Sensitizing acute myeloid leukemia cells to induced differentiation by inhibiting the RIP1/RIP3 pathway.

Tumor necrosis factor-α (TNF-α)-induced RIP1/RIP3 (receptor-interacting protein kinase 1/receptor-interacting protein kinase 3)-mediated necroptosis has been proposed as an alternative strategy for treating apoptosis-resistant leukemia. However, we found that most acute myeloid leukemia (AML) cells, especially M4 and M5 subtypes, produce TNF and show basal level activation of RIP1/RIP3/MLKL signaling, yet do not undergo necroptosis. TNF, through RIP1/RIP3 signaling, prevents degradation of SOCS1, a key negative regulator of interferon-γ (IFN-γ) signaling. Using both pharmacologic and genetic assays, we show here that inactivation of RIP1/RIP3 resulted in reduction of SOCS1 protein levels and partial differentiation of AML cells. AML cells with inactivated RIP1/RIP3 signaling show increased sensitivity to IFN-γ-induced differentiation. RIP1/RIP3 inactivation combined with IFN-γ treatment significantly attenuated the clonogenic capacity of both primary AML cells and AML cell lines. This combination treatment also compromised the leukemogenic ability of murine AML cells in vivo. Our studies suggest that inhibition of RIP1/RIP3-mediated necroptotic signaling might be a novel strategy for the treatment of AML when combined with other differentiation inducers.

Impact of hospital transfer on surgical outcomes of intestinal atresia.

BACKGROUND: Examine effects of hospital transfer into a quaternary care center on surgical outcomes of intestinal atresia. METHODS: Children <1 yo principally diagnosed with intestinal atresia were identified using the Kids' Inpatient Database (2012). Exposure variable was patient transfer status. Outcomes measured were inpatient mortality, hospital length of stay (LOS) and discharge status. Linearized standard errors, design-based F tests, and multivariable logistic regression were performed. RESULTS: 1672 weighted discharges represented a national cohort. The highest income group and those with private insurance had significantly lower odds of transfer (OR:0.53 and 0.74, p < 0.05). Rural patients had significantly higher transfer rates (OR: 2.73, p < 0.05). Multivariate analysis revealed no difference in mortality (OR:0.71, p = 0.464) or non-home discharge (OR: 0.79, p = 0.166), but showed prolonged LOS (OR:1.79, p < 0.05) amongst transferred patients. CONCLUSIONS: Significant differences in hospital LOS and treatment access reveal a potential healthcare gap. Post-acute care resources should be improved for transferred patients.

Epigallocatechin-3-Gallate Upregulates miR-221 to Inhibit Osteopontin-Dependent Hepatic Fibrosis.

Osteopontin (OPN) promotes hepatic fibrosis, and developing therapies targeting OPN expression in settings of hepatic injury holds promise. The polyphenol epigallocatechin-3-gallate (EGCG), found in high concentrations in green tea, downregulates OPN expression through OPN mRNA degradation, but the mechanism is unknown. Previous work has shown that microRNAs can decrease OPN mRNA levels, and other studies have shown that EGCG modulates the expression of multiple microRNAs. In our study, we first demonstrated that OPN induces hepatic stellate cells to transform into an activated state. We then identified three microRNAs which target OPN mRNA: miR-181a, miR-10b, and miR-221. In vitro results show that EGCG upregulates all three microRNAs, and all three microRNAs are capable of down regulating OPN mRNA when administered alone. Interestingly, only miR-221 is necessary for EGCG-mediated OPN mRNA degradation and miR-221 inhibition reduces the effects of EGCG on cell function. In vivo experiments show that thioacetamide (TAA)-induced cell cytotoxicity upregulates OPN expression; treatment with EGCG blocks the effects of TAA. Furthermore, chronic treatment of EGCG in vivo upregulates all three microRNAs equally, suggesting that in more chronic treatment all three microRNAs are involved in modulating OPN expression. We conclude that in in vitro and in vivo models of TAA-induced hepatic fibrosis, EGCG inhibits OPN-dependent injury and fibrosis. EGCG works primarily by upregulating miR-221 to accelerate OPN degradation. EGCG may therefore have utility as a protective agent in settings of liver injury.

Urothelial cells undergo epithelial-to-mesenchymal transition after exposure to muscle invasive bladder cancer exosomes.

Bladder cancer, the fourth most common noncutaneous malignancy in the United States, is characterized by high recurrence rate, with a subset of these cancers progressing to a deadly muscle invasive form of disease. Exosomes are small secreted vesicles that contain proteins, mRNA and miRNA, thus potentially modulating signaling pathways in recipient cells. Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells. EMT has been implicated in the initiation of metastasis for cancer progression. We investigated the ability of bladder cancer-shed exosomes to induce EMT in urothelial cells. Exosomes were isolated by ultracentrifugation from T24 or UMUC3 invasive bladder cancer cell conditioned media or from patient urine or bladder barbotage samples. Exosomes were then added to the urothelial cells and EMT was assessed. Urothelial cells treated with bladder cancer exosomes showed an increased expression in several mesenchymal markers, including α-smooth muscle actin, S100A4 and snail, as compared with phosphate-buffered saline (PBS)-treated cells. Moreover, treatment of urothelial cells with bladder cancer exosomes resulted in decreased expression of epithelial markers E-cadherin and ß-catenin, as compared with the control, PBS-treated cells. Bladder cancer exosomes also increased the migration and invasion of urothelial cells, and this was blocked by heparin pretreatment. We further showed that exosomes isolated from patient urine and bladder barbotage samples were able to induce the expression of several mesenchymal markers in recipient urothelial cells. In conclusion, the research presented here represents both a new insight into the role of exosomes in transition of bladder cancer into invasive disease, as well as an introduction to a new platform for exosome research in urothelial cells.

BCAS2 promotes prostate cancer cells proliferation by enhancing AR mRNA transcription and protein stability.

BACKGROUND: We showed previously that breast carcinoma amplified sequence 2 (BCAS2) functions as a negative regulator of p53. We also found that BCAS2 is a potential AR-associated protein. AR is essential for the growth and survival of prostate carcinoma. Therefore we characterised the correlation between BCAS2 and AR. METHODS: Protein interactions were examined by GST pull-down assay and co-immunoprecipitation. Clinical prostate cancer (PCa) specimens were evaluated by immunohistochemical assay. AR transcriptional activity and LNCaP cell growth were assessed by luciferase assay and MTT assay, respectively. RESULTS: BCAS2 expression was significantly increased in PCa. BCAS2 stabilised AR protein through both hormone-dependent and -independent manners. There are at least two mechanisms for BCAS2-mediated AR protein upregulation: One is p53-dependent. The p53 is suppressed by BCAS2 that results in increasing AR mRNA and protein expression. The other is via p53-independent inhibition of proteasome degradation. As BCAS2 can form a complex with AR and HSP90, it may function with HSP90 to stabilise AR protein from being degraded by proteasome. CONCLUSIONS: In this study, we show that BCAS2 is a novel AR-interacting protein and characterise the correlation between BCAS2 and PCa. Thus we propose that BCAS2 could be a diagnostic marker and therapeutic target for PCa.

Osteopontin mediates an MZF1-TGF-ß1-dependent transformation of mesenchymal stem cells into cancer-associated fibroblasts in breast cancer.

Interactions between tumor cells and cancer-associated fibroblasts (CAFs) in the tumor microenvironment significantly influence cancer growth and metastasis. Transforming growth factor-ß (TGF-ß) is known to be a critical mediator of the CAF phenotype, and osteopontin (OPN) expression in tumors is associated with more aggressive phenotypes and poor patient outcomes. The potential link between these two pathways has not been previously addressed. Utilizing in vitro studies using human mesenchymal stem cells (MSCs) and MDA-MB231 (OPN+) and MCF7 (OPN-) human breast cancer cell lines, we demonstrate that OPN induces integrin-dependent MSC expression of TGF-ß1 to mediate adoption of the CAF phenotype. This OPN-TGF-ß1 pathway requires the transcription factor, myeloid zinc finger 1 (MZF1). In vivo studies with xenotransplant models in NOD-scid mice showed that OPN expression increases cancer growth and metastasis by mediating MSC-to-CAF transformation in a process that is MZF1 and TGF-ß1 dependent. We conclude that tumor-derived OPN engenders MSC-to-CAF transformation in the microenvironment to promote tumor growth and metastasis via the OPN-MZF1-TGF-ß1 pathway.

Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice.

BACKGROUND: Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehog-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis. METHODS: Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-ß, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. RESULTS: OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-ß signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis. CONCLUSIONS: OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis.

Microstructure and crystallization kinetics analysis of the (In15Sb85)(100-x)Zn(x) phase change recording thin films.

The (In15Sb85)(100-x)Zn(x) films (x = 0 - 17.4) were deposited on nature oxidized Si wafer and glass substrate at room temperature by magnetron co-sputtering of Sb target and InZn composite target. The thermal property of the films was examined by a homemade reflectivity thermal analyzer. Microstructures of the films were analyzed by transmission electron microscope (TEM). We examined the effects of Zn addition on the thermal property, crystallization kinetics, and crystallization mechanism of the In15Sb85 recording film. As x = 0 - 17.4, thermal analysis shows that the (In15Sb85)(100-x)Zn(x) films have two phase transition temperature ranges which are 189 degrees C-215 degrees C and 300 degrees C-350 degrees C. It is found that the activation energy is increased with Zn content. This indicates that the thermal stability of amorphous state is improved by doping Zn. The optical contrasts of the films are all larger than 15%, as x = 0 - 6.2, indicating that the films have the potential in blue laser optical recording media application.

Crystallization mechanisms of phase change (GeSbSn)(100-x)Co(x) optical recording films.

In this study, the (GeSbSn)(100-x0Co(x) films (x = 0-13.3) were deposited on natural oxidized silicon wafer and glass substrate by dc magnetron co-sputtering of GeSbSn and Co targets. The ZnS-SiO2 films were used as protective layers. The thicknesses of the (GeSbSn)(100-x)Co(x) films and protective layer were 100 nm and 30 nm, respectively. We investigated the effects of Co addition on the thermal property, crystallization kinetics, and crystallization mechanism of the GeSbSn recording film. The crystallization temperatures of (GeSbSn)(100-x)Co(x) films were decreased with Co content. It was found that the activation energy of the (GeSbSn)(100-x)Co(x) films will decrease from 1.53 eV to 0.55 eV as Co content increased from 0 at.% to 13.3 at.%.

Effect of Ag underlayer on microstructures and perpendicular magnetic properties of CoPt nanocomposite thin films.

CoPt/Ag films were prepared by magnetron sputtering on glass substrates and subsequent annealing. The dependence of degree of ordering and magnetic properties on Ag film thickness and annealing conditions were investigated. It was found that the Ag underlayer played a dominant role in inducing the (001) texture of the CoPt film after annealing. CoPt films with a thickness about 20 nm and Ag underlayers with a thickness about 70 nm are easy to obtain a large degree of ordering and a perpendicular magnetic anisotropy after annealing at 700 degrees C for 30 min. CoPt/Ag films with out-of-plane coercivity (Hc (perpendicular)) in the range of 13.5-14.0 kOe and a out-of-plane squareness (S(perpendicular)) of 0.97 were obtained after annealing at 700 degrees C for 30 min. Ag underlayer is beneficial to enhance the Hc(perpendicular)and S(perpendicular) of CoPt film significantly. The degree of ordering and perpendicular magnetic properties of the CoPt films which deposited on Ag underlayer are larger than those of the single layer CoPt films.

Molecular and immunocytochemical identification of coxsackievirus A-24 variant from the acute haemorrhagic conjunctivitis outbreak in Taiwan in 2007.

AIMS: Acute haemorrhagic conjunctivitis (AHC) is a highly contagious conjunctivitis associated with enteroviruses. Coxsackie A-24 variant (CA-24v) and enterovirus-70 (EV-70) are the two major causative agents. During October 2007, an epidemic of AHC occurred in Taiwan, affecting more than 11 000 people. The aim of this study was to determine the aetiological agent associated with the outbreak in patients diagnosed with AHC and treated at the Cathay General Hospital, Taipei (CGHT) and Cathay General Hospital Sijhih (CGHS), Taiwan during October 2007. METHODS: Virus isolates were obtained from six patients (four from CGHS and two from CGH), and a total of seven specimens (one throat and one rectal, and five eye swabs) were collected. The specimens were inoculated onto the MRC-5 cell lines. The viral isolation was confirmed by performing real-time reverse transcription-polymerase chain reaction (RT-PCR) and indirect immunofluorescence assay (IFA). RESULTS: The conjunctival, throat, and rectal swabs collected in this study were all tested positive for a variant of CA-24. All seven viral isolates were characterized as a variant of CA-24 and confirmed by IFA and real-time RT-PCR. CONCLUSIONS: The findings suggest that the outbreak of AHC that occurred during October 2007 in the northern area of Taiwan was caused by a variant of coxsackie A-24. Further phylogenic analysis is underway to further classify this CA-24v strain.

Supply and demand for liver transplant surgery: are we training enough surgeons?

The purpose of our study is to determine whether the current level of transplant fellow training is sufficient to meet the future demand for liver transplantation in the United States. Historical data from the Nationwide Inpatient Samples (NIS) for the years 1998 through 2003 were used to construct an estimate of the annual number of liver transplant procedures currently being performed in the United States, and the number projected for each year through 2020. Estimates for the current and future number of surgeons performing liver transplant procedures were also constructed using the same database. The NIS database was used because current national transplant registries do not include information on the number of surgeons performing liver transplant procedures. Using historical data derived from the NIS database, we project that the estimated number of liver transplant procedures per surgeon will remain relatively stable through 2020, with each surgeon performing an average of 12.9 procedures in 2020 compared to 12.9 currently. We conclude that the relationship between demand for liver transplantation in the United States and the supply of liver transplant surgeons will remain stable over the next 15 years.

One-year results with extended-release tacrolimus/MMF, tacrolimus/MMF and cyclosporine/MMF in de novo kidney transplant recipients.

Once-daily tacrolimus extended-release formulation (Prograf XL, formerly referred to as MR or MR4) was compared with the twice-a-day tacrolimus formulation (TAC) and cyclosporine microemulsion (CsA), all administered in combination with mycophenolate mofetil (MMF), corticosteroids and basiliximab induction, in a phase 3, randomized (1:1:1), open-label trial in 638 de novo kidney transplant recipients. In combination with MMF and corticosteroids, XL had an efficacy profile comparable to TAC and CsA. XL/MMF and TAC/MMF were statistically noninferior at 1-year posttransplantation to CsA/MMF for the primary efficacy endpoint, efficacy failure (death, graft loss, biopsy-confirmed acute rejection (BCAR) or lost to follow-up). One-year patient and graft survival were 98.6% and 96.7% in the XL/MMF group, 95.7% and 92.9% in TAC/MMF group and 97.6% and 95.7% in CsA/MMF group. The safety profile of XL in comparison with CsA was similar to that observed with TAC in this study and consistent with previously published reports of TAC in comparison with CsA. The results support the safety and efficacy of tacrolimus in combination with MMF, corticosteroids and basiliximab induction, as well as XL as a safe and effective once-daily dosing alternative.

Obesity predicts increased overall complications following pancreas transplantation.

PURPOSE: We sought to evaluate the role of recipient body mass index (BMI) on postoperative complications in patients receiving pancreas transplants. METHODS: A single-institution retrospective study of 145 consecutive patients undergoing either simultaneous kidney pancreas (SPK) or pancreas after kidney (PAK) transplantation from January 1997 through December 2003. Variables analyzed included: age, sex, BMI, number of prior transplants, cytomegalovirus status of donor and recipient, postoperative insulin resistance, complications, and overall patient and graft survival. Differences in continuous variables and dichotomous variables were evaluated using two-tailed t test and Fisher exact test, respectively. Univariate and multivariate logistic regression analyses were employed to identify predictors of overall complications following surgery. RESULTS: Obesity was defined by a BMI > or = 30. Of the 145 patients, 33 (23%) had a BMI > or = 30 and 112 (77%) had a BMI < 30. There was no significant difference in age or sex between obese and nonobese patients (P = .98 and P = .56, respectively). The type of transplantation, SPK or PAK, did not affect the complication rate (P = .36). Overall complications (infection, dehiscence, evisceration, ventral hernia, allograft failure, gangrene, necrotizing fasciitis, postoperative bleeding, or death) were significantly higher in the obese group (81% vs 40%, P < .001). Obesity was specifically associated with increased frequency of dehiscence, ventral hernia, intra-abdominal infection, gangrene, necrotizing fasciitis, and repeat laparotomy. Obese patients also had a threefold higher rate of graft pancreatitis/enteric leak. Multivariate logistic regression analysis identified age > or = 50 and BMI > or = 30 as independent predictors of overall complications following surgery (odds ratio 4.0, P = .014 and OR 6.8, P < .001, respectively). There was no difference identified between groups with regards to allograft failure, posttransplant insulin resistance, and death. CONCLUSION: Obese patients are at increased risk of overall complications following pancreas transplantation. Specifically, obese patients experience higher frequency of dehiscence, ventral hernia, intra-abdominal infection, gangrene, and necrotizing fasciitis. This study demonstrates the need for careful postoperative monitoring in the obese patient.

Pharmacokinetics and biochemical effects of hepapoietin in patients with chronic liver disease.

BACKGROUND: Hepapoietin is a naturally occurring cytokine that promotes hepatocyte growth. Animal studies have suggested that hepapoietin and hepatocyte growth factor have a potential role in the prevention and management of liver diseases. However, human studies have been lacking. AIM: To evaluate the safety and pharmacokinetics of single escalating doses of hepapoietin in patients with chronic liver disease. METHODS: An open-label, single escalating dose trial with five different doses of hepapoietin (1, 3, 10, 30 and 100 mg) was performed. Adults with chronic, compensated, non-viral liver disease were included. Liver function tests were obtained before dosing, 24 h after hepapoietin administration and on days 4, 7, 30 and 45. All patients were followed for 45 days. RESULTS: Twenty-five subjects received hepapoietin, with five subjects each at 1, 3, 10, 30 and 100 mg of hepapoietin. Significant decreases occurred in total bilirubin, ammonia, partial thromboplastin time and cholesterol levels overall, and both high-density and low-density lipoprotein cholesterol showed a downward trend. An increase in albumin was observed at the 30 mg dose level. Slight decreases in haemoglobin and red blood cell levels were observed at day 4, but returned to normal levels immediately thereafter. Child-Pugh scores from day 0 to day 7 were improved in 24%, stable in 64% and worse in 12% of patients. Hepatic encephalopathy displayed changes from day 0 to day 45 with improvement in 16%, no change in 80% and worsening in 4%. CONCLUSIONS: Hepapoietin in doses up to 100 mg is safe for use in humans. Potential benefits are suggested by significant decreases in bilirubin, ammonia, partial thromboplastin time and cholesterol levels and an increase in albumin. Further studies with multiple dosing regimens are needed to identify the clinical utility of hepapoietin in the management of chronic liver disease.

Nitric oxide inhibits expression of cytochrome B in endotoxin-stimulated murine macrophages.

In LPS-mediated states of sepsis, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production inhibit cellular respiration and mitochondrial electron transport. NO has been demonstrated to inhibit mitochondrial respiration by nitrosylation of the iron-sulfur centers of aconitase, complex I (NADH-ubiquinone oxidoreductase), complex II (succinate-ubiquinone oxidoreductase), and complex IV (cytochrome c oxidase). However, little is known of the effect of NO on expression of critical proteins in the electron transport chain. In ANA-1 murine macrophages, LPS-mediated NO synthesis decreases Cyt b protein expression and steady-state mRNA levels. Mitochondrial run-on analysis demonstrates unaltered Cyt b mitochondrial gene transcription. In this study utilizing LPS-stimulated ANA-1 murine macrophages, we demonstrate that expression of the mitochondrial protein, Cyt b, is significantly decreased as the result of a unique and previously unknown, NO-dependent posttranscriptional regulatory mechanism. (c)2001 Elsevier Science.

Outcome of extra-anatomic vascular reconstruction in orthotopic liver transplantation.

BACKGROUND: Portal venous and hepatic arterial reconstruction are critical to successful outcomes in orthotopic liver transplantation (OLT). With portal vein thrombosis or inadequate hepatic arterial inflow, extra-anatomic vascular reconstruction is required. However, the clinical outcomes following extra-anatomic vascular reconstruction are largely unknown. METHODS: To determine the outcomes associated with extra-anatomic vascular reconstruction, we performed a retrospective review of 205 OLT recipients transplanted between 1995 and 2000. RESULTS: Extra-anatomic portal venous inflow was based upon the recipient superior mesenteric vein using donor iliac vein graft in a retrogastric position (n = 12). Extra-anatomic arterial inflow was based on recipient infrarenal aorta using donor iliac artery graft through the transverse mesocolon (n = 25). OLT with routine anatomic vascular construction served as control (n = 168). Extra-anatomic vascular reconstruction was not associated with increased morbidity, mortality, operating room time, length of stay, or thrombosis. CONCLUSION: We conclude that extra-anatomic vascular conduits are associated with excellent long-term outcomes and provide acceptable alternatives for vascular reconstruction in OLT.