the collagen structure remained preserved. No remaining nuclei stained positive for Gal or NeuGc, and expression of these oligosaccharides on collagen was also greatly decreased compared to expression on fresh corneas. Human IgM/IgG binding to decellularized corneal tissue was considerably reduced compared to fresh corneal tissue. The cultured CECs formed a confluent monolayer on the surface of decellularized tissue. CONCLUSION: Though incomplete, the significant reduction in the cellular component of the decellularized cornea should be associated with a significantly reduced in vivo immune response compared to fresh corneas

Expression of NeuGc on Pig Corneas and Its Potential Significance in Pig Corneal Xenotransplantation

PURPOSE: Pigs expressing neither galactose-α1,3-galactose (Gal) nor N-glycolylneuraminic acid (NeuGc) take xenotransplantation one step closer to the clinic. Our aims were (1) to document the lack of NeuGc expression on corneas and aortas and cultured endothelial cells [aortic endothelial cells (AECs); corneal (CECs)] of GTKO/NeuGcKO pigs, and (2) to investigate whether the absence of NeuGc reduced human antibody binding to the tissues and cells. METHODS: Wild-type (WT), GTKO, and GTKO/NeuGcKO pigs were used for the study. Human tissues and cultured cells were negative controls. Immunofluorescence staining was performed using anti-Gal and anti-NeuGc antibodies, and human IgM and IgG binding to tissues was determined. Flow cytometric analysis was used to determine Gal and NeuGc expression on cultured CECs and AECs and to measure human IgM/IgG binding to these cells. RESULTS: Both Gal and NeuGc were detected on WT pig corneas and aortas. Although GTKO pigs expressed NeuGc, neither humans nor GTKO/NeuGcKO pigs expressed Gal or NeuGc. Human IgM/IgG binding to corneas and aortas from GTKO and GTKO/NeuGcKO pigs was reduced compared with binding to WT pigs. Human antibody binding to GTKO/NeuGcKO AECs was significantly less than that to GTKO AECs, but there was no significant difference in binding between GTKO and GTKO/NeuGcKO CECs. CONCLUSIONS: The absence of NeuGc on GTKO aortic tissue and AECs is associated with reduced human antibody binding, and possibly will provide a better outcome in clinical xenotransplantation using vascularized organs. For clinical corneal xenotransplantation, the absence of NeuGc expression on GTKO/NeuGcKO pig corneas may not prove an advantage over GTKO corneas

Diagnostic Utility of LEF1 Immunostain in Cytology Specimens of Solid Pseudopapillary Neoplasm of Pancreas

Solid pseudopapillary neoplasm (SPN) of the pancreas is a rare neoplasm. Diagnosis of SPN requires an integrated approach with aid of radiology, biopsy, cytology, and immunohistochemical stains. Although morphological features in combination with nuclear positivity of β-catenin IHC have been the gold standard of SPN diagnosis, but overlapping morphology and immunohistochemical findings with other entities in differential diagnoses such as pancreatic neuroendocrine tumors and pancreatic ductal adenocarcinoma make the diagnosis of SPN difficult particularly in limited cytology specimens. Lymphoid enhancer-binding factor 1 (LEF1), a key player in the Wnt signaling pathway, has shown promising diagnostic utility in SPN in recent literatures. &lt;b&gt;&lt;i&gt;Methods:&lt;/i&gt;&lt;/b&gt; In this retrospective study, we evaluated the diagnostic utility of LEF1 IHC in SPN in cytology specimens. LEF1 IHC was performed and compared with β-catenin, synaptophysin, and chromogranin immunostains in 13 SPN and 23 pancreatic neuroendocrine tumors (PanNETs) cytology cases with retrievable cell blocks. &lt;b&gt;&lt;i&gt;Results:&lt;/i&gt;&lt;/b&gt; LEF1 was positive in 13 of 13 (100%) SPNs and was negative in all PanNETs (0%). &lt;b&gt;&lt;i&gt;Conclusion:&lt;/i&gt;&lt;/b&gt; LEF1 shows 100% sensitivity and specificity in cytology specimens for SPN and can be valuable immuno­stain in the diagnosis of SPN in cytology cell blocks. </jats:p