Fn14•Trail effectively inhibits hepatocellular carcinoma growth.

BACKGROUND: New strategies for the treatment of hepatocellular carcinoma (HCC) are needed, given that currently available chemotherapeutics are inefficient. Since tumor growth reflects the net balance between pro-proliferative and death signaling, agents shifting the equilibrium toward the latter are of considerable interest. The TWEAK:Fn14 signaling axis promotes tumor cell proliferation and tumor angiogenesis, while TRAIL:TRAIL-receptor (TRAIL-R) interactions selectively induce apoptosis in malignant cells. Fn14•TRAIL, a fusion protein bridging these two pathways, has the potential to inhibit tumor growth, by interfering with TWEAK:Fn14 signaling, while at the same time enforcing TRAIL:TRAIL-R-mediated apoptosis. Consequently, Fn14•TRAIL\u27s capacity to inhibit HCC growth was tested. RESULTS: Fn14•TRAIL induced robust apoptosis of multiple HCC cell lines, while sparing non-malignant hepatocyte cell lines. Differential susceptibility to this agent did not correlate with expression levels of TRAIL, TRAIL-R, TWEAK and Fn14 by these lines. Fn14•TRAIL was more potent than soluble TRAIL, soluble Fn14, or a combination of the two. The requirement of both of Fn14•TRAIL\u27s molecular domains for function was established using blocking antibodies directed against each of them. Subcutaneous injection of Fn14•TRAIL abrogated HCC growth in a xenograft model, and was well tolerated by the mice. CONCLUSIONS: In this study, Fn14•TRAIL, a multifunctional fusion protein originally designed to treat autoimmunity, was shown to inhibit the growth of HCC, both in vitro and in vivo. The demonstration of this fusion protein\u27s potent anti-tumor activity suggests that simultaneous targeting of two signaling axes by a single fusion can serve as a basis for highly effective anti-cancer therapies

HTT-lowering reverses Huntington's disease immune dysfunction caused by NFκB pathway dysregulation

The peripheral immune response is altered in Huntington's disease, but the underlying mechanisms are unclear. Using RNA interference to lower huntingtin levels in leucocytes from patients, Träger et al. reverse disease-associated phenotypes including cytokine elevation and transcriptional dysregulation, and argue for a direct effect of mutant huntingtin on NFκΒ signallin

A Dominant Language Constellations case-study on language use and the affective domain

Globalisation, international mobility, and new technologies make current multilingualism qualitatively different to not only mono- and bi-lingualism but also to any of its historical incarnations. As a new linguistic dispensation (Aronin & Singleton, 2008, 2012; Aronin, 2015), current multilingualism is understood to be complex, suffusive, liminal, and super-diverse; four essential properties which necessitate alternative foci in multilingual research. From this view, multilinguals are the ‘glue’ that binds cultures and societies, it is therefore essential to focus on their socioculturally-situated multilingual practices if we are to better understand the protagonists of this considerable social responsibility. In this regard, two concepts of profound interest are multilinguality (Aronin & Ó Laoire, 2004) and, its expression/realisation in concrete time-frames and socio-cultural contexts, dominant language constellations (DLC - Aronin, 2006; 2016). With this in mind, the current study examines the individual DLC of a Moroccan immigrant living in the Valencian Community in Spain. In line with earlier research calling for more varied self-report data (Todeva & Cenoz, 2009; Canagarajah & Wurr, 2011; Gorter & Cenoz, 2011), the study uses semi-structured interviews to investigate relationships between the DLC and the affective domain; specifically, attitudes, emotion, and identity. The resulting qualitative data explores the following questions: How does a multilingual speaker use their DLC to navigate specific sociolinguistic contexts? What influence does the DLC have on the expression of identity and emotions in concrete daily situations? What role does the DLC play in the formation of language attitudes? Moreover, these issues are framed within an acculturation context to articulate what Canagarajah and Wurr (2011) call ‘voices from the periphery’. While accepting that a case-study limits any attempt at generalisation, it is hoped that this research focus may contribute by providing another small piece to the overall puzzle of multilingual practices realised in concrete social and cultural contexts

AAV5-miHTT gene therapy demonstrates suppression of mutant huntingtin aggregation and neuronal dysfunction in a rat model of Huntington's disease.

Huntington's disease (HD) is a fatal progressive neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene. To date, there is no treatment to halt or reverse the course of HD. Lowering of either total or only the mutant HTT expression is expected to have therapeutic benefit. This can be achieved by engineered micro (mi)RNAs targeting HTT transcripts and delivered by an adeno-associated viral (AAV) vector. We have previously showed a miHTT construct to induce total HTT knock-down in Hu128/21 HD mice, while miSNP50T and miSNP67T constructs induced allele-selective HTT knock-down in vitro. In the current preclinical study, the mechanistic efficacy and gene specificity of these selected constructs delivered by an AAV serotype 5 (AAV5) vector was addressed using an acute HD rat model. Our data demonstrated suppression of mutant HTT messenger RNA, which almost completely prevented mutant HTT aggregate formation, and ultimately resulted in suppression of DARPP-32-associated neuronal dysfunction. The AAV5-miHTT construct was found to be the most efficient, although AAV5-miSNP50T demonstrated the anticipated mutant HTT allele selectivity and no passenger strand expression. Ultimately, AAV5-delivered-miRNA-mediated HTT lowering did not cause activation of microglia or astrocytes suggesting no immune response to the AAV5 vector or therapeutic precursor sequences. These preclinical results suggest that using gene therapy to knock-down HTT may provide important therapeutic benefit for HD patients and raised no safety concerns, which supports our ongoing efforts for the development of an RNA interference-based gene therapy product for HD

Safe and Efficient Silencing with a Pol II, but not a Pol lII, Promoter Expressing an Artificial miRNA Targeting Human Huntingtin

Huntington\u27s disease is a devastating, incurable neurodegenerative disease affecting up to 12 per 100,000 patients worldwide. The disease is caused by a mutation in the Huntingtin (Htt) gene. There is interest in reducing mutant Huntingtin by targeting it at the mRNA level, but the maximum tolerable dose and long-term effects of such a treatment are unknown. Using a self-complementary AAV9 vector, we delivered a mir-155-based artificial miRNA under the control of the chicken β-actin or human U6 promoter. In mouse brain, the artificial miRNA reduced the human huntingtin mRNA by 50%. The U6, but not the CβA promoter, produced the artificial miRNA at supraphysiologic levels. Embedding the antisense strand in a U6-mir-30 scaffold reduced expression of the antisense strand but increased the sense strand. In mice treated with scAAV9-U6-mir-155-HTT or scAAV9-CβA-mir-155-HTT, activated microglia were present around the injection site 1 month post-injection. Six months post-injection, mice treated with scAAV9-CβA-mir-155-HTT were indistinguishable from controls. Those that received scAAV9-U6-mir-155-HTT showed behavioral abnormalities and striatal damage. In conclusion, miRNA backbone and promoter can be used together to modulate expression levels and strand selection of artificial miRNAs, and in brain, the CβA promoter can provide an effective and safe dose of a human huntingtin miRNA

Allele-Selective Suppression of Mutant Huntingtin in Primary Human Blood Cells

Post-transcriptional gene silencing is a promising therapy for the monogenic, autosomal dominant, Huntington\u27s disease (HD). However, wild-type huntingtin (HTT) has important cellular functions, so the ideal strategy would selectively lower mutant HTT while sparing wild-type. HD patients were genotyped for heterozygosity at three SNP sites, before phasing each SNP allele to wild-type or mutant HTT. Primary ex vivo myeloid cells were isolated from heterozygous patients and transfected with SNP-targeted siRNA, using glucan particles taken up by phagocytosis. Highly selective mRNA knockdown was achieved when targeting each allele of rs362331 in exon 50 of the HTT transcript; this selectivity was also present on protein studies. However, similar selectivity was not observed when targeting rs362273 or rs362307. Furthermore, HD myeloid cells are hyper-reactive compared to control. Allele-selective suppression of either wild-type or mutant HTT produced a significant, equivalent reduction in the cytokine response of HD myeloid cells to LPS, suggesting that wild-type HTT has a novel immune function. We demonstrate a sequential therapeutic process comprising genotyping and mutant HTT-linkage of SNPs, followed by personalised allele-selective suppression in a small patient cohort. We further show that allele-selectivity in ex vivo patient cells is highly SNP-dependent, with implications for clinical trial target selection

Rapid diagnosis of experimental meningitis by bacterial heat production in cerebrospinal fluid

BACKGROUND: Calorimetry is a nonspecific technique which allows direct measurement of heat generated by biological processes in the living cell. We evaluated the potential of calorimetry for rapid detection of bacterial growth in cerebrospinal fluid (CSF) in a rat model of bacterial meningitis. METHODS: Infant rats were infected on postnatal day 11 by direct intracisternal injection with either Streptococcus pneumoniae, Neisseria meningitidis or Listeria monocytogenes. Control animals were injected with sterile saline or heat-inactivated S. pneumoniae. CSF was obtained at 18 hours after infection for quantitative cultures and heat flow measurement. For calorimetry, 10 microl and 1 microl CSF were inoculated in calorimetry ampoules containing 3 ml trypticase soy broth (TSB). RESULTS: The mean bacterial titer (+/- SD) in CSF was 1.5 +/- 0.6 x 108 for S. pneumoniae, 1.3 +/- 0.3 x 106 for N. meningitidis and 3.5 +/- 2.2 x 104 for L. monocytogenes. Calorimetric detection time was defined as the time until heat flow signal exceeded 10 microW. Heat signal was detected in 10-microl CSF samples from all infected animals with a mean (+/- SD) detection time of 1.5 +/- 0.2 hours for S. pneumoniae, 3.9 +/- 0.7 hours for N. meningitidis and 9.1 +/- 0.5 hours for L. monocytogenes. CSF samples from non-infected animals generated no increasing heat flow (<10 microW). The total heat was the highest in S. pneumoniae ranging from 6.7 to 7.5 Joules, followed by L. monocytogenes (5.6 to 6.1 Joules) and N. meningitidis (3.5 to 4.4 Joules). The lowest detectable bacterial titer by calorimetry was 2 cfu for S. pneumoniae, 4 cfu for N. meningitidis and 7 cfu for L. monocytogenes. CONCLUSION: By means of calorimetry, detection times of <4 hours for S. pneumoniae and N. meningitidis and <10 hours for Listeria monocytogenes using as little as 10 microl CSF were achieved. Calorimetry is a new diagnostic method allowing rapid and accurate diagnosis of bacterial meningitis from a small volume of CSF

Artificial miRNAs Reduce Human Mutant Huntingtin Throughout the Striatum in a Transgenic Sheep Model of Huntington's Disease.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT, but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-length human HTT with 73 CAG repeats. AAV9 was used to deliver unilaterally to HD sheep striatum an artificial miRNA targeting exon 48 of the human HTT mRNA under control of two alternative promoters: U6 or CβA. The treatment reduced human mutant (m) HTT mRNA and protein 50-80% in the striatum at 1 and 6 months post injection. Silencing was detectable in both the caudate and putamen. Levels of endogenous sheep HTT protein were not affected. There was no significant loss of neurons labeled by DARPP32 or NeuN at 6 months after treatment, and Iba1-positive microglia were detected at control levels. It is concluded that safe and effective silencing of human mHTT protein can be achieved and sustained in a large-animal brain by direct delivery of an AAV carrying an artificial miRNA