S -Adenosyl- l -homocysteine in brain

Administration of methionine sulfoximine (MSO) to rats and mice significantly decreased cerebral levels of S -adenosyl- l -homocysteine (AdoHcy). Concurrent administration of methionine prevented this decrease and, when methionine was given alone, significantly elevated AdoHcy levels resulted in both species. Regionally, AdoHcy levels varied from 20 nmol/g in rat cerebellum and spinal cord to about 60 nmol/g in hypothalamus and midbrain. MSO decreased AdoHcy in all regions tested except striatum, midbrain, and spinal cord. AdoMet/AdoHcy ratios (methylation index) varied from 0.48 in hypothalamus to 2.4 in cerebellum, and MSO administration decreased these ratios in all regions except hypothalamus. AdoHcy hydrolase activity was lowest in hypothalamus, highest in brainstem and, generally, varied inversely with regional AdoHcy levels. MSO decreased AdoHcy hydrolase activity in all regions except hypothalamus and spinal cord. Cycloleucine administration resulted in significantly decreased levels of mouse brain AdoHcy, whereas the administration of dihydroxyphenylalanine (DOPA) failed to affect AdoHcy levels. It is concluded that (a) cerebral AdoHcy levels are more tightly regulated than are those of AdoMet after MSO administration, (b) slight fluctuations of AdoHcy levels may be important in regulating AdoHcy hydrolase activity and hence AdoHcy catabolism in vivo, (c) the AdoMet/AdoHcy ratio reflects the absolute AdoMet concentration rather than the transmethylation flux, (d) the decreased AdoMet levels in midbrain, cortex, and striatum after MSO with no corresponding decrease in AdoHcy suggest an enhanced AdoMet utilization, hence an increased transmethylation in the MSO preconvulsant state.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45411/1/11064_2004_Article_BF00966019.pd

Modelling neurofibromatosis type 1 tibial dysplasia and its treatment with lovastatin

<p>Abstract</p> <p>Background</p> <p>Bowing and/or pseudarthrosis of the tibia is a known severe complication of neurofibromatosis type 1 (NF1). Mice with conditionally inactivated neurofibromin (Nf1) in the developing limbs and cranium (Nf1Prx1) show bowing of the tibia caused by decreased bone mineralisation and increased bone vascularisation. However, in contrast to NF1 patients, spontaneous fractures do not occur in Nf1Prx1 mice probably due to the relatively low mechanical load. We studied bone healing in a cortical bone injury model in Nf1Prx1 mice as a model for NF1-associated bone disease. Taking advantage of this experimental model we explore effects of systemically applied lovastatin, a cholesterol-lowering drug, on the Nf1 deficient bone repair.</p> <p>Methods</p> <p>Cortical injury was induced bilaterally in the <it>tuberositas tibiae </it>in Nf1Prx1 mutant mice and littermate controls according to a method described previously. Paraffin as well as methacrylate sections were analysed from each animal. We divided 24 sex-matched mutant mice into a lovastatin-treated and an untreated group. The lovastatin-treated mice received 0.15 mg activated lovastatin by daily gavage. The bone repair process was analysed at three consecutive time points post injury, using histological methods, micro computed tomography measurements and <it>in situ </it>hybridisation. At each experimental time point, three lovastatin-treated mutant mice, three untreated mutant mice and three untreated control mice were analysed. The animal group humanely killed on day 14 post injury was expanded to six treated and six untreated mutant mice as well as six control mice.</p> <p>Results</p> <p>Bone injury repair is a complex process, which requires the concerted effort of numerous cell types. It is initiated by an inflammatory response, which stimulates fibroblasts from the surrounding connective tissue to proliferate and fill in the injury site with a provisional extracellular matrix. In parallel, mesenchymal progenitor cells from the periost are recruited into the injury site to become osteoblasts. In Nf1Prx1 mice bone repair is delayed and characterised by the excessive formation and the persistence of fibro-cartilaginous tissue and impaired extracellular matrix mineralisation. Correspondingly, expression of Runx2 is downregulated. High-dose systemic lovastatin treatment restores Runx2 expression and accelerates new bone formation, thus improving cortical bone repair in Nf1Prx1 tibia. The bone anabolic effects correlate with a reduction of the mitogen activated protein kinase pathway hyper-activation in Nf1-deficient cells.</p> <p>Conclusion</p> <p>Our data suggest the potential usefulness of lovastatin, a drug approved by the US Food and Drug Administration in 1987 for the treatment of hypercholesteraemia, in the treatment of Nf1-related fracture healing abnormalities. The experimental model presented here constitutes a valuable tool for the pre-clinical stage testing of candidate drugs, targeting Nf1-associated bone dysplasia.</p

E6 and E7 from Beta Hpv38 Cooperate with Ultraviolet Light in the Development of Actinic Keratosis-Like Lesions and Squamous Cell Carcinoma in Mice

Cutaneous beta human papillomavirus (HPV) types appear to be involved in the development of non-melanoma skin cancer (NMSC); however, it is not entirely clear whether they play a direct role. We have previously shown that E6 and E7 oncoproteins from the beta HPV type 38 display transforming activities in several experimental models. To evaluate the possible contribution of HPV38 in a proliferative tissue compartment during carcinogenesis, we generated a new transgenic mouse model (Tg) where HPV38 E6 and E7 are expressed in the undifferentiated basal layer of epithelia under the control of the Keratin 14 (K14) promoter. Viral oncogene expression led to increased cellular proliferation in the epidermis of the Tg animals in comparison to the wild-type littermates. Although no spontaneous formation of tumours was observed during the lifespan of the K14 HPV38 E6/E7-Tg mice, they were highly susceptible to 7,12-dimethylbenz(a)anthracene (DMBA)/12-0-tetradecanoylphorbol-13-acetate (TPA) two-stage chemical carcinogenesis. In addition, when animals were exposed to ultraviolet light (UV) irradiation, we observed that accumulation of p21WAF1 and cell-cycle arrest were significantly alleviated in the skin of Tg mice as compared to wild-type controls. Most importantly, chronic UV irradiation of Tg mice induced the development of actinic keratosis-like lesions, which are considered in humans as precursors of squamous cell carcinomas (SCC), and subsequently of SCC in a significant proportion of the animals. In contrast, wild-type animals subjected to identical treatments did not develop any type of skin lesions. Thus, the oncoproteins E6 and E7 from beta HPV38 significantly contribute to SCC development in the skin rendering keratinocytes more susceptible to UV-induced carcinogenesis

A Canadian Critical Care Trials Group project in collaboration with the international forum for acute care trialists - Collaborative H1N1 Adjuvant Treatment pilot trial (CHAT): study protocol and design of a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Swine origin influenza A/H1N1 infection (H1N1) emerged in early 2009 and rapidly spread to humans. For most infected individuals, symptoms were mild and self-limited; however, a small number developed a more severe clinical syndrome characterized by profound respiratory failure with hospital mortality ranging from 10 to 30%. While supportive care and neuraminidase inhibitors are the main treatment for influenza, data from observational and interventional studies suggest that the course of influenza can be favorably influenced by agents not classically considered as influenza treatments. Multiple observational studies have suggested that HMGCoA reductase inhibitors (statins) can exert a class effect in attenuating inflammation. The Collaborative H1N1 Adjuvant Treatment (CHAT) Pilot Trial sought to investigate the feasibility of conducting a trial during a global pandemic in critically ill patients with H1N1 with the goal of informing the design of a larger trial powered to determine impact of statins on important outcomes.</p> <p>Methods/Design</p> <p>A multi-national, pilot randomized controlled trial (RCT) of once daily enteral rosuvastatin versus matched placebo administered for 14 days for the treatment of critically ill patients with suspected, probable or confirmed H1N1 infection. We propose to randomize 80 critically ill adults with a moderate to high index of suspicion for H1N1 infection who require mechanical ventilation and have received antiviral therapy for ≤ 72 hours. Site investigators, research coordinators and clinical pharmacists will be blinded to treatment assignment. Only research pharmacy staff will be aware of treatment assignment. We propose several approaches to informed consent including a priori consent from the substitute decision maker (SDM), waived and deferred consent. The primary outcome of the CHAT trial is the proportion of eligible patients enrolled in the study. Secondary outcomes will evaluate adherence to medication administration regimens, the proportion of primary and secondary endpoints collected, the number of patients receiving open-label statins, consent withdrawals and the effect of approved consent models on recruitment rates.</p> <p>Discussion</p> <p>Several aspects of study design including the need to include central randomization, preserve allocation concealment, ensure study blinding compare to a matched placebo and the use novel consent models pose challenges to investigators conducting pandemic research. Moreover, study implementation requires that trial design be pragmatic and initiated in a short time period amidst uncertainty regarding the scope and duration of the pandemic.</p> <p>Trial Registration Number</p> <p><a href="http://www.controlled-trials.com/ISRCTN45190901">ISRCTN45190901</a></p

Differential Regulation of Cutaneous Oncoprotein HPVE6 by wtp53, Mutant p53R248W and ΔNp63α is HPV Type Dependent

UV exposure and p53 mutations are major factors in non-melanoma skin cancer, whereas a role for HPV infections has not been defined. Previous data demonstrated the wtp53-mediated degradation of cutaneous HPV20E6 by caspase-3. ΔNp63α and hot-spot mutant p53R248W conveyed a protective effect on HPV20E6 under these conditions. We demonstrate a differential regulation by wtp53 of the E6 genes of cutaneous types HPV4, HPV5, HPV7, HPV27, HPV38, HPV48, HPV60 and HPV77. Caspase- or proteasome-mediated down-regulation was HPV type dependent. Mutant p53R248W up-regulated expression of all these E6 proteins as did ΔNp63α except for HPV38E6 which was down-regulated by the latter. None of these cellular proteins affected HPV41E6 expression. Ectopic expression of both mutp53R248W and ΔNp63α in the normal NIKS keratinocyte cell line harbouring endogenous p53 and p63however led to a down-regulation of HPV20E6. We demonstrate that HPV20E6 expression in these cells is modulated by additional, yet unidentified, cellular protein(s), which are not necessarily involved in apoptosis or autophagy. We further demonstrate proliferation of HPV20E6-expressing keratinocytes. Levels of proteins involved in cell cycle control, cyclin-D1, cdk6 and p16INK4a, phosphorylated pRB, as well as c-Jun and p-c-Jun, were all increased in these cells. HPV20E6 did not compete for the interaction between p16INK4a with cyclin-D1 or cdk6. Phosphorylation of pRB in the HPV20E6 expressing cells seems to be sufficient to override the cytokenetic block induced by the p16INK4a/pRB pathway. The present study demonstrates the diverse influence of p53 family members on individual cutaneous HPVE6 proteins. HPV20E6 expression also resulted in varying protein levels of factors involved in proliferation and differentiation

Landscape of alcohol-related hepatocellular carcinoma in the last 15 years highlights the need to expand surveillance programs

Background &amp; Aims: Alcohol abuse and metabolic disorders are leading causes of hepatocellular carcinoma (HCC) worldwide. Alcohol-related aetiology is associated with a worse prognosis compared with viral agents, because of the lower percentage of patients diagnosed with HCC under routine surveillance and a higher burden of comorbidity in alcohol abusers. This study aimed to describe the evolving clinical scenario of alcohol-related HCC over 15 years (2006–2020) in Italy. Methods: Data from the Italian Liver Cancer (ITA.LI.CA) registry were used: 1,391 patients were allocated to three groups based on the year of HCC diagnosis (2006–2010; 2011–2015; 2016–2020). Patient characteristics, HCC treatment, and overall survival were compared among groups. Survival predictors were also investigated. Results: Approximately 80% of alcohol-related HCCs were classified as cases of metabolic dysfunction-associated fatty liver disease. Throughout the quinquennia, &lt;50% of HCCs were detected by surveillance programmes. The tumour burden at diagnosis was slightly reduced but not enough to change the distribution of the ITA.LI.CA cancer stages. Intra-arterial and targeted systemic therapies increased across quinquennia. A modest improvement in survival was observed in the last quinquennia, particularly after 12 months of patient observation. Cancer stage, HCC treatment, and presence of oesophageal varices were independent predictors of survival. Conclusions: In the past 15 years, modest improvements have been obtained in outcomes of alcohol-related HCC, attributed mainly to underuse of surveillance programmes and the consequent low amenability to curative treatments. Metabolic dysfunction-associated fatty liver disease is a widespread condition in alcohol abusers, but its presence did not show a pivotal prognostic role once HCC had developed. Instead, the presence of oesophageal varices, an independent poor prognosticator, should be considered in patient management and refining of prognostic systems. Impact and Implications: Alcohol abuse is a leading and growing cause of hepatocellular carcinoma (HCC) worldwide and is associated with a worse prognosis compared with other aetiologies. We assessed the evolutionary landscape of alcohol-related HCC over 15 years in Italy. A high cumulative prevalence (78%) of metabolic dysfunction-associated fatty liver disease, with signs of metabolic dysfunction, was observed in HCC patients with unhealthy excessive alcohol consumption. The alcohol + metabolic dysfunction-associated fatty liver disease condition tended to progressively increase over time. A modest improvement in survival occurred over the study period, likely because of the persistent underuse of surveillance programmes and, consequently, the lack of improvement in the cancer stage at diagnosis and the patients’ eligibility for curative treatments. Alongside the known prognostic factors for HCC (cancer stage and treatment), the presence of oesophageal varices was an independent predictor of poor survival, suggesting that this clinical feature should be carefully considered in patient management and should be included in prognostic systems/scores for HCC to improve their performance

Material deprivation affects the management and clinical outcome of hepatocellular carcinoma in a high-resource environment

none94Aim: This study investigated how material deprivation in Italy influences the stage of hepatocellular carcinoma (HCC) at diagnosis and the chance of cure. Methods: 4114 patients from the Italian Liver Cancer database consecutively diagnosed with HCC between January 2008 and December 2018 were analysed about severe material deprivation (SMD) rate tertiles of the region of birth and region of managing hospitals, according to the European Statistics on Income and Living Conditions. The main outcomes were HCC diagnosis modalities (during or outside surveillance), treatment adoption and overall survival. Results: In more deprived regions, HCC was more frequently diagnosed during surveillance, while the incidental diagnosis was prevalent in the least deprived. Tumour characteristics did not differ among regions. The proportion of patients undergoing potentially curative treatments progressively decreased as the SMD worsened. Consequently, overall survival was better in less deprived regions. Patients who moved from most deprived to less deprived regions increased their probability of receiving potentially curative treatments by 1.11 times (95% CI 1.03 to 1.19), decreasing their mortality likelihood (hazard ratio 0.78 95% CI 0.67 to 0.90). Conclusions: Socioeconomic status measured through SMD does not seem to influence HCC features at diagnosis but brings a negative effect on the chance of receiving potentially curative treatments. Patient mobility from the most deprived to the less deprived regions increased the access to curative therapies, with the ultimate result of improving survival.openCucchetti A.; Gramenzi A.; Johnson P.; Giannini E.G.; Tovoli F.; Rapaccini G.L.; Marra F.; Cabibbo G.; Caturelli E.; Gasbarrini A.; Svegliati-Baroni G.; Sacco R.; Zoli M.; Morisco F.; Di Marco M.; Mega A.; Foschi F.G.; Biasini E.; Masotto A.; Nardone G.; Raimondo G.; Azzaroli F.; Vidili G.; Brunetto M.R.; Farinati F.; Trevisani F.; Avanzato F.; Biselli M.; Caraceni P.; Garuti F.; Neri A.; Santi V.; Pellizzaro F.; Imondi A.; Sartori A.; Penzo B.; Sanmarco A.; Granito A.; Muratori L.; Piscaglia F.; Sansone V.; Forgione A.; Dajti E.; Marasco G.; Ravaioli F.; Cappelli A.; Golfieri R.; Mosconi C.; Renzulli M.; Cela E.M.; Facciorusso A.; Cacciato V.; Casagrande E.; Moscatelli A.; Pellegatta G.; de Matthaeis N.; Allegrini G.; Lauria V.; Ghittoni G.; Pelecca G.; Chegai F.; Coratella F.; Ortenzi M.; Missale G.; Olivani A.; Inno A.; Marchetti F.; Busacca A.; Camma C.; Di Martino V.; Maria Rizzo G.E.; Franze M.S.; Saitta C.; Sauchella A.; Berardinelli D.; Bevilacqua V.; Borghi A.; Gardini A.C.; Conti F.; Dall'Aglio A.C.; Ercolani G.; Adotti V.; Arena U.; Di Bonaventura C.; Campani C.; Dragoni G.; Gitto S.; Laffi G.; Coccoli P.; Malerba A.; Guarino M.; Capasso M.; Oliveri F.; Romagnoli V.Cucchetti, A.; Gramenzi, A.; Johnson, P.; Giannini, E. G.; Tovoli, F.; Rapaccini, G. L.; Marra, F.; Cabibbo, G.; Caturelli, E.; Gasbarrini, A.; Svegliati-Baroni, G.; Sacco, R.; Zoli, M.; Morisco, F.; Di Marco, M.; Mega, A.; Foschi, F. G.; Biasini, E.; Masotto, A.; Nardone, G.; Raimondo, G.; Azzaroli, F.; Vidili, G.; Brunetto, M. R.; Farinati, F.; Trevisani, F.; Avanzato, F.; Biselli, M.; Caraceni, P.; Garuti, F.; Neri, A.; Santi, V.; Pellizzaro, F.; Imondi, A.; Sartori, A.; Penzo, B.; Sanmarco, A.; Granito, A.; Muratori, L.; Piscaglia, F.; Sansone, V.; Forgione, A.; Dajti, E.; Marasco, G.; Ravaioli, F.; Cappelli, A.; Golfieri, R.; Mosconi, C.; Renzulli, M.; Cela, E. M.; Facciorusso, A.; Cacciato, V.; Casagrande, E.; Moscatelli, A.; Pellegatta, G.; de Matthaeis, N.; Allegrini, G.; Lauria, V.; Ghittoni, G.; Pelecca, G.; Chegai, F.; Coratella, F.; Ortenzi, M.; Missale, G.; Olivani, A.; Inno, A.; Marchetti, F.; Busacca, A.; Camma, C.; Di Martino, V.; Maria Rizzo, G. E.; Franze, M. S.; Saitta, C.; Sauchella, A.; Berardinelli, D.; Bevilacqua, V.; Borghi, A.; Gardini, A. C.; Conti, F.; Dall'Aglio, A. C.; Ercolani, G.; Adotti, V.; Arena, U.; Di Bonaventura, C.; Campani, C.; Dragoni, G.; Gitto, S.; Laffi, G.; Coccoli, P.; Malerba, A.; Guarino, M.; Capasso, M.; Oliveri, F.; Romagnoli, V

Effect of methionine sulfoximine on methylation of guanine residues in astroglial transfer ribonucleic acids

Culture-grown astrocytes derived from 3-day-old rat brain were incubated in the presence of [ 3 H]guanosine and of the convulsant agent l -methionine- dl -sulfoximine (MSO). The resulting [ 3 H]tRNA was purified from control and MSO-exposed cells at several time points during the incubation and was hydrolyzed to [ 3 H]guanine and four [ 3 H]methyl guanines which were separated by high pressure liquid chromatography. Three of the four [ 3 H]methyl guanines were more highly labeled in the [ 3 H]tRNA of the MSO-exposed cells, relative to that of the control cells throughout the entire incubation period. The findings extend to cultured astrocytes, the stimulatory effect of MSO on the methylation of neural tRNA guanines, previouly observed both in vitro using [ 14 C] S -adenosyl- l -methionine and in vivo using [ methyl 3 -H] l -methionine.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45427/1/11064_2004_Article_BF00964832.pd