Differentiation of Uc-MSCs into insulin secreting islet-like clusters by trypsin through TGF-beta signaling pathway.

Differentiation of human umbilical cord mesenchymal stem cells (Uc-MSCs) into islet-like clusters which are capable of synthesizing and secreting insulin can potentially serve as donors for islet transplantation in the patient deficiency in islet ß cell function both in type 1 or type 2 diabetic patients. Therefore, we developed an easy and higher efficacy approach by trypsinazing the Uc-MSCs and followed culture in differentiation medium to induce of Uc-MSCs differentiation into islet-like clusters, and the potential mechanism that in the early stage of differentiation was also investigated by using RNA-sequencing and bioinformatics. Results show that induction efficacy was reached to 98% and TGF-ß signaling pathway may play critical role in the early stage differentiation, it was further confirmed that the retardant effect of differentiation progress either in cell morphology or in islet specific genes expression can be observed upon blocking the activation of TGF-ß signaling pathway using specific inhibitor of LY2109761 (TßRI/II kinase inhibitor). Our current study, for the first time, development a protocol for differentiation of Uc-MSCs into islet-like clusters, and revealed the importance of TGF-ß signaling pathway in the early stage of differentiation of Uc-MSCs into islet-like clusters. Our study will provide alternative approach for clinical treatment of either type I or type II diabtes mellitus with dysfunctional pancreatic islets.

Developing a predictive model for clinically significant prostate cancer by combining age, PSA density, and mpMRI.

PURPOSE: The study aimed to construct a predictive model for clinically significant prostate cancer (csPCa) and investigate its clinical efficacy to reduce unnecessary prostate biopsies. METHODS: A total of 847 patients from institute 1 were included in cohort 1 for model development. Cohort 2 included a total of 208 patients from institute 2 for external validation of the model. The data obtained were used for retrospective analysis. The results of magnetic resonance imaging were obtained using Prostate Imaging Reporting and Data System version 2.1 (PI-RADS v2.1). Univariate and multivariate analyses were performed to determine significant predictors of csPCa. The diagnostic performances were compared using the receiver operating characteristic (ROC) curve and decision curve analyses. RESULTS: Age, prostate-specific antigen density (PSAD), and PI-RADS v2.1 scores were used as predictors of the model. In the development cohort, the areas under the ROC curve (AUC) for csPCa about age, PSAD, PI-RADS v2.1 scores, and the model were 0.675, 0.823, 0.875, and 0.938, respectively. In the external validation cohort, the AUC values predicted by the four were 0.619, 0.811, 0.863, and 0.914, respectively. Decision curve analysis revealed that the clear net benefit of the model was higher than PI-RADS v2.1 scores and PSAD. The model significantly reduced unnecessary prostate biopsies within the risk threshold of > 10%. CONCLUSIONS: In both internal and external validation, the model constructed by combining age, PSAD, and PI-RADS v2.1 scores exhibited excellent clinical efficacy and can be utilized to reduce unnecessary prostate biopsies.

Effects of Huatan Tongluo decoction on vascular endothelial growth factor receptor 2 expression in synovial tissues of rats with collagen-induced arthritis.

OBJECTIVE: To determine the therapeutic effect and potential mechanism of Huatan Tongluo decoction on rats with collagen-induced arthritis. METHODS: Forty specific pathogen-free Wistar rats were selected, and 10 were randomly selected as the control (group 1). The remaining rats were injected intradermally with emulsified type II bovine collagen at the tail base and back, followed by a booster 7 d post first immunization. After establishing collagen-induced arthritis (CIA), rats were randomly divided into three groups (n = 10). The rats were treated orally for 30 d as follows: group 1, saline; group 2, model (saline); group 3, tripterygium polyglycoside (TP; 7.81 mg/kg, positive control); group 4, Huatan Tongluo decoction (HTTL; 7.5 g/kg). Body weight, ankle swelling and arthritis index were measured over the course of the study. The rats were sacrificed 30 d after treatment. Morphological changes in the synovium were observed by hematoxylin and eosin staining. Pannus formation and synovial thickness in the left ankle were observed by color Doppler ultrasoundVascular endothelial growth factor (VEGF) and VEGFR2 protein levels were measured by immunohistochemistry. VEGF/VEGFR2 mRNA levels were measured by real-time quantitative polymerase chain reaction. RESULTS: Compared with the model group, a significantly lower arthritis index was observed in the positive control group (P < 0.05) and HTTL group (P < 0.01), after treatment. Both positive control and HTTL reduced intra-articular pannus formation and synovial thickening. Furthermore, VEGF mRNA, and VEGFR2 protein and mRNA levels were significantly downregulated (P < 0.05) in the treatment groups. CONCLUSION: Inhibition of the expression of VEGF and VEGFR2 in synovial tissues and the formation of pannus and synovial hyperplasia may be part of the mechanism of HTTL for relieving the symptoms of rheumatoid arthritis in CIA rats.

Pathogenic Events in a Nonhuman Primate Model of Oral Poliovirus Infection Leading to Paralytic Poliomyelitis.

Despite a great deal of prior research, the early pathogenic events in natural oral poliovirus infection remain poorly defined. To establish a model for study, we infected 39 macaques by feeding them single high doses of the virulent Mahoney strain of wild type 1 poliovirus. Doses ranging from 107 to 109 50% tissue culture infective doses (TCID50) consistently infected all the animals, and many monkeys receiving 108 or 109 TCID50 developed paralysis. There was no apparent difference in the susceptibilities of the three macaque species (rhesus, cynomolgus, and bonnet) used. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia, and virus was isolated from tonsils, gut mucosa, and draining lymph nodes. Viral replication proteins were detected in both epithelial and lymphoid cell populations expressing CD155 in the tonsil and intestine, as well as in spinal cord neurons. Necrosis was observed in these three cell types, and viral replication in the tonsil/gut was associated with histopathologic destruction and inflammation. The sustained response of neutralizing antibody correlated temporally with resolution of viremia and termination of virus shedding in oropharynges and feces. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), extending previous studies of poliovirus pathogenesis in humans. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis and to assess the efficacy of candidate antiviral drugs and new vaccines.IMPORTANCE Early pathogenic events of poliovirus infection remain largely undefined, and there is a lack of animal models mimicking natural oral human infection leading to paralytic poliomyelitis. All 39 macaques fed with single high doses ranging from 107 to 109 TCID50 Mahoney type 1 virus were infected, and many of the monkeys developed paralysis. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia; tonsil, mesentery lymph nodes, and intestinal mucosa served as major target sites of viral replication. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), thereby supplementing historical reconstructions of poliovirus pathogenesis. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis, candidate antiviral drugs, and the efficacy of new vaccines.

MADD knock-down enhances doxorubicin and TRAIL induced apoptosis in breast cancer cells.

The Map kinase Activating Death Domain containing protein (MADD) isoform of the IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.

Down-modulation of expression, or dephosphorylation, of IG20/MADD in tumor necrosis factor-related apoptosis-inducing ligand-resistant thyroid cancer cells makes them susceptible to treatment with this ligand.

BACKGROUND: The IG20/MADD gene is overexpressed in thyroid cancer tissues and cell lines, and can contribute to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance. The ability of the MADD protein to resist TRAIL-induced apoptosis is dependent upon its phosphorylation by Akt. Interestingly, while TRAIL induces a significant reduction in the levels of phospho-Akt (pAkt) and phospho-MADD (pMADD) in TRAIL-sensitive cells, it fails to do so in TRAIL-resistant cells. In this study, we investigated if MADD phosphorylation by Akt was contributing to TRAIL resistance in thyroid cancer cells. METHODS: We determined the susceptibility of different thyroid cancer cell lines to TRAIL-induced apoptosis by fluorescence-activated cell sorting (FACS) analysis. We tested for various TRAIL resistance factors by FACS analyses or for IG20/MADD expression by quantitative reverse transcription-polymerase chain reaction. We determined the levels of pAkt and pMADD upon TRAIL treatment in thyroid cancer cells by Western blotting. We tested if down-modulation of IG20/MADD gene expression using shRNA or phosphorylation using a dominant negative Akt (DN-Akt) or pretreatment with LY294002, a PI3 kinase inhibitor, could help overcome TRAIL resistance. RESULT: BCPAP and TPC1 cells were susceptible, while KTC1 and FTC133 cells were resistant, to TRAIL-induced apoptosis. The differential susceptibility to TRAIL was not related to the levels of expression of death receptors, decoy receptors, or TRAIL. KTC1 and FTC133 cells showed higher levels of IG20/MADD expression relative to BCPAP and TPC1, and were rendered susceptible to TRAIL treatment upon IG20/MADD knockdown. Interestingly, upon TRAIL treatment, the pAkt and pMADD levels were reduced in TRAIL-sensitive BCPAP and TPC1 cells, while they remained unchanged in the resistant KTC1 and FTC133 cells. While expression of a constitutively active Akt in BCPAP and TPC1 cells rendered them resistant to TRAIL, pretreating KTC1 and FTC133 cells with LY294002 rendered them TRAIL-sensitive. Moreover, expression of a DN-Akt in KTC1 and FTC133 cells reduced the levels of pAkt and pMADD and sensitized them to TRAIL-induced apoptosis. CONCLUSION: Our results show that pMADD is an important TRAIL resistance factor in certain thyroid cancer cells and suggest that down-modulation of either IG20/MADD expression or phosphorylation can render TRAIL-resistant thyroid cancer cells sensitive to TRAIL.

Knockdown of MADD and c-FLIP overcomes resistance to TRAIL-induced apoptosis in ovarian cancer cells.

OBJECTIVE: The clinical utility of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the treatment of established human malignancies is limited by the development of resistance to TRAIL. We hypothesized that knockdown of map-kinase activating death domain containing protein (MADD), a TRAIL-resistance factor, may overcome TRAIL resistance in ovarian cancer cells. STUDY DESIGN: MADD expression in resected ovarian cancer specimens and cell lines was quantified with the use of polymerase chain reaction. Sensitivity of ovarian cancer cell lines to TRAIL, with or without MADD knockdown, was assessed. RESULTS: MADD is expressed at relatively higher levels in human malignant ovarian cancer tissues and cell lines, compared with normal ovarian tissues. The cell lines OVCA429 and OVCAR3 were susceptible, and cell lines CAOV-3 and SKOV-3 were resistant to TRAIL. MADD knockdown in CAOV-3 cells, but not in SKOV-3 cells, conferred TRAIL sensitivity. Knockdown of cellular Fas-associated death domain-like interleukin-1 beta-converting enzyme-inhibitory protein (c-FLIP) in SKOV-3 cells increased spontaneous and TRAIL-induced apoptosis, which was further increased on MADD knockdown. CONCLUSION: MADD/c-FLIP(L) knockdown can render TRAIL-resistant ovarian cancer cells susceptible to TRAIL.