Electron microscopic observation revealed the remarkable change in microfilament cytoskeleton (AandB) and elongation of cytoplasmic processes along its apicobasal axis into subepithelial stroma (B)

Electron microscopic observation revealed the remarkable change in microfilament cytoskeleton (AandB) and elongation of cytoplasmic processes along its apicobasal axis into subepithelial stroma (B).C:An abundant collagen bundle adjacent to the myoepithelium.D:Elongated processes of myoepithelia, extending into the subepithelial stroma.E:Residual myoepithelium in cGVHD lacrimal gland.AEfrom patient 13. ocular cGVHD epithelia gain the mesenchymal phenotype and the capacity to migrate into the subepithelial stroma. Our findings suggest that EMT may be partially responsible for the conjunctival and lacrimal gland fibrosis found in patients with cGVHD. Allogeneic hematopoietic stem cell Etoricoxib D4 transplantation is usually a potentially curative treatment for hematological malignancies. However, chronic graft-versus-host disease (cGVHD) is usually a major complication after allogeneic hematopoietic stem Etoricoxib D4 cell transplantation (HSCT), which has hampered the success of HSCT.1Although numerous advances have been made for treating acute GVHD, the pathogenesis of cGVHD remains largely unknown and effective therapy has not been established. We previously studied the pathogenesis of cGVHD by focusing on the ocular surface and lacrimal gland and found that excessive fibrosis and a subset of fibroblasts contribute to the pathogenesis of ocular cGVHD.2,3 Recently, several studies have reported that epithelial-mesenchymal transition (EMT) contributes to various fibrotic diseases of the kidney,4lung,5and liver.6For example, 40% of fibroblasts in kidney fibrosis arise from epithelial cells via local EMT triggered by inflammatory Etoricoxib D4 stress.7EMT is involved in embryonic development, organ fibrosis, and also cancer metastasis. 8EMT is usually characterized by the loss of apical/basal cell polarity and loss of cell-to-cell adhesions, followed by the acquisition of a mesenchymal phenotype, ie, migration and invasion ability and expression of mesenchymal markers. EMT is brought on by various stimuli including irradiation,9hypoxia,10reactive oxygen species,11inflammatory cytokines such as transforming growth factor- and fibroblast growth factor,8disruption of basal lamina, and exposure of cytoplasm to extracellular matrix.12These triggers of EMT also participate in conjunction with the pathogenesis of cGVHD after HSCT. In a clinical setting, total body irradiation before HSCT and migrating inflammatory cells after HSCT generate substantial proinflammatory cytokines.1This cytokine storm then acts on T cells in the graft, prompting them to attack host antigens.13In addition, reactive oxygen species-mediated organ injury was reported in bone marrow transplant recipients.14The purpose of this study was to elucidate whether EMT is involved in mucosal and exocrine gland cGVHD. == Materials and Methods == == Patients == Etoricoxib D4 We studied 13 allogeneic HSCT recipients who had clinically significant dry eye15and were diagnosed with cGVHD.16Conjunctive biopsies from 11 patients with cGVHD(Table 1)and lacrimal gland biopsies from 9 patients with cGVHD(Table 2)were taken for diagnostic purposes in all patients. Biopsy specimens were compared with controls consisting of 5 normal conjunctival tissue samples, one non-GVHD conjunctival tissue sample from a HSCT patient suspected of a malignant tumor of the conjunctiva, and 5 lacrimal gland samples from patients with Sjgrens syndrome serving as controls. Written informed consent was obtained in advance from all patients in accordance with the principles expressed in the Declaration of Helsinki. This study was approved by the Keio University Institutional Review Boards. == Table 1. == Demographics and Medication of Patients with Conjunctival GVHD F, female; M, male; APL, acute promyelocytic leukemia; CML, chronic myeloid leukemia; ALL, acute lymphocytic leukemia; MM, multiple myeloma; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; PSL, prednisolone; Cys A, cyclosporine A; FK506, tacrolimus; AT, artificial tears frequently/day; HA, topical hyaluronic acid 5 times/day; VA, topical vitamin A 5 times/day; AS, autologous serum 5 times/day; S, topical steroid 4 times/day; B, topical antibiotics 3 times/day. Per day. Alternative day. == Table 2. == Demographics and Medication of Patients with Lacrimal Gland cGVHD TBI, total body irradiation; F, female; M, male; APL, acute promyelocytic leukemia; CML, chronic myeloid leukemia; ALL, acute lymphoblastic leukemia; MM, multiple myeloma; MDS, myelodysplastic syndrome; PSL, prednisolone; Cys A, cyclosporine A; FK506, tacrolimus; AT, artificial tears frequently/day; HA, topical hyaluronic acid 5 times/day; VA, topical vitamin A 5 times/day; AS, autologous serum 5 times/day; S, topical steroid 4 times/day; B, topical antibiotics 3 times/day. Per day. Alternative day. == Histology and Immunohistochemistry == Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded tissue sections as described previously. In brief, antigen unmasking for p63 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), HIF1A heat shock protein (HSP) 47 (Stress Gen Biotechnologies Corp, Victoria, BC, Canada), and Snail (Abgent, San Diego, CA) was performed by autoclaving the sections at 120C for 20 minutes in 10 mmol/L sodium citrate buffer, pH 6.0, to detect intranuclear and intracytoplasmic.