技术资料

Despite implementation of virtual crossmatches,flow cytometry crossmatches (FCXM) are still used by many transplant centers to determine immunological risk before kidney transplantation. To determine if common profiles of patients prone to false positive FCXM exist,we examined the demographics and native diseases of kidney patients tested with autologous FCXM (n = 480). Improvements to FCXM and cell isolation methods significantly reduced the positive rate from 15.1{\%} to 5.3{\%}. Patients with native diseases considered 'immunological' (vasculitis,lupus,IgA nephropathy) had more positive autologous FCXM (OR = 3.36,p = 0.003) vs. patients with all other diseases. Patients who were tested using our updated method (n = 321) still showed that these immunological diseases were a significant predictor for positive autologous FCXM (OR = 4.79,p = 0.006). Interestingly,patients on peritoneal dialysis (PD) also had significantly more positive autologous FCXM than patients on hemodialysis or waiting for pre-emptive kidney transplants (OR = 3.27,p = 0.02). These findings were confirmed in patients who had false positive allogeneic FCXM. Twenty of 24 (83.3{\%}) patients with false positive allogeneic FCXM tested with updated method either had immunological diseases originally or were on PD. Our findings are helpful when interpreting an unexpected positive FCXM,especially for transplantation from deceased donors. View Publication

Shiga toxigenic Escherichia coli (STEC) are responsible for a worldwide foodborne disease,which is characterized by severe bloody diarrhea and hemolytic uremic syndrome (HUS). Subtilase cytotoxin (SubAB) is a novel AB5 toxin,which is produced by Locus for Enterocyte Effacement (LEE)-negative STEC. Cleavage of the BiP protein by SubAB induces endoplasmic reticulum (ER) stress,followed by induction of cytotoxicity in vitro or lethal severe hemorrhagic inflammation in mice. Here we found that steroids and diacylglycerol (DAG) analogues (e.g.,bryostatin 1,Ingenol-3-angelate) inhibited SubAB cytotoxicity. In addition,steroid-induced Bcl-xL expression was a key step in the inhibition of SubAB cytotoxicity. Bcl-xL knockdown increased SubAB-induced apoptosis in steroid-treated HeLa cells,whereas SubAB-induced cytotoxicity was suppressed in Bcl-xL overexpressing cells. In contrast,DAG analogues suppressed SubAB activity independent of Bcl-xL expression at early time points. Addition of Shiga toxin 2 (Stx2) with SubAB to cells enhanced cytotoxicity even in the presence of steroids. In contrast,DAG analogues suppressed cytotoxicity seen in the presence of both toxins. Here,we show the mechanism by which steroids and DAG analogues protect cells against SubAB toxin produced by LEE-negative STEC. View Publication

Graphene quantum dots (GQDs) have gained significant attention in various biomedical applications. The physicochemical properties of these nanoparticles,including toxic effects,are largely determined by their surface modifications. Previous studies have demonstrated high in vitro cytotoxicity of the hydroxylated GQDs (OH-GQDs). The focus of this study was on the intestinal toxicity of OH-GQDs. Briefly,C57BL/6J mice were given daily oral gavage of 0.05,0.5 or 5 mg/kg OH-GQD for 7 days,and the indices of intestinal damage were evaluated. Higher doses of the OH-GQDs caused significant intestinal injuries,such as enhanced intestinal permeability,shortened villi and crypt loss. The number of Lgr5+ intestinal stem cells also decreased dramatically upon OH-GQDs exposure,which also inhibited the Ki67+ proliferative progenitor cells. In addition,an increased number of crypt cells harboring the oxidized DNA base 8-OHdG and $\gamma$H2AX foci were also detected in the intestines of OH-GQD-treated mice. Mechanistically,the OH-GQDs up-regulated both total and phosphorylated p53. Consistent with this,the average number of TUNEL+ and cleaved caspase-3+ apoptotic intestinal epithelial cells were significantly increased after OH-GQDs treatment. Finally,a 3-dimensional organoid culture was established using isolated crypts,and OH-GQDs treatment significantly reduced the size of the surviving intestinal organoids. Taken together,the intestinal toxicity of the OH-GQDs should be taken into account during biomedical applications. View Publication

Neurogenin3 (NEUROG3) is required for endocrine lineage formation of the pancreas and intestine. Patients with NEUROG3 mutations are born with congenital malabsorptive diarrhea due to complete loss of enteroendocrine cells,whereas endocrine pancreas development varies in an allele-specific manner. These findings suggest a context-dependent requirement for NEUROG3 in pancreas versus intestine. We utilized human tissue differentiated from NEUROG3-/- pluripotent stem cells for functional analyses. Most disease-associated alleles had hypomorphic or null phenotype in both tissues,whereas the S171fsX68 mutation had reduced activity in the pancreas but largely null in the intestine. Biochemical studies revealed NEUROG3 variants have distinct molecular defects with altered protein stability,DNA binding,and gene transcription. Moreover,NEUROG3 was highly unstable in the intestinal epithelium,explaining the enhanced sensitivity of intestinal defects relative to the pancreas. These studies emphasize that studies of human mutations in the endogenous tissue context may be required to assess structure-function relationships. View Publication

Osteoporotic osteoarthritis (OPOA) is a common bone disease mostly in the elderly,but the relationship between Osteoporotic (OP) and osteoarthritis (OA) is complex. It has been shown that knee loading can mitigate OA symptoms. However,its effects on OPOA remain unclear. In this study,we characterized pathological linkage of OP to OA,and evaluated the effect of knee loading on OPOA. We employed two mouse models (OA and OPOA),and conducted histology,cytology,and molecular analyses. In the OA and OPOA groups,articular cartilage was degenerated and Osteoarthritis Research Society International score was increased. Subchondral bone underwent abnormal remodeling,the differentiation of bone marrow mesenchymal stem cells (BMSCs) to osteoblasts and chondrocytes was reduced,and migration and adhesion of pre-osteoclasts were enhanced. Compared to the OA group,the pathological changes of OA in the OPOA group were considerably aggravated. After knee loading,however,cartilage degradation was effectively prevented,and the abnormal remodeling of subchondral bone was significantly inhibited. The differentiation of BMSCs was also improved,and the expression of Wnt/$\beta$-catenin was elevated. Collectively,this study demonstrates that osteoporosis aggravates OA symptoms. Knee loading restores OPOA by regulating subchondral bone remodeling,and may provide an effective method for repairing OPOA. View Publication