A major goal in gene therapy is to develop efficient gene transfer protocols that allow tissue-specific, long-term and tightly regulated expression of the desired transgene. This objective is ...becoming more attainable through the co-evolution of gene transfer vectors and regulation systems. The ideal vector should efficiently transduce non-dividing cells with minimal toxicity, thus endowing the system with persistent transgene expression. The helper-dependent adenovirus vectors meet these requirements, as demonstrated in various studies in the literature. The most promising regulation system is the tet-on system, which has low basal transcriptional activity and high inducibility. To explore the regulated transgene expression in the context of a helper-dependent vector, we constructed the HD-TET-IFN vector, containing the mIFNα gene under the control of the tetracycline inducible transactivator rtTA2s-S2. Mice injected with HD-TET-IFN showed high levels of serum mIFNα only upon transcriptional activation. The transgene expression was reinducible to the same high level up to 3 months p.i., and the amount of expressed cytokine could be regulated by dosing doxycycline. Transcriptional activation of mIFNα induced by doxycycline resulted in prolonged survival and reduced liver damage in HD-TET-IFN-injected mice challenged with a lethal dose of coronavirus. Activation of antiviral genes mediated by doxycycline-dependent mIFNα expression was also observed at low HD-TET-IFN doses. The possibility of controlling gene expression by the combination of HD vectors and the latest tet-on transactivator also holds promise for studying gene function in other animal models.
BackgroundImmunotherapy based on anti CTLA-4 (αCTLA-4) and anti PD1 (αPD1) is being tested in combination with different therapeutic approaches including other immunotherapy approaches such as ...neoantigen cancer vaccines (NCV). Here we explored, in two cancer murine models, different therapeutic combinations of αCTLA-4 and/or αPD1 with a plasmid DNA vaccine expressing neoantigens and delivered by electroporation (EP).Materials and MethodsTo evaluate the impact of NCV in the MC38 and in the CT26 tumor model three plasmid vaccines were generated with or without CD4 epitopes. Therapeutic DNA vaccines were delivered by EP in different therapeutic protocols including large tumors. Flow cytometry was utilized to measure CD8, CD4, T-reg, and B cells as well as neoantigen-specific immune responses, which were also measured by IFN-γ ELIspot.ResultsImmune responses were augmented in combination with αCTLA4 but not with αPD1 in the MC38 tumor model with significantly impacting tumor growth. Similarly, neoantigen-specific T cell immune responses were observed in the CT26 tumor model where large tumors regressed in all mice treated with αCTLA-4 and NCV. In line with previous evidence, we observed an increased switched memory B cells in the spleen of mice treated with αCTLA-4 alone or in combination with NCV.ConclusionsThese results support the use of NCV delivered by DNA-EP with αCTLA-4 and suggest a new combined therapy for clinical testing.Disclosure InformationF. Palombo: A. Employment (full or part-time); Significant; Neomatrix biotech. E. Salvatori: A. Employment (full or part-time); Significant; Takis biotech. L. Lione: A. Employment (full or part-time); Significant; Takis biotech. M. Compagnone: A. Employment (full or part-time); Significant; Neomatrix biotech. A. Conforti: A. Employment (full or part-time); Significant; Evvivax. L. Aurisicchio: A. Employment (full or part-time); Significant; Neomatrix biotech, Takis biotech, Evvivax.
Preclinical science research focuses on the study of physiological systems regulating body functions, and how they are dysregulated in disease, in a non-human setting. For example, cells in a dish, ...computer simulations or animals. Scientific procedures traditionally involve a specialist scientist developing a hypothesis and subsequently testing it using an experimental set-up. The results are then disseminated to the wider scientific community, following peer review and only at the last stage the news will reach the general, lay public. In the last few years, some research funding institutions have promoted a different model, with the direct involvement of members of the public in the research co-creation, from the hypothesis development, to the grant revision, project monitoring and results communication. We personally experienced this model and brought it to a further level by producing a movie. Animal research is a very controversial topic as, while still being necessary for the investigation of body functions, it brings about issues related to the ethics, the regulation and the practical execution of experimental procedures on animals. Here we discuss the different stages of the ideation, production and outcomes of the movie ‘Of Mice and Dementia’, a filmed conversation on animal experimentation in dementia research. The conversation was between scientists and lay people with a direct experience of dementia.
Inducible nitric oxide synthase (iNOS) has previously been shown to contribute to atherosclerotic lesion formation and protein nitration. Micro attenuated total reflection (ATR)-Fourier transform ...infrared (FTIR) spectroscopic imaging was applied ex vivo to analyse lesions in atherosclerotic (ApoE-/-) mice. Histologies of cardiovascular tissue of ApoE-/- mice that contain the gene for iNOS and ApoE-/- mice without iNOS (ApoE-/-iNOS-/- mice) were examined. Spectroscopic imaging of the aortic root revealed that iNOS did not affect the composition of the tunica media; furthermore, irrespective of iNOS presence, lipid esters were found to form the atherosclerotic plaque. ApoE-/- mouse aortic root lesions exhibited a more bulky atheroma that extended into the medial layer; signals characteristic of triglycerides and free fatty acids were apparent here. In ApoE-/-iNOS-/- mouse specimens, lesions composed of free cholesterol were revealed. ATR-FTIR spectra of the intimal plaque from the two mouse strains showed higher lipid concentrations in ApoE-/- mice, indicating that iNOS contributes to lesion formation. The reduction of lesion prevalence in ApoE-/-iNOS-/- mice compared with ApoE-/- mice is consistent with previous data. Moreover, the analysis of the plaque region revealed a change in the spectral position of the amide I band, which may be indicative of protein nitration in the ApoE-/- mouse, correlating with a more ordered (beta-sheet) structure, while a less ordered structure was apparent for the ApoE-/-iNOS-/- mouse, in which protein nitration is attenuated. These results indicate that micro ATR-FTIR spectroscopic imaging with high spatial resolution is a valuable tool for investigating differences in the structure and chemical composition of atherosclerotic lesions of ApoE-/- and ApoE-/-iNOS-/- mice fed a high-fat Western diet and can therefore be applied successfully to the study of mouse models of atherosclerosis.
A new dark sector antibaryon, denoted ψ_{D}, could be produced in decays of B mesons. This Letter presents a search for B^{+}→ψ_{D}+p (and the charge conjugate) decays in e^{+}e^{-} annihilations at ...10.58 GeV, using data collected in the BABAR experiment. Data corresponding to an integrated luminosity of 398 fb^{-1} are analyzed. No evidence for a signal is observed. Branching fraction upper limits in the range from 10^{-7}-10^{-5} are obtained at 90% confidence level for masses of 1.0<m_{ψ_{D}}<4.3 GeV/c^{2}. The result is also reinterpreted to provide the first limits on a supersymmetric model with R-parity violation and a light neutralino.
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In human cells, mismatch recognition is mediated by a heterodimeric complex, hMutSα, comprised of two members of the MutS homolog (MSH) family of proteins, hMSH2 and GTBP 1,2. Correspondingly, ...tumour-derived cell lines defective in hMSH2 and GTBP have a mutator phenotype 3,4, and extracts prepared from these cells lack mismatch-binding activity 1. However, although hMSH2 mutant cell lines showed considerable microsatellite instability in tracts of mononucleotide and dinucleotide repeats 4,5, only mononucleotide repeats were somewhat unstable in GTBP mutants 4,6. These findings, together with data showing that extracts of cells lacking GTBP are partially proficient in the repair of two-nucleotide loops 2, suggested that loop repair can be GTBP-independent. We show here that hMSH2 can also heterodimerize with a third human MSH family member, hMSH3, and that this complex, hMutSβ, binds loops of one to four extrahelical bases. Our data further suggest that hMSH3 and GTBP are redundant in loop repair, and help explain why only mutations in hMSH2, and not in GTBP or hMSH3, segregate with hereditary non-polyposis colorectal cancer (HNPCC) 7.
The properties of the water-saturated 1-octanol solution in the 10−75 °C temperature range have been investigated by means of different scattering spectroscopies. The results of Raman, ...depolarized-Rayleigh, and Rayleigh−Brillouin experiments have been compared to the corresponding results recently obtained for the pure alcohol in our laboratory. Spectroscopic findings obtained for the temperature behavior of vibrational, rotational, and translational degrees of freedom are analyzed according to a single, unifying picture of this liquid system. Differences and analogies between pure and water-saturated 1-octanol suggest the key for interpreting the structural properties connected to the amphiphilic nature of n-alcohols. In a wider perspective, the same arguments can be applied to the study of larger and more complicated systems such as phospholipids for which 1-octanol represents a simple model.
In extracts of human cells, base–base mismatches and small insertion/deletion loops are bound primarily by hMutSα, a heterodimer of hMSH2 and hMSH6 (also known as GTBP or p160). Recombinant hMutSα ...bound a G/T mismatch‐containing oligonucleotide with an apparent dissociation constant Kd = 2.6 nM, while its affinity for a homoduplex substrate was >20‐fold lower. In the presence of ATP, hMutSα dissociated from mismatched oligonucleotide substrates, and this reaction was attenuated by mutating the conserved lysine in the ATP‐binding domains of hMSH6, hMSH2 or both to arginine. Surprisingly, this reaction required only ATP binding, not hydrolysis. The ATPase activity of hMutSα variants carrying the Lys→Arg mutation in hMSH2 or in hMSH6 was severely affected, but these mutants were still proficient in mismatch binding and were able to complement, albeit to different extents, mismatch repair‐deficient cell extracts. The mismatch binding‐proficient, ATPase‐deficient double mutant was inactive in the complementation assay and its presence in repair‐proficient extracts was inhibitory. We conclude that although the ATPase activity of hMutSα is dispensible for mismatch binding, it is required for mismatch correction.
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