A RelA(p65) Thr505 phospho-site mutation reveals an important mechanism regulating NF-κB-dependent liver regeneration and cancer.

Post-translational modifications of nuclear factor (NF)-κB subunits provide a mechanism to differentially regulate their activity in response to the many stimuli that induce this pathway. However, the physiological significance of these modifications is largely unknown, and it remains unclear if these have a critical role in the normal and pathological functions of NF-κB in vivo. Among these, phosphorylation of the RelA(p65) Thr505 residue has been described as an important regulator of NF-κB activity in cell lines, but its physiological significance was not known. Therefore, to learn more about the role of this pathway in vivo, we generated a knockin mouse with a RelA T505A mutation. Unlike RelA knockout mice, the RelA T505A mice develop normally but exhibit aberrant hepatocyte proliferation following liver partial hepatectomy or damage resulting from carbon tetrachloride (CCl4) treatment. Consistent with these effects, RelA T505A mice exhibit earlier onset of cancer in the N-nitrosodiethylamine model of hepatocellular carcinoma. These data reveal a critical pathway controlling NF-κB function in the liver that acts to suppress the tumour-promoting activities of RelA.

Pancreatic progenitor-derived hepatocytes are viable and functional in a 3D high density bioreactor culture system.

The rat pancreatic progenitor cell line B-13 is of interest for research on drug metabolism and toxicity since the cells trans-differentiate into functional hepatocyte-like cells (B-13/H) when treated with glucocorticoids. In this study we investigated the trans-differentiation and liver-specific functions of B-13/H cells in a three-dimensional (3D) multi-compartment bioreactor, which has already been successfully used for primary liver cell culture. Undifferentiated B-13 cells were inoculated into the bioreactor system and exposed to dexamethasone to promote hepatic trans-differentiation (B-13/HT). In a second approach, pre-differentiated B-13 cells were cultured in bioreactors for 15 days to evaluate the maintenance of liver-typical functions (B-13/HP). During trans-differentiation of B-13 cells into hepatocyte-like cells in the 3D bioreactor system (approach B-13/HT), an increase in glucose metabolism and in liver-specific functions (urea and albumin synthesis; cytochrome P450 [CYP] enzyme activity) was observed, whereas amylase - characteristic for exocrine pancreas and undifferentiated B-13 cells - decreased over time. In bioreactors with pre-differentiated cells (approach B-13/HP), the above liver-specific functions were maintained over the whole culture period. Results were confirmed by gene expression and protein analysis showing increased expression of carbamoyl-phosphate synthase 1 (CPS-1), albumin, CYP2E1, CYP2C11 and CYP3A1 with simultaneous loss of amylase. Immunohistochemical studies showed the formation of 3D structures with expression of liver-specific markers, including albumin, cytokeratin (CK) 18, CCAAT/enhancer-binding protein beta (CEBP-ß), CYP2E1 and multidrug resistance protein 2 (MRP2). In conclusion, successful culture and trans-differentiation of B-13 cells in the 3D bioreactor was demonstrated. The requirement for only one hormone and simple culture conditions to generate liver-like cells makes this cell type useful for in vitro research using 3D high-density culture systems.

NFκB1 is a suppressor of neutrophil-driven hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) develops on the background of chronic hepatitis. Leukocytes found within the HCC microenvironment are implicated as regulators of tumour growth. We show that diethylnitrosamine (DEN)-induced murine HCC is attenuated by antibody-mediated depletion of hepatic neutrophils, the latter stimulating hepatocellular ROS and telomere DNA damage. We additionally report a previously unappreciated tumour suppressor function for hepatocellular nfkb1 operating via p50:p50 dimers and the co-repressor HDAC1. These anti-inflammatory proteins combine to transcriptionally repress hepatic expression of a S100A8/9, CXCL1 and CXCL2 neutrophil chemokine network. Loss of nfkb1 promotes ageing-associated chronic liver disease (CLD), characterized by steatosis, neutrophillia, fibrosis, hepatocyte telomere damage and HCC. Nfkb1(S340A/S340A)mice carrying a mutation designed to selectively disrupt p50:p50:HDAC1 complexes are more susceptible to HCC; by contrast, mice lacking S100A9 express reduced neutrophil chemokines and are protected from HCC. Inhibiting neutrophil accumulation in CLD or targeting their tumour-promoting activities may offer therapeutic opportunities in HCC.

The potential contribution of tumour-related factors to the development of FOLFOX-induced sinusoidal obstruction syndrome.

BACKGROUND: Chemotherapy-associated liver injury (CALI) has been linked to increased morbidity and poorer disease-specific outcomes in patients undergoing resection of colorectal liver metastases (CRLM). The aim of this study was to assess the contribution of tumour-related factors to the development of FOLFOX-induced liver injury. METHODS: We assessed the effect of FOLFOX treatment on the murine liver either in the presence or absence of CRLM to evaluate the contribution of both chemotherapy and tumour death to the development of CALI. RESULTS: In the presence of liver metastases, there was increased hepatic expression of plasminogen activator inhibitor-1 (146-fold; P<0.01) and vWF (2.4-fold; P<0.01) transcript as compared with sham-operated controls. In addition, we detected large clusters of megakaryocytes in the spleen of FOLFOX-treated tumour-bearing animals. The livers of FOLFOX-treated animals also showed changes in matrix remodelling genes such as TGFß (P<0.01), MMP2 (P<0.001), TIMP1 (P<0.001) and Pro-Collagen I (P<0.05) which was exacerbated in the presence of tumour. These genes have previously been demonstrated to have a key role in FOLFOX-induced liver injury. CONCLUSION: It appears that the toxicity of FOLFOX chemotherapy is enhanced by tumour-related factors.

Pathogenesis of FOLFOX induced sinusoidal obstruction syndrome in a murine chemotherapy model.

BACKGROUND & AIMS: Sinusoidal obstruction syndrome (SOS) following oxaliplatin based chemotherapy can have a significant impact on post-operative outcome following resection of colorectal liver metastases. To date no relevant experimental models of oxaliplatin induced SOS have been described. The aim of this project was to establish a rodent model which could be utilised to investigate mechanisms underlying SOS to aid the development of therapeutic strategies. METHODS: C57Bl/6 mice, maintained on a purified diet, were treated with intra-peritoneal FOLFOX (n=10), or vehicle (n=10), weekly for five weeks and culled one week following final treatment. Sections of the liver and spleen were fixed in formalin and paraffin embedded for histological analysis. The role of oxidative stress on experimental-induced SOS was determined by dietary supplementation with butylated hydroxyanisole and N-acetylcysteine. RESULTS: FOLFOX treatment was associated with the development of sinusoidal dilatation and hepatocyte atrophy on H&E stained sections of the liver in keeping with SOS. Immunohistochemistry for p21 demonstrated the presence of replicative senescence within the sinusoidal endothelium. FOLFOX induced endothelial damage leads to a pro-thrombotic state within the liver associated with upregulation of PAI-1 (p<0.001), vWF (p<0.01) and Factor X (p<0.001), which may contribute to the propagation of liver injury. Dietary supplementation with the antioxidant BHA prevented the development of significant SOS. CONCLUSIONS: We have developed the first reproducible model of chemotherapy induced SOS that reflects the pathogenesis of this disease in patients. It appears that the use of antioxidants alongside oxaliplatin based chemotherapy may be of value in preventing the development of SOS in patients with colorectal liver metastases.

BAG-1 interacts with the p50-p50 homodimeric NF-κB complex: implications for colorectal carcinogenesis.

Understanding the mechanisms that promote aberrant tumour cell survival is critical for the determination of novel strategies to combat colorectal cancer (CRC). We have recently shown that the anti-apoptotic protein BAG-1, highly expressed in pre-malignant and CRC tissue, can potentiate cell survival through regulating NF-κB transcriptional activity. In this study, we identify a novel complex between BAG-1 and the p50-p50 NF-κB homodimers, implicating BAG-1 as a co-regulator of an atypical NF-κB pathway. Importantly, the BAG-1-p50 complex was detected at gene regulatory sequences including the epidermal growth factor receptor (EGFR) and COX-2 (PTGS2) genes. Suppression of BAG-1 expression using small interfering RNA was shown to increase EGFR and suppress COX-2 expression in CRC cells. Furthermore, mouse embryonic fibroblasts derived from the NF-κB1 (p105/p50) knock-out mouse were used to demonstrate that p50 expression was required for BAG-1 to suppress EGFR expression. This was shown to be functionally relevant as attenuation of BAG-1 expression increased ligand activated phosphorylation of EGFR in CRC cells. In summary, this paper identifies a novel role for BAG-1 in modulating gene expression through interaction with the p50-p50 NF-κB complexes. Data presented led us to propose that BAG-1 can act as a selective regulator of p50-p50 NF-κB responsive genes in colorectal tumour cells, potentially important for the promotion of cell survival in the context of the fluctuating tumour microenvironment. As BAG-1 expression is increased in the developing adenoma through to metastatic lesions, understanding the function of the BAG-1-p50 NF-κB complexes may aid in identifying strategies for both the prevention and treatment of CRC.

Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis.

Myofibroblasts are critical cellular elements of wound healing generated at sites of injury by transdifferentiation of resident cells. A paradigm for this process is conversion of hepatic stellate cells (HSC) into hepatic myofibroblasts. Treatment of HSC with DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-azadC) blocked transdifferentiation. 5-azadC also prevented loss of IkappaBalpha and PPARgamma expression that occurs during transdifferentiation to allow acquisition of proinflammatory and profibrogenic characteristics. ChIP analysis revealed IkappaBalpha promoter is associated with transcriptionally repressed chromatin that converts to an active state with 5-azadC treatment. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. siRNA knockdown of MeCP2 elevated IkappaBalpha promoter activity, mRNA and protein expression in myofibroblasts. MeCP2 interacts with IkappaBalpha promoter via a methyl-CpG-dependent mechanism and recruitment into a CBF1 corepression complex. We conclude that MeCP2 and DNA methylation exert epigenetic control over hepatic wound healing and fibrogenesis.

Naltrexone, an opioid receptor antagonist, attenuates liver fibrosis in bile duct ligated rats.

AIM: The aim of this study was to investigate the hypothesis that the opioid system is involved in the development of hepatic fibrosis. METHODS: The effect of naltrexone (an opioid receptor antagonist) on hepatic fibrosis in bile duct ligated (BDL) or sham rats was assessed by histology and hepatic hydroxyproline levels. Liver matrix metalloproteinase 2 (MMP-2) was measured by zymography, and alpha smooth muscle actin (alpha-SMA) and CD45 (leucocyte common antigen) by immunohistochemistry. The redox state of the liver was assessed by hepatic glutathione (GSH)/oxidised glutathione (GSSG) and S-nitrosothiol levels. Subtypes of opioid receptors in cultured hepatic stellate cells (HSCs) were characterised by reverse transcriptase-polymerase chain reaction, and the effects of selective delta opioid receptor agonists on cellular proliferation, tissue inhibitor of metalloproteinase 1 (TIMP-1), and procollagen I expression in HSCs determined. RESULTS: Naltrexone markedly attenuated the development of hepatic fibrosis as well as MMP-2 activity (p<0.01), and decreased the number of activated HSCs in BDL rats (p<0.05). The development of biliary cirrhosis altered the redox state with a decreased hepatic GSH/GSSG ratio and increased concentrations of hepatic S-nitrosothiols, which were partially or completely normalised by treatment with naltrexone, respectively. Activated rat HSCs exhibited expression of delta1 receptors, with increased procollagen I expression, and increased TIMP-1 expression in response to delta(1) and delta(2) agonists, respectively. CONCLUSIONS: This is the first study to demonstrate that administration of an opioid antagonist prevents the development of hepatic fibrosis in cirrhosis. Opioids can influence liver fibrogenesis directly via the effect on HSCs and regulation of the redox sensitive mechanisms in the liver.

The role and regulation of hepatic stellate cell apoptosis in reversal of liver fibrosis.

Liver fibrosis and its end-stage disease cirrhosis are major world health problems arising from chronic injury of the liver by a variety of etiological factors including viruses, alcohol and drug abuse, the metabolic syndrome, autoimmune disease and hereditary disorders of metabolism. Fibrosis is a progressive pathological process in which wound-healing myofibroblasts of the liver respond to injury by promoting replacement of the normal hepatic tissue with a scar-like matrix composed of cross-linked collagen. Until recently it was believed that this process was irreversible. However emerging experimental and clinical evidence is starting to show that even cirrhosis is potentially reversible. Key to this is the discovery that reversion of fibrosis is accompanied by clearance of hepatic stellate cells (HSC) by apoptosis. Furthermore, proof-of-concept studies in rodents have demonstrated that experimental augmentation of HSC apoptosis will promote the resolution of fibrosis. Consequently there is now considerable interest in determining the molecular events that regulate HSC apoptosis and the discovery of drugs that will stimulate HSC apoptosis in a selective manner. This review will consider the regulatory role played by growth factors (e.g. NGF, IGF-1, TGFbeta), death receptor ligands (TRAIL, FAS), components and regulators of extracellular matrix (integrins, collagen, matrix metalloproteinases and their tissue inhibitors) and signal transduction proteins and transcription factors (Rho/Rho kinase, Jun N-terminal Kinase (JNK), IkappaKinase (IKK), NF-kappa B). The potential for known pharmacological agents such as gliotoxin, sulfasalazine, benzodiazepine ligands, curcumin and tanshinone I to induce HSC apoptosis and therefore to be used therapeutically will be explored.

Novel sulfasalazine analogues with enhanced NF-kB inhibitory and apoptosis promoting activity.

The NF-kB transcription factor plays a key role in the regulation of apoptosis by modulating expression of a wide range of cell death control molecules. NF-kB also plays an important role in human diseases by promoting inappropriate cell survival. Small molecule inhibitors of NF-kB are therefore likely to provide novel therapeutic opportunities. Sulfasalazine (SFZ) is a synthetic anti-inflammatory comprising an aminosalicylate, 5-amino salicylic acid (5-ASA), linked to an antibiotic, sulfapyridine (SPY). SFZ, but not 5-ASA or SPY, inhibits activation of NF-kB. We synthesised a small number of SFZ analogues and determined their ability to inhibit NF-kB activity and promote apoptosis in chronic lymphocytic leukaemia and hepatic stellate cells, where NF-kB plays an important role in cell survival. Remarkably, 3 of the 6 analogues synthesised were significantly more effective (up to 8-fold) inhibitors of NF-kB dependent transcription and this increased activity was associated with enhanced apoptosis. Therefore, it is possible to readily improve the NF-kB inhibiting activity of SFZ and analogues of SFZ may be attractive therapeutic agents for malignancies and chronic liver disease where NF-kB is thought to play a significant role.

Herpesvirus saimiri-based vector biodistribution using noninvasive optical imaging.

Herpesvirus saimiri (HVS) is capable of infecting a range of human cell types with high efficiency and the viral genome persists as high copy number, circular, nonintegrated episomes which segregate to progeny upon cell division. This allows the HVS-based vector to stably transduce a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Here we assess the dissemination of HVS-based vectors in vivo following intravenous and intraperitoneal administration. Bioluminescence imaging of an HVS-based vector expressing luciferase demonstrates that the virus can infect and establish a persistent latent infection in a variety of mouse tissues. Moreover, the long-term in vivo maintenance of the HVS genome as a nonintegrated circular episome provided sustained expression of luciferase over a 10-week period. A particularly high level of transgene expression in the liver and the ability of HVS to infect and persist in hepatic stellate cells suggest that HVS-based vectors may have potential for the treatment of inherited and acquired liver diseases.

Histidine-stimulated divalent metal uptake in human erythrocytes and in the erythroleukaemic cell line HEL.92.1.7.

The uptake of 65Zn by human erythrocytes was investigated in the presence of high (40 mM) and low (5 mM) concentrations of histidine and 0-500 microM cobalt, nickel, manganese and zinc. Varying concentrations of metal mono- and bis-histidine complexes will be formed and the inhibition of 65Zn uptake could be correlated with the calculated complex concentrations to investigate competition between metals. For each metal, the calculated concentrations of bis-histidine complex giving 50% inhibition of 65Zn uptake were similar at both 5 mM and 40 mM histidine. Manganese-bis-histidine appeared to have a much higher affinity for the binding site than the other metal-bis-histidine complexes, which had similar affinities to each other. Studies of the inhibition of histidine-stimulated 54Mn uptake by the addition of manganese confirmed that manganese-bis-histidine does act as a substrate for the transporter in a similar fashion to the other metals studied. In addition, human erythroleukaemic cells (HEL cells) were used as a model for erythroid precursor cells. L-histidine, but not D-histidine, stimulated 65Zn uptake in a saturable fashion. The other metals competed with zinc in a similar manner to that seen in erythrocytes, and the affinity for manganese-bis-histidine was much greater than for the bis-histidine complexes of the other three metals. Both the capacity for metal transport per cell, and the affinity of the transporter for the metal-bis-histidine complexes, were much greater in the HEL cells than in the erythrocyte. It is suggested that histidine-stimulated metal transport may play a role in the supply of metals to maturing erythroid cells.

Patient and operator dose during fluoroscopic examination of swallow mechanism.

Dose-area product (DAP) measurements were made for 21 patients undergoing a modified barium swallow. The procedures were performed by a radiologist and speech and language therapist, to characterize swallowing disorders in patients with head or spinal injury, stroke, other neurological conditions or simple globus symptoms, in order to inform feeding strategies. The DAP values were used to estimate effective dose to the patient, in order to provide a measure of the radiation risk associated with the procedure. Whole body doses to operators, together with equivalent doses to extremities and eyes were also measured to inform the employer's risk assessment. Median DAP for the series was 3.5 (3.1-5.2) Gycm(2) with a corresponding effective dose to the patient of 0.85 (0.76-1.3) mSv, and a low associated risk, mainly of cancer induction, of about 1 in 16 000. The organ receiving the greatest dose was the thyroid, with a calculated median equivalent dose of 13.9 (12.3-20.7) mSv. Median screening time was 3.7 (2.5-4.3) min. Mean operator doses were 0.5 mSv equivalent dose (eyes), 0.9 mSv (extremities), and less than 0.3 mSv whole body dose. Extrapolating for an annual workload of 50 patients per year, this work will lead to annual operator doses of less than 0.6 mSv whole body dose, and approximately 1 mSv equivalent dose (eyes) and 1.8 mSv (extremities), against corresponding legal dose limits of 20 mSv, 150 mSv and 500 mSv, respectively.

Improvement in activities of daily living in elderly following treatment for post-bereavement depression.

OBJECTIVE: To determine if elderly bereaved depressed subjects display difficulty with activities of daily living (ADL) and if their ADL difficulty improves with psychopharmacologic intervention. METHOD: The Assessment of Motor and Process Skills (AMPS), an ADL assessment measuring ADL motor and ADL process skills, was administered to a pilot sample of elderly persons with post-bereavement depression prior to psychopharmacologic intervention and subsequently during treatment response. RESULTS: In the pretreatment phase, subjects displayed difficulty with ADL motor and ADL process skills that significantly improved during the treatment response phase. CONCLUSION: Results suggest that elderly bereaved depressed individuals demonstrated ADL difficulty that responds positively to psychopharmacologic intervention.

Influence of a stearic acid-rich structured triacylglycerol on postprandial lipemia, factor VII concentrations, and fibrinolytic activity in healthy subjects.

BACKGROUND: An elevated postprandial lipid concentration is believed to be atherogenic and to increase the risk of thrombosis. OBJECTIVE: The objective was to test whether the consumption of a stearic acid-rich structured triacylglycerol has adverse effects on postprandial fibrinolytic activity and lipemia, factor VII coagulant (FVII:c) activity, and activated FVII (FVIIa) concentrations. DESIGN: A randomized crossover design was used to compare the effects on middle-aged healthy men (n = 17) and women (n = 18) of meals enriched with cocoa butter, high-oleate sunflower oil (oleate), or a structured triacylglycerol containing stearic acid. RESULTS: The mean increases from fasting in plasma triacylglycerol 3 h after the oleate, cocoa butter, and structured triacylglycerol meals were 1.36 (95% CI: 1.17, 1.56), 1.39 (1.17,1.63), and 0.65 (0.50, 0.82) mmol/L, respectively. Tissue plasminogen activator activity increased and plasminogen activator type 1 activity decreased after all 3 meals. Plasma FVII:c increased after the oleate and cocoa butter meals but not after the structured triacylglycerol meal. The values 6 h after the oleate and cocoa butter meals were 11.3% (7.0%, 15.6%) and 9.9% (4.7%, 15.2%), respectively, and were significantly different (P < 0.0001 and P = 0.001, respectively) from the value after the triacylglycerol meal [2.1% (-1.1%, 5.3%)]. Plasma FVIIa increased after all 3 meals, more so after the oleate and cocoa butter meals than after the structured triacylglycerol meal. CONCLUSION: The consumption of stearic acid in the form of a structured triacylglycerol leads to less of an increase in plasma triacylglycerol and in FVII:c than does a meal enriched in cocoa butter or oleate.

A validation study of the daily activities questionnaire: an activities of daily living assessment for people with Alzheimer's disease.

The Daily Activities Questionnaire (DAQ) was developed to assess activities of daily living (ADL) independence in people with Alzheimer's disease. After administering it to 276 people diagnosed with Alzheimer's disease, we examined the quality of the rating scale and its structure using a Rasch measurement approach. Results indicated that the original 10-point rating scale should be restructured to a 5-point rating scale to improve the quality of the instrument. In addition, we found that all but two ADL items defined the same construct and could be combined into a single summary measure of ADL independence. The remaining items were positioned along a hierarchical continuum, with IADL tasks more difficult than PADL tasks. Furthermore, the tasks were logically ordered by difficulty. We therefore report that the DAQ is a valid scale and conclude that it is a viable measure of ADL independence for studies of Alzheimer's disease.

Postprandial effects of an oleic acid-rich oil compared with butter on clotting factor VII and fibrinolysis in healthy men.

BACKGROUND: Factor VII coagulant activity (FVII:c) is associated with an increased risk of fatal ischemic heart disease, is correlated with plasma triacylglycerol concentration, and increases after a meal rich in long-chain fatty acids. OBJECTIVE: We planned to compare effects of meals rich in oleate and butter fat with those of a low-fat meal on FVII:c and fibrinolytic activity. DESIGN: A crossover design was used to compare the postprandial effects on coagulant and fibrinolytic activities in 12 men of 3 high-fat (95 g) meals--high oleate, butter, and oleate + medium-chain triacylglycerols (oleate+MCT)--with an isoenergetic low-fat meal (18 g MCT). The oleate+MCT blend was used to mimic the ratio of long-chain to shorter-chain fatty acids in butter. RESULTS: Neither the amount nor type of fat consumed influenced plasminogen activator inhibitor 1 or t-plasminogen activator activities or D-dimer concentration. FVII:c increased by 12.5% (95% CI: 4.6%, 20.5%) after the high-fat meals at 3 h and by 6.7% (95% CI: 1.6%, 11.7%) at 7 h and changed 7 h after the low-fat meal by -14.3% (95% CI: -3.3%, -25.4%). The responses to the high-fat meals did not differ. Measurements of activated FVII (FVIIa), FVII zymogen, and activated FXII (FXIIa) concentrations made after the low-fat and high-oleate meals showed a significant increase in FVIIa only after the high-oleate meal. CONCLUSIONS: The results of this study confirm that FVII:c falls after a low-fat meal and suggests that postprandial activation of FVII occurs rapidly after a fat-rich meal without involving an increase in FXIIa.