Droplet Digital PCR-Based Chimerism Analysis for Primary Immunodeficiency Diseases.

OBJECTIVE: In the current study, we aimed to accurately evaluate donor/recipient or male/female chimerism in samples from patients who underwent hematopoietic stem cell transplantation (HSCT). METHODS: We designed the droplet digital polymerase chain reaction (ddPCR) for SRY and RPP30 to detect the male/female chimerism. We also developed mutation-specific ddPCR for four primary immunodeficiency diseases. RESULTS: The accuracy of the male/female chimerism analysis using ddPCR was confirmed by comparing the results with those of conventional methods (fluorescence in situ hybridization and short tandem repeat-PCR) and evaluating dilution assays. In particular, we found that this method was useful for analyzing small samples. Thus, this method could be used with patient samples, especially to sorted leukocyte subpopulations, during the early post-transplant period. Four mutation-specific ddPCR accurately detected post-transplant chimerism. CONCLUSION: ddPCR-based male/female chimerism analysis and mutation-specific ddPCR were useful for all HSCT, and these simple methods contribute to following the post-transplant chimerism, especially in disease-specific small leukocyte fractions.

Expression of ß-catenin in regenerating renal tubules of cisplatin-induced kidney failure in rats.

BACKGROUND: ß-Catenin is a multi-functional protein involved in nephrogenesis and also plays important roles in renal injury. Here, the expression of ß-catenin was investigated in the proximal renal tubular epithelial cells in cisplatin (CDDP)-induced acute kidney injury (AKI) and chronic kidney injury (CKI), because CDDP-induced renal lesions were characterized by proximal renal tubular epithelial degeneration/regeneration and subsequent interstitial fibrosis. METHODS: F344 rats were treated with CDDP. The expression of ß-catenin and proliferative (Ki67) or fibrogenic [vimentin, α-smooth action (α-SMA)] markers was analyzed by immunolabeling. RESULTS: ß-Catenin, vimentin and Ki67 were not seen in the proximal renal tubules of control rats. Interestingly, in CDDP-induced AKI, the regenerating proximal renal tubular epithelial cells reacting strongly with Ki67 expressed membranous or cytoplasmic ß-catenin and also showed a positive reaction to vimentin but not to α-SMA. In CDDP-induced CKI, the epithelial cells of abnormally dilated or atrophied renal tubules did not react to ß-catenin or Ki67, but showed positive reactions to vimentin and α-SMA. ß-Catenin mRNAs were significantly increased in AKI and significantly decreased in CKI. CONCLUSION: Newly expressed ß-catenin in the proximal renal tubules after AKI may participate in functional regeneration. In CKI, epithelial cells of abnormal renal tubules did not express ß-catenin but reacted to vimentin, and α-SMA might indicate the epithelial-mesenchymal transition (EMT) formation, because α-SMA is usually expressed in myofibroblasts forming via EMT. The presence or absence of ß-catenin expression would become a marker for the EMT phenomenon in progressive renal fibrosis.

Immunolocalization of ß-catenin, E-cadherin and N-cadherin in neonate and adult rat kidney.

ß-catenin, E-cadherin and N-cadherin are adhesion molecules that play important roles in organogenesis, tissue homeostasis, renal epithelial integrity and polarity. The present study demonstrated their immunolocalization in adult and neonate rat kidney. Membranous or cytoplasmic expression of ß-catenin, E-cadherin and N-cadherin were seen in adult and developing renal tubular epithelial cells. Particularly, in adult kidney, E-cadherin and ß-catenin were intensively expressed in distal renal tubules, whereas N-cadherin was expressed in proximal renal tubules. In neonate rat kidney on 1 and 4 days old, developing renal tubular epithelial cells were mainly reacted with E-cadherin and very weakly expressed N-cadherin; ß-catenin was expressed in developing renal tubules and mesenchymal blastemal cells. Interestingly, ß-catenin-positive renal tubular epithelial cells simultaneously expressed E-cadherin in the kidney of adult and developing rats. Collectively, the adhesion molecules were differentially distributed in the renal tubules of adult rats and ß-catenin and E-cadherin are predominant adhesion molecules in developing kidney. The present findings would provide the basic information of evaluating renal tubular toxicity using rats, in addition to renal genesis, in terms of adhesion molecules.