透明质酸修饰的绿原酸脂质体对U14宫颈癌荷瘤小鼠的抑制作用

目的:对透明质酸(HA)靶向绿原酸(CA)脂质体(HA-CA脂质体)进行处方筛选,以及对U14宫颈癌小鼠的抑制作用实验。方法:筛选制备HA-CA脂质体的方法,并以磷脂比、药脂比、PBS的p H为单因素考察指标通过正交实验筛选最优处方;采用透析袋法考察HA-CA的体外释放;Bal b/c小鼠右腋皮下接种U14宫颈癌瘤株,连续尾静脉注射给药14 d后,摘取瘤体称重,并计算肿瘤生长抑制。结果:采用薄膜分散法制备脂质体,最优处方为磷脂比为4:1,药脂比为1:30,PBS的p H为7.4。HA-CA脂质体与CA脂质体释放曲线基本一致,都具有一定的缓释效果。48 h时,HA-CA脂质体和CA脂质体的累计释放度分别为78.39%、83.01%。HA-CA脂质体对U14宫颈癌小鼠的抑瘤率为60.39%,与阳性对照组环磷酰胺相当,高于CA和CA脂质体。结论:HA-CA脂质体由于其具有主动靶向配体HA的修饰,使其抑制U14宫颈癌裸鼠的效果明显高于CA和CA脂质体。     

Inhibition of murine hepatic tumor growth by liposomes containing a lipophilic muramyl dipeptide

Summary We have investigated the ability of liposomes containing a lipophilic muramyl dipeptide, N-acetylmuramyl-l-alanyl-d-isoglutamine glycerol dipalmitate (MDP-GDP) to activate Kupffer cell tumoricidal activity in situ and to inhibit the growth of experimental hepatic micrometastases of tumor cell line H-59, a liver-homing variant of the Lewis lung carcinoma. Liposomes prepared from distearoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DSPC/DMPG) and containing MDP-GDP (1 mol and 2 g, respectively) were efficiently taken up by the liver after i.v. administration. A single i.v. injection of DSPC/DMPG liposomes containing MDP-GDP was capable of inducing Kupffer cell tumoricidal activity against H-59 tumor cells as measured in vitro. Control liposomes or 100 g free MDP were ineffective in inducing Kupffer cell tumoricidal activity in situ. Two treatment regimens were evaluated in vivo: firstly, C57BL/6 mice were injected with tumor cell line H-59 and subsequently treated with multiple injections of liposomal MDP-GDP. Secondly, treatment with liposomal MDP-GDP was initiated prior to tumor cell injection and continued after tumor cell injection. The ability of liposomes containing MDP-GDP to reduce the number of hepatic micrometastases using the first protocol was related to the tumor cell inoculum, significant inhibition being observed at lower liver tumor burdens (<25 tumor nodules). Pretreatment of the mice prior to tumor cell challenge followed by treatment afterwards greatly enhanced the efficacy of liposomal MDP-GDP and brought about a highly significant inhibition of the growth of experimental metastases even at high liver tumor burdens (>50 nodules).     

Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma

Background

This study evaluated the safety and immune responses to an autologous dendritic cell vaccine pulsed with class I peptides from tumor-associated antigens (TAA) expressed on gliomas and overexpressed in their cancer stem cell population (ICT-107).

Methods

TAA epitopes included HER2, TRP-2, gp100, MAGE-1, IL13Rα2, and AIM-2. HLA-A1- and/or HLA-A2-positive patients with glioblastoma (GBM) were eligible. Mononuclear cells from leukapheresis were differentiated into dendritic cells, pulsed with TAA peptides, and administered intradermally three times at two-week intervals.

Results

Twenty-one patients were enrolled with 17 newly diagnosed (ND-GBM) and three recurrent GBM patients and one brainstem glioma. Immune response data on 15 newly diagnosed patients showed 33 % responders. TAA expression by qRT-PCR from fresh-frozen tumor samples showed all patient tumors expressed at least three TAA, with 75 % expressing all six. Correlations of increased PFS and OS with quantitative expression of MAGE1 and AIM-2 were observed, and a trend for longer survival was observed with gp100 and HER2 antigens. Target antigens gp100, HER1, and IL13Rα2 were downregulated in recurrent tumors from 4 HLA-A2+ patients. A decrease in or absence of CD133 expression was seen in five patients who underwent a second resection. At a median follow-up of 40.1 months, six of 16 ND-GBM patients showed no evidence of tumor recurrence. Median PFS in newly diagnosed patients was 16.9 months, and median OS was 38.4 months.

Conclusions

Expression of four ICT-107 targeted antigens in the pre-vaccine tumors correlated with prolonged overall survival and PFS in ND-GBM patients. The goal of targeting tumor antigens highly expressed on glioblastoma cancer stem cells is supported by the observation of decreased or absent CD133 expression in the recurrent areas of gadolinium-enhanced tumors.     

Doxorubicin Encapsulated in Long-Circulating Thermosensitive Liposomes

Abstract

Doxorubicin (DXR) was encapsulated in long-circulating, thermosensitive liposomes (TSL, 180-200 nm in mean diameter), prepared from dipalmitoyl phosphatidyl choline (DPPC)/distearoyl phosphatidyl choline (DSPC) (9:1, m/m) and either 3 mol% of amphipathic polyethylene glycol (PEG) with 1000 in average molecular weight or 6 mol% of ganglioside GMI (GMI), with 95-98% entrapping efficiency by the pH gradient method. 57% or 45% of the entrapped DXR was released from PEG/DPPC/DSPC or G_M1/DPPC/DSPC liposomes, respectively, by incubation with 20% serum at 42°C for 5 min. Inclusion of PEG or G_M1 endowed TSL with prolonged circulation ability, resulting in increased blood levels of liposomes and decreased reticuloendothelial system (RES) uptake over 6 hours after injection. Concomitantly, high DXR level in blood was kept for long time.

Accumulation of DXR into tumor tissue of tumor-bearing mice (mouse colon carcinoma 26) by local hyperthermia after injection of DXR-long-circulating TSL was 2 times or 7 times higher than that after treatment with DXR-TSL liposomes or free DXR in combination with hyperthermia, respectively. Furthermore, the systemic treatment with DXR-long-circulating TSL and hyperthermia resulted in effective tumor growth retardation and increased survival time. These results indicate that the combination of long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.     

Liposome-Trapped Nystatin in Growth Inhibition of Aspergillus niger

Abstract

Liposome-trapped nystatin caused growth inhibition of the wild type A. niger and its nystatin-resistant mutant at concentrations at which the free drug or free drug plus empty liposomes was significantly less effective. In the case of the mutant, negatively charged liposomes made from phosphatidylcholine (PC)/ phosphatidyiethanolamine (PE)ldicetyl phosphate (DCP) (1:1:0.22) showed the highest efficiency (75%). However, neutral liposomes made from PC/PE (1:1) caused 68% growth inhibition of the wild type and were most effective in this system. Growth inhibition studies as a function of time and increasing concentrations of nystatin entrapped in the above liposome systems showed significantly greater inhibition of both the wild type and the mutant. This indicates that superior efficacy is due to entrapment of nystatin in liposomes. the increased efficiency of negatively charged liposomes in the case of the mutant may be due to electrostatic attraction that favors the interaction between the liposomal membrane and the fungal cell surface in this case.     

Enhanced Delivery and Antitumor Effect of Doxorubicin Encapsulated in Long-Circulating Liposomes

Abstract

Long-circulating liposomes containing amphipathic polyethyleneglycol (PEG) or ganglioside GM1 (GM1) have been tested for their utility as enhanced delivery system of doxorubicin (DXR) in vivo. DXR was entrapped into liposomes by pH gradient method.

The long-circulating LUV (200 nm in size) composed of DSPC/CH (1:1, m/m) and either 6 mol% of DSPE-PEG1000 or GM1 entrapped DXR with >95% in trapping efficiency. DXR-long-circulating LUVs were administered to leukemic (LI210) mice via the tail vein at a dose of 5mg DXR/kg. The high blood concentration was kept for long time, and significantly increased survival time was observed as compared with free DXR and DXR-LUV. The data indicated that DXR was slowly released from long-circulating LUV during that stayed in bloodstream for long time. Administration of DXR-long-circulating SUV (100 nm) to the colon 26 bearing mice produced the increased DXR level in tumor compared with bear SUV or free drug did, respectively, and resulted in effective tumor growth retardation and increased survival time. DXR was delivered to tumor by accumulation of SUVs themselves.

Long-circulating thermosensitive liposomes (TSL) were prepared from DPPC /DSPC (9:1, m/m) and 3-6 mol% of PEG1000 or GM1. DXR was entrapped with >95% in trapping efficiency. Accumulation of DXR into tumor tissue by local hyperthermia after injection of DXR-long-circulating TSL to colon 26 bearing mice was significantly higher man that of DXR-bare TSL or free DXR, and resulted in effective tumor growth retardation and increased survival time. It was suggested that the entrapped DXR was efficiently released from long-circulating TSL by hyperthermia at the tumor site and entered the tumor tissue by simple diffusion.     

Receptor-Specific Delivery of Liposomes Via Folate-Peg-Chol

Abstract

A novel lipophilic conjugate of folate, folate-PEG-Chol, was synthesized and evaluated for receptor-mediated targeting of liposomes to tumor cells. Liposomes composed of DSPC/Chol/PEG-DSPE/folate-PEG-Chol (60/ 34/5/1, m/m) were taken up by cultured folate receptor-bearing KB cells via a saturable mechanism. Cellular binding of these liposomes could be competitively inhibited by free folic acid with an IC₅₀ of 0.39 mM, indicating an extraordinarily high binding affinity. Fluorescence micrographs of KB cells treated with targeted liposomes encapsulating calcein showed that they were distributed both on the cell surface and in intracellular vesicular compartments. Targeted liposomes carrying doxorubicin were shown to be 38 times more toxic to KB cells than non-targeted control liposomes. A biodistribution study in receptor-positive tumor-bearing C57BL/6 mice showed no significant differences between the tumor uptake of folate-PEG-liposomes and non-targeted control liposomes. This study has demonstrated that cholesterol could be used as an alternative to phospholipids as an effective anchor for incorporation of a targeting ligand into liposomes.     

The Design of a Pharmaceuttcally Acceptable Liposomal Formulation of Recombinant Interleukin-2 (Ril-2) for Locoregional Anticancer Immunotherapy

Abstract

Liposomes composed of egg-phosphatidylcholine and egg-phosphati-dylglycerol containing recombinant interleukin-2 (rIL-2) were prepared and characterised for use in locoregional anticancer immunotherapy. During the encapsulation studies it was observed that the protein precipitated. Therefore, the impact of the precipitation phenomenon on the characteristics and in vivo performance of rIL-2 liposomes was studied. Recombinant IL-2 was diluted in various aqueous media and the amount of precipitated protein determined. Also, the in vitro bioactivity, chemical stability, and in vivo antitumor efficacy of liposomes prepared with precipitated rIL-2 or non-precipitated rIL-2 were assessed.

Massive protein precipitation (60 up ± 95% of total protein) was observed upon dilution of rIL-2 in salt-containing media, but not upon dilution in 5% glucose or water. Liposomes prepared with precipitated rIL-2 were shown to release 50% of the entrapped rIL-2 over a three-day period at 37° in protein-containing media. Loss of rIL-2 bioactivity and chemical integrity was observed during storage at 4° over a 4-week period. Locoregional administration of precipitated rIL-2 in the guinea pig Line 10 tumor model resulted in significantly more tumor growth inhibition than administration of non-precipitated rIL-2. Liposomes containing non-precipitated rIL-2 were found to elicit similar antitumor effects as precipitated rIL-2. The results point to the importance of proper characterisation of new rIL-2 formulations as the physical properties of formulated rIL-2 may strongly influence its bioactivity.     

Tissue distribution of 99mic-labeled liposomes prepared from synthetic amphiphiles containing amino acid residues

Abstract

The tissue distribution of ^99mTc-labeled liposomes prepared from synthetic amphiphiles containing amino acid residues was investigated for application to radiopharmaceuticals. The amphiphiles used were N,N-didodecyl-N α-[6-(trimethylammoniohexanoyl]-L-ala-ninamide bromide (N+C₅Ala2C₁₂), N,N-didodecyl-N_α-{6-[dimethyl(2-carboxyethyl)ammonio]hexanoyl}-L-alaninamide bromide (CAC₂N⁺C₅Ala2C₁₂) and S-{l-carboxy-2-([2,3-bis (he xadecyloxy)propoxy]carbony1)ethyl}homocy ste ine. These liposomes were stable in saline and 50% serum at 37° for at least 24h in comparison with the liposomes prepared from phosphatidylcholine and cholesterol (1:1). Most of the radioactivity of N+C₅Ala2C₁₂ and CAC₂N+C₅Ala2C₁₂ liposomes was firmly bound to Ehrlich ascites tumor cells in vitro. But the accumulation of three liposomes into the tumor of Ehrlich solid tumor-bearing mice after intravenous injection was low and most of the liposomes was taken up highly in liver and spleen which belong to the reticuloendothelial system (RES). Some approaches were made to reduce the RES uptake of N+C5Ala2C12 liposomes as follows: (1) the pretreatment of dextran sulfate depressed the uptake of the liposomes in the liver accompanied by increasing uptake in tumor and other tissues except stomach, (2) the modification of the liposomes with n-dodecyl glucoside or n-dodecyl sucrose depressed the uptake in liver and spleen, resulting in an increase in blood and other tissues such as tumor, duodenum and kidney, (3) the modification of the liposomes with ganglioside GM3 or GM1 reduced the uptake in liver and spleen, but increased scarcely the uptake in blood and tumor because of the rapid excretion into urine, (4) the intraperitoneal injection reduced the uptake of the liposomes in liver and increased significantly the accumulation in pancreas.     

Synthesis and biological evaluation of new 4-thiazolidinone derivatives

A series of new 2-aryl-4-thiazolidinones (3 and 4) was synthesized from 2-hydroxy-2,2-diphenyl-N’-[(substituted phenyl)methylene]acetohydrazides (2) and mercaptoacetic acid or 2-mercaptopropionic acid. The antimycobacterial activity of these compounds was determined and several leads with 95–99% inhibition at 6.25 μg/mL test concentration were identified. In addition, antitumor activities were measured against several tumor cell lines, and significant growth inhibition was observed for compound 4p. Taken together, 2-aryl-4-thiazolidinones were shown to be promising scaffolds for both antimycobacterial and tumor-targeting compounds.     

Tumor-specific idiotype vaccines. I. Generation and characterization of internal image tumor antigen

The concept of idiotype vaccines against tumor-associated antigens (TAA) was tested in the DBA/2 L1210 lymphoma subline, L1210/GZL. Monoclonal antibodies against a TAA that cross-reacts with the envelope glycoprotein gp52 of the mammary tumor virus were used to make hybridoma anti-idiotype antibodies (Ab2). In this report we describe the characterization of monoclonal anti-idiotypic antibodies against the combining site of 11C1 (Ab1), which recognizes a shared determinant of gp52 of mouse mammary tumor virus (MMTV) and the TAA of L1210/GZL. Hybridomas expressing the internal image of gp52 were screened by an idiotype inhibition assay. Mice sensitized with radiated L1210/GZL cells produced specific delayed type hypersensitivity (DTH) against the Ab2 hybridoma. Five Ab2 hybridomas were selected and were used to immunize DBA/2 mice. Such immunized animals showed specific DTH reaction against a challenge with the L1210/GZL tumor cells. Similar results were obtained in mice immunized with purified Ab2. Fluorescence-activated cell sorter analysis demonstrated that fluorescence staining of L1210/GZL cells by 11C1 can be completely inhibited with preabsorption on Ab2 hybridoma cells. Mice immunized with 2F10 and 3A4 coupled to keyhole limpet hemocyanin (KLH) contained antibodies binding to MMTV. But only in mice immunized with 2F10-KLH was significant inhibition of L1210/GZL tumor growth observed. Collectively, these results indicate that certain anti-idiotypic antibodies can mimic the MMTV gp52 antigen, as well as the gp52-like epitope expressed on the L1210/GZL tumor cells. These properties of anti-idiotypic antibodies mimicking TAA could be exploited for making idiotype vaccines against tumors.     

Polaprezinc prevents ongoing thioacetamide-induced liver fibrosis in rats

AimsCirrhotic patients commonly have a liver zinc deficiency, which may aggravate liver fibrosis due to the lack of antioxidative effects of zinc. This study examined the ability of polaprezinc, N-(3-aminopropionyl)-l-histidinato zinc, to prevent fibrosis in a rat model of thioacetamide (TAA)-induced hepatic fibrosis.Main methodsLiver cirrhosis was induced by orally administering TAA for 20 weeks. The rats were cotreated with one of the following for the last 10 weeks of TAA treatment: (1) polaprezinc (50 or 200 mg/kg/day); (2) l-carnosine (155 mg/kg/day), which contained equal amounts of l-carnosine as 200 mg/kg/day polaprezinc; (3) zinc sulfate (112 mg/kg/day) or (4) zinc-l-aspartic complex (317.8 mg/kg/day). Both zinc supplementations contained equal amounts of zinc as high-dose polaprezinc.Key findingsHepatic zinc levels fell significantly in rats treated with TAA for 20 weeks. Cotreating with high-dose polaprezinc and zinc-l-aspartic complex for 10 weeks prevented hepatic zinc loss. Hepatic hydroxyproline and tissue inhibitor of metalloproteinases-1 (TIMP-1) were significantly higher in rats treated with TAA for 20 weeks than 10 weeks, whereas polaprezinc prevented changes in these fibrosis markers and reduced hepatic transforming growth factor-β1 protein concentration, macroscopic and histologic changes. TAA caused oxidative stress-related changes in the liver that were prevented by high-dose polaprezinc and partially by zinc-l-aspartic complex. Treatment with l-carnosine, low-dose polaprezinc or zinc sulfate for 10 weeks did not affect liver fibrosis progression or oxidative stress-related changes.SignificancePolaprezinc may prevent ongoing fibrosis by preventing zinc depletion, oxidative stress and fibrosis markers in cirrhotic livers.     

重组腺相关病毒介导的RNAi靶向治疗作用研究

目的:采用前期成功构建的靶向沉寂CDK2基因的重组腺相关病毒r AAV-sh RNA-CDK2转染人肝癌Hep G2细胞,研究其对人肝癌细胞增殖的抑制作用。方法:取人肝癌Hep G2细胞于裸鼠前肢腋下接种,构建裸鼠皮下移植瘤模型,将成瘤裸鼠随机分为三组:肿瘤组、NC对照组、r AAV-sh RNA-CDK2给药组。各试验组均通过尾静脉注射给药,每隔五天用游标卡尺测量肿瘤的长径(a)、短径(b),计算肿瘤体积。根据每组裸鼠移植瘤体积的平均值,绘制移植瘤生长曲线。于给药24 h后处死,称取瘤重,计算抑瘤率,应用实时荧光定量PCR和Western blot方法检测各组肝癌组织中CDK2基因m RNA和蛋白的表达量,观察r AAV-sh RNA-CDK2对肝癌组织CDK2表达的影响;结果:r AAV-sh RNA-CDK2能够显著抑制肝癌Hep G2细胞的增殖,其抑瘤率为72.18%;并能够下调肝癌组织中CDK2基因m RNA与蛋白表达量;结论:r AAV-sh RNA-CDK2实现了体内靶向治疗肝癌的目的,并确定静脉定量给药方式。     

Dodecaborate lipid liposomes as new vehicles for boron delivery system of neutron capture therapy

closo-Dodecaborate lipid liposomes were developed as new vehicles for boron delivery system (BDS) of neutron capture therapy. The current approach is unique because the liposome shell itself possesses cytocidal potential in combination with neutron irradiation. The liposomes composed of closo-dodecaborate lipids DSBL and DPBL displayed high cytotoxicity with thermal neutron irradiation. The closo-dodecaborate lipid liposomes were taken up into the cytoplasm by endocytosis without degradation of the liposomes. Boron concentration of 22.7 ppm in tumor was achieved by injection with DSBL-25% PEG liposomes at 20 mg B/kg. Promising BNCT effects were observed in the mice injected with DSBL-25% PEG liposomes: the tumor growth was significantly suppressed after thermal neutron irradiation (1.8 × 10¹² neutrons/cm²).     

Discovery of a series of small molecules as potent histone deacetylase inhibitors

Abstract

A series of small molecules were designed and synthesized based on our previous virtual screening approach, which was performed to discover potent histone deacetylase inhibitors (HDACIs) with novel structures. The derived compounds were tested by Hela cell nucleus extract for enzyme inhibition assay. Tumor cell growth inhibition assays were performed using a series of tumor cell lines. Molecule 4h has the best performance among these compounds with enzyme inhibition IC₅₀ of 0.14?μM and tumor cell growth inhibition IC₅₀ of 1.85 (U937), 2.02 (HL60), 2.67 (K562). Docking studies showed that multiple H-bonds and hydrophobic interactions make 4h binding to the active site of HDAC. 4h has the advantage of low molecular weight, so a variety of structural modifications can be performed in our further studies.     

Induction of alloreactive cytotoxic T lymphocytes by intra-splenic immunization with allogeneic class I Major Histocompatibility Complex DNA and DC-chol cationic liposomes

Abstract

A simple strategy for designing a cancer immunotherapeutic system involves modification of tumor cells from tumor-bearing animals in vivo in such a way that the host can evoke a specific immune response against them. We have expressed allogeneic class I major histocompatibility complex (MHC) molecules on tumor cells, through ex vivo DNA-mediated gene transfer. These molecules are potent immuno-modulators for the stimulation of strong immune reactions against certain malignancies. In order to achieve efficient gene delivery to tumor cells in vivo we have compared the efficiencies of gene transfer into mammalian tumor cells by the biolistic particle delivery system and cationic liposomes. In this report, we have demonstrated that cationic liposomes prepared by DC-chol and DOPE gives the best efficiency of transfection for tumor cells in vivo. We also showed that a strong anti-H-2K^b allo-reactive cytotoxic T lymphocyte (CTL) response could be generated following in vivo immunization of AKR/J mouse spleens with the H-2K^b gene and DC-chol cationic liposomes. The direct immunization of mouse spleens to induce cell-mediated immunity against exogenous antigens may allow alternative treatment strategies for cancer immunotherapy.     

T cells redirected to interleukin-13Rα2 with interleukin-13 mutein–chimeric antigen receptors have anti-glioma activity but also recognize interleukin-13Rα1

Background aimsOutcomes for patients with glioblastoma remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting interleukin (IL)13Rα2, human epidermal growth factor receptor 2, epidermal growth factor variant III or erythropoietin-producing hepatocellular carcinoma A2 has shown promise for the treatment of glioma in preclinical models. On the basis of IL13Rα2 immunotoxins that contain IL13 molecules with one or two amino acid substitutions (IL13 muteins) to confer specificity to IL13Rα2, investigators have constructed CARS with IL13 muteins as antigen-binding domains. Whereas the specificity of IL13 muteins in the context of immunotoxins is well characterized, limited information is available for CAR T cells.MethodsWe constructed four second-generation CARs with IL13 muteins with one or two amino acid substitutions, and evaluated the effector function of IL13-mutein CAR T cells in vitro and in vivo.ResultsT cells expressing all four CARs recognized IL13Rα1 or IL13Rα2 recombinant protein in contrast to control protein (IL4R) as judged by interferon-γ production. IL13 protein produced significantly more IL2, indicating that IL13 mutein–CAR T cells have a higher affinity to IL13Rα2 than to IL13Rα1. In cytotoxicity assays, CAR T cells killed IL13Rα1- and/or IL13Rα2-positive cells in contrast to IL13Rα1- and IL13Rα2-negative controls. Although we observed no significant differences between IL13 mutein–CAR T cells in vitro, only T cells expressing IL13 mutein–CARs with an E13K amino acid substitution had anti-tumor activity in vivo that resulted in a survival advantage of treated animals.ConclusionsOur study highlights that the specificity/avidity of ligands is context-dependent and that evaluating CAR T cells in preclinical animal model is critical to assess their potential benefit.     

Deletion of kif3a in CK19 positive cells leads to primary cilia loss,biliary cell proliferation and cystic liver lesions in TAA-treated mice

Background & aimsLoss of primary cilia in epithelial cells is known to cause cystic diseases of the liver and kidney. We have previously shown that during experimental and human cirrhosis that primary cilia were predominantly expressed on biliary cells in the ductular reaction. However, the role of primary cilia in the pathogenesis of the ductular reaction is not fully understood.MethodsPrimary cilia were specifically removed in biliary epithelial cells (BECs) by the administration of tamoxifen to Kif3a^f/f;CK19^CreERT mice at week 2 of a 20-week course of TAA treatment. Biliary progenitor cells were isolated and grown as organoids from gallbladders. Cells and tissue were analysed using histology, immunohistochemistry and Western blot assays.ResultsAt the end of 20 weeks TAA administration, primary cilia loss in liver BECs resulted in multiple microscopic cystic lesions within an unaltered ductular reaction. These were not seen in control mice who did not receive TAA. There was no effect of biliary primary cilia loss on the development of cirrhosis. Increased cellular proliferation was seen within the cystic structures associated with a decrease in hepatocyte lobular proliferation. Loss of primary cilia within biliary organoids was initially associated with reduced cell passage survival but this inhibitory effect was diminished in later passages. ERK but not WNT signalling was enhanced in primary cilia loss-induced cystic lesions in vivo and its inhibition reduced the expansion of primary cilia deficient biliary progenitor cells in vitro.ConclusionsTAA-treated kif3a BEC-specific knockout mice had an unaltered progression to cirrhosis, but developed cystic lesions that showed increased proliferation.     

Racial disparities in total ankle arthroplasty utilization and outcomes

IntroductionThe objective of this study was to examine the racial disparities in total ankle arthroplasty (TAA) utilization and outcomes.MethodsWe used the National Inpatient Sample (NIS) to study the time-trends. Race was categorized as White and Black. Utilization rates were calculated for the U.S. general population per 100,000. Hospital length of stay, discharge disposition and mortality after TAA were assessed. We used the Cochran Armitage trend test to assess time-trends from 1998 to 2011 and chi-square test to compare TAA utilization. We used analysis of variance or chi-squared test to compare the characteristics of Whites and Blacks undergoing TAA and logistic regression to compare mortality, length of stay and discharge to home versus medical facility.ResultsThe mean ages for Whites undergoing TAA were 62 years and for Blacks was 52 years. Significant racial disparities were noted in TAA utilization rates (/100,000) in 1998, 0.14 in Whites vs. 0.07 in Blacks (P < 0.0001; 2-fold) and in 2011, 1.17 in Whites vs. 0.33 in Blacks (P < 0.0001; 4-fold). Racial disparities in TAA utilization increased significantly from 1998 to 2011 (P < 0.0001). There was a trend towards statistical significance for the difference in the length of hospital stay in Blacks vs. Whites (52.9% vs. 44.3% with length of hospital stay higher than the median; P = 0.08). Differences in the proportion discharged to an inpatient medical facility after TAA, 16.6% Blacks vs. 13.4% Whites, were not significant (P = 0.36).ConclusionsThis study demonstrated significant racial disparities with lower TAA utilization and suboptimal outcomes in Blacks compared to Whites. Further studies are needed to understand the mediators of these disparities and to assess whether these mediators can be targeted to reduce racial disparities in TAA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0589-2) contains supplementary material, which is available to authorized users.