Targeting mTOR and Src restricts hepatocellular carcinoma growth in a novel murine liver cancer model.

Liver cancer is a poor prognosis cancer with limited treatment options. To develop a new therapeutic approach, we derived HCC cells from a known model of murine hepatocellular carcinoma (HCC). We treated adiponectin (APN) knock-out mice with the carcinogen diethylnitrosamine, and the resulting tumors were 7-fold larger than wild-type controls. Tumors were disassociated from both genotypes and their growth characteristics evaluated. A52 cells from APN KO mice had the most robust growth in vitro and in vivo, and presented with pathology similar to the parental tumor. All primary tumors and cell lines exhibited activity of the mammalian target of Rapamycin (mTOR) and Src pathways. Subsequent combinatorial treatment, with the mTOR inhibitor Rapamycin and the Src inhibitor Dasatinib reduced A52 HCC growth 29-fold in vivo. Through protein and histological analyzes we observed activation of these pathways in human HCC, suggesting that targeting both mTOR and Src may be a novel approach for the treatment of HCC.

Community football teams for people with intellectual disabilities in secure settings: "They take you off the ward, it was like a nice day, and then you get like medals at the end".

BACKGROUND: People with learning disabilities (LD) are particularly vulnerable to mental health and behavioural difficulties, and it has been shown that regular exercise can improve psychosocial well-being as well as physical fitness. This research aims to explore the experiences of men with LD detained in secure settings who have engaged in community football training programmes and identify the benefits of such provision. METHOD: Interviews were conducted with eight patients in a forensic LD service, discussing their experiences of participating in community football. Template analysis was undertaken on the transcripts. RESULTS: Two master themes were identified: physical fitness and psychosocial benefits. As the analysis progressed, new emerging themes were identified around role identity and achievement, as well as extending and refining some of the themes from the original template including fun and belonging. Some anticipated themes were removed from the template entirely. CONCLUSION: The psychosocial benefits of organised community sports programmes far outweigh the physical health benefits. Careful consideration must be given to where on a treatment and rehabilitation pathway non-traditional therapeutic interventions such as sports programmes are offered as an adjunct to specific risk reduction interventions for people with LD in secure settings.

Intensive Interaction Training for Paid Carers: 'Looking, Looking and Find Out When They Want to Relate to You'.

BACKGROUND: Intensive interaction (II) is a communication approach useful for working with people with severe intellectual disabilities. Health and social care providers offer II training courses to paid carers working in local services with the goal of improving social communication for their clients. MATERIALS AND METHODS: Eight paid carers who had been trained in II were interviewed 2-3 years after training, to explore how their practice had changed, and whether any changes were sustainable. Interviews were analysed using interpretative phenomenological analysis. RESULTS: Analysis revealed a master theme of emotional and practical endurance, with empowerment, better understanding and perceived barriers to implementation comprising subthemes. CONCLUSIONS: Despite actual and perceived barriers to the enduring use of II, all participants spoke positively of the approach and were continuing to use II in practice.

MBOAT7 rs641738 increases risk of liver inflammation and transition to fibrosis in chronic hepatitis C.

Cirrhosis likely shares common pathophysiological pathways despite arising from a variety of liver diseases. A recent GWAS identified rs641738, a polymorphism in the MBOAT7 locus, as being associated with the development of alcoholic cirrhosis. Here we explore the role of this variant on liver inflammation and fibrosis in two cohorts of patients with chronic hepatitis C. In 2,051 patients, rs641738 associated with severe hepatic inflammation and increased risk of fibrosis, as well as fast fibrosis progression. At functional level, rs641738 associated with MBOAT7 transcript and protein levels in liver and blood, and with serum inflammatory, oxidative stress and macrophage activation markers. MBOAT7 was expressed in immune cell subsets, implying a role in hepatic inflammation. We conclude that the MBOAT7 rs641738 polymorphism is a novel risk variant for liver inflammation in hepatitis C, and thereby for liver fibrosis.

Treg depletion attenuates the severity of skin disease from ganglionic spread after HSV-2 flank infection.

Regulatory T cells (Tregs) attenuate lesion severity and disease after HSV ocular or genital infection, but their role in cutaneous infection remains unclear. Treg depletion (anti-CD25 mAb in C57BL/6 mice or diphtheria toxin (DT) in DEREG mice) prior to tk-deficient HSV-2 flank infection significantly decreased skin lesion severity, granulocyte receptor-1(Gr-1(+)) cell number, and chemokine (KC) expression in the secondary skin, but significantly increased immune effectors and chemokine expression (MCP-1, KC, VEGF-A) in the draining LN, and activated, interferon-γ producing CD8(+)T cells in the ganglia. Treg depletion also significantly reduced HSV-2 DNA in the ganglia. Thus, Tregs increase the severity of recurrent skin lesions, and differentially alter chemokine expression and immune effector homing in the skin and LN after cutaneous infection, and limit CD8(+) T cell responses in the ganglia. Our data suggests that effects of Treg manipulation on recurrent herpes lesions should be considered when developing Treg mediated therapeutics.

Herpes simplex virus infects skin gamma delta T cells before Langerhans cells and impedes migration of infected Langerhans cells by inducing apoptosis and blocking E-cadherin downregulation.

The role individual skin dendritic cell (DC) subsets play in the immune response to HSV remains unclear. We investigated the effect of HSV on DC virus uptake, viability, and migration after cutaneous infection in vitro and in vivo. HSV increased the emigration of skin DCs from whole skin explants over 3 d postinfection (p.i.) compared with mock controls, but the kinetics of emigration was influenced by the skin DC subset. Uninfected (bystander) Langerhans cells (LCs) were the major emigrant DC subset at 24 h p.i., but thereafter, large increases in infected CD103(+)langerin(+) dermal DC (dDC) and uninfected langerin(-) dDC emigration were also observed. LC infection was confirmed by the presence of HSV glycoprotein D (gD) and was associated with impaired migration from cultured skin. Langerin(+) dDC also expressed HSV gD, but infection did not impede migration. We then followed the virus in live MacGreen mice in which LCs express GFP using a fluorescent HSV-1 strain by time-lapse confocal microscopy. We observed a sequential infection of epidermal cells, first in keratinocytes and epidermal gammadelta T cells at 6 h p.i., followed by the occurrence of HSVgD(+) LCs at 24 h p.i. HSV induced CCR7 upregulation on all langerin(+) DC, including infected LCs, and increased production of skin TNF-alpha and IL-1beta. However, a large proportion of infected LCs that remained within the skin was apoptotic and failed to downregulate E-cadherin compared with bystander LCs or mock controls. Thus, HSV infection of LCs is preceded by infection of gammadelta T cells and delays migration.

Cyclin-dependent kinase-like 5 (CDKL5) mutation screening in Rett syndrome and related disorders.

Rett syndrome (RTT) is a severe neurodevelopmental disorder affecting females almost exclusively and is characterized by a wide spectrum of clinical manifestations. Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene have been found in up to 95% of classical RTT cases and a lesser proportion of atypical cases. Recently, mutations in another X-linked gene, CDKL5 (cyclin-dependent kinase-like 5) have been found to cause atypical RTT, in particular the early onset seizure (Hanefeld variant) and one female with autism. In this study we screened several cohorts of children for CDKL5 mutations, totaling 316 patients, including individuals with a clinical diagnosis of RTT but who were negative for MECP2 mutations (n=102), males with X-linked mental retardation (n=9), patients with West syndrome (n=52), patients with autism (n=59), patients with epileptic encephalopathy (n=33), patients with Aicardi syndrome (n=7) and other patients with intellectual disability with or without seizures (n=54). In all, seven polymorphic variations and four de novo mutations (c.586C>T [p.S196L]; c.58G>C [p.G20R]; c.2504delC [p.P835fs]; deletion of exons 1-3) were identified, and in all instances of the latter the clinical phenotype was that of an epileptic encephalopathy. These results suggest that pathogenic CDKL5 mutations are unlikely to be identified in the absence of severe early-onset seizures and highlight the importance of screening for large intragenic and whole gene deletions.

Headloop suppression PCR and its application to selective amplification of methylated DNA sequences.

Selective amplification in PCR is principally determined by the sequence of the primers and the temperature of the annealing step. We have developed a new PCR technique for distinguishing related sequences in which additional selectivity is dependent on sequences within the amplicon. A 5' extension is included in one (or both) primer(s) that corresponds to sequences within one of the related amplicons. After copying and incorporation into the PCR product this sequence is then able to loop back, anneal to the internal sequences and prime to form a hairpin structure-this structure is then refractory to further amplification. Thus, amplification of sequences containing a perfect match to the 5' extension is suppressed while amplification of sequences containing mismatches or lacking the sequence is unaffected. We have applied Headloop PCR to DNA that had been bisulphite-treated for the selective amplification of methylated sequences of the human GSTP1 gene in the presence of up to a 10(5)-fold excess of unmethylated sequences. Headloop PCR has a potential for clinical application in the detection of differently methylated DNAs following bisulphite treatment as well as for selective amplification of sequence variants or mutants in the presence of an excess of closely related DNA sequences.