Cancer control by adaptive immunity involves a number of defined death and clearance mechanisms. However, efficient inhibition of exponential cancer growth by T cells and interferon-γ (IFN-γ) requires additional undefined mechanisms that arrest cancer cell proliferation. Here we show that the combined action of the T-helper-1-cell cytokines IFN-γ and tumour necrosis factor (TNF) directly induces permanent growth arrest in cancers. To safely separate senescence induced by tumour immunity from oncogene-induced senescence, we used a mouse model in which the Simian virus 40 large T antigen (Tag) expressed under the control of the rat insulin promoter creates tumours by attenuating p53- and Rb-mediated cell cycle control. When combined, IFN-γ and TNF drive Tag-expressing cancers into senescence by inducing permanent growth arrest in G1/G0, activation of p16INK4a (also known as CDKN2A), and downstream Rb hypophosphorylation at serine 795. This cytokine-induced senescence strictly requires STAT1 and TNFR1 (also known as TNFRSF1A) signalling in addition to p16INK4a. In vivo, Tag-specific T-helper 1 cells permanently arrest Tag-expressing cancers by inducing IFN-γ- and TNFR1-dependent senescence. Conversely, Tnfr1(-/-)Tag-expressing cancers resist cytokine-induced senescence and grow aggressively, even in TNFR1-expressing hosts. Finally, as IFN-γ and TNF induce senescence in numerous murine and human cancers, this may be a general mechanism for arresting cancer progression.

SummaryNeonates show an impaired anti-microbial host defence, but the underlying immune mechanisms are not understood fully. Myeloid-derived suppressor cells (MDSCs) represent an innate immune cell subset characterized by their capacity to suppress T cell immunity. In this study we demonstrate that a distinct MDSC subset with a neutrophilic/granulocytic phenotype (Gr-MDSCs) is highly increased in cord blood compared to peripheral blood of children and adults. Functionally, cord blood isolated Gr-MDSCs suppressed T cell proliferation efficiently as well as T helper type 1 (Th1), Th2 and Th17 cytokine secretion. Beyond T cells, cord blood Gr-MDSCs controlled natural killer (NK) cell cytotoxicity in a cell contact-dependent manner. These studies establish neutrophilic Gr-MDSCs as a novel immunosuppressive cell subset that controls innate (NK) and adaptive (T cell) immune responses in neonates. Increased MDSC activity in cord blood might serve as key fetomaternal immunosuppressive mechanism impairing neonatal host defence. Gr-MDSCs in cord blood might therefore represent a therapeutic target in neonatal infections.

Chemically modified mRNA is capable of inducing therapeutic levels of protein expression while circumventing the threat of genomic integration often associated with viral vectors. We utilized this novel therapeutic tool to express the regulatory T cell transcription factor, FOXP3, in a time-and site-specific fashion in murine lung, in order to prevent allergic asthma in vivo. We show that modified Foxp3 mRNA rebalanced pulmonary T helper cell responses and protected from allergen-induced tissue inflammation, airway hyperresponsiveness, and goblet cell metaplasia in 2 asthma models. This protection was conferred following delivery of modified mRNA either before or after the onset of allergen challenge, demonstrating its potential as both a preventive and a therapeutic agent. Mechanistically, FOXP3 induction controlled Th2 and

In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency (2015) Nature Biotechnology, 33 (6), pp. 584-586.In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiencyTo the Editor:Nuclease-mediated genome editing holds great potential to knock out or repair diseasecausing genes. An ideal nuclease delivery vehicle is short-lived, does not integrate into the genome, and can enter target cells efficiently. These requirements have not yet been achieved simultaneously by any nuclease delivery vector. We and others have used modified mRNA, which is non-integrating and provides a transient pulse of protein expression, as an alternative to traditional viral vectors [1][2][3][4][5] . This approach allowed us to deliver therapeutic proteins in mouse models of Surfactant Protein B (SP-B) deficiency 3 and experimental asthma 4 . Here we apply it to deliver site-specific nucleases, demonstrating the value of nuclease-encoding chemically modified (nec) mRNA as a tool for in vivo genome editing. We chose a well-established transgenic mouse model of SP-B deficiency 6 in which SP-B cDNA is under the control of a tetracycline-inducible promoter 7 . Administration of doxycycline drives SP-B expression levels similar to those in wild-type mice (Supplementary Fig. 1), whereas cessation of doxycycline leads to phenotypic changes similar to those of the human disease, including thickened alveolar walls, heavy cellular infiltration, increased macrophages and neutrophils, interstitial edema, augmented cytokines in the lavage, a decline in lung function, and fatal respiratory distress leading to death within days 8,9 . We inserted a constitutive CAG promoter immediately upstream of the SP-B cDNA to allow doxycycline-independent expression and prolonged life in treated mice.First, we customized a panel of ZFNs and TALENs targeting the transgenic SP-B cassette ( Fig. 1a and Supplementary Fig. 2). We chose TALEN #1 (T1) and ZFN #3 (Z3) owing to their high activity and proximity to the desired site of promoter integration (Figs. 1a,b; amino acid sequences in Supplementary Fig. 4) and compared delivery by plasmid 1 DNA and mRNA. mRNA delivery resulted in higher levels of double-strand break (DSB)-induction ( Fig. 1c and Supplementary Fig. 3; P < 0.05) and homology-directed repair (HDR) ( Fig. 1d, P < 0.05). As Z3 mRNA was more efficient than T1 mRNA in both cases, Z3 was chosen for further experimentation. Comparison with a Z3-encoding AAV serotype 6 vector (AAV6) ("Z3 AAV") shows the relatively transient expression of Z3 mRNA (Fig. 1e), limiting the time during which off-target cleavage activity could occur.To optimize Z3 expression in the mouse lung, we administered a panel of 3xFLAG-tagged Z3 mRNAs with various modification schemes 2,5,10 , with or without complexation to biocompatible, biodegradable nanoparticles (NPs) made of chitosan-coated poly (lactic-coglycolic) acid (chit-PLGA) 11,12 . Following intratracheal (i.t.) delivery, NP-complexation significantly increased mRNA expression levels ( Supplem...

Stimulating the immune system to attack cancer is a promising approach, even for the control of advanced cancers. Several cytokines that promote interferon-γ-dominated immune responses show antitumor activity, with interleukin 12 (IL-12) being of major importance. Here, we used an antibody-IL-12 fusion protein (NHS-IL12) that binds histones of necrotic cells to treat human sarcoma in humanized mice. Following sarcoma engraftment, NHS-IL12 therapy was combined with either engineered IL-7 (FcIL-7) or IL-2 (IL-2MAB602) for continuous cytokine bioavailability. NHS-IL12 strongly induced innate and adaptive antitumor immunity when combined with IL-7 or IL-2. NHS-IL12 therapy significantly improved survival of sarcoma-bearing mice and caused long-term remissions when combined with IL-2. NHS-IL12 induced pronounced cancer cell senescence, as documented by strong expression of senescence-associated p16INK4a and nuclear translocation of p-HP1γ, and permanent arrest of cancer cell proliferation. In addition, this cancer immunotherapy initiated the induction of myogenic differentiation, further promoting the hypothesis that efficient antitumor immunity includes mechanisms different from cytotoxicity for efficient cancer control in vivo.

Enzyme immobilization is one of the most important techniques for industrial applications. It makes the immobilized enzyme more stable and advantageous than the free form in different aspects. α-Amylase was immobilized on 4% cyanuric chloride-activated amidoximated acrylic fabric at pH 7.0 with (79%) maximum efficiency. A field emission scanning electron microscope and Fourier transform infrared were used to confirm the immobilization process. Even after being recycled 10 times, the immobilized enzyme lost just 28% of its initial activity. Owing to immobilization, the pH of the soluble α-amylase was shifted from 6.0 to 6.5. The immobilized α-amylases showed thermal stability at 60°C, and became more resistant to heavy metal ions. The km values of the immobilized and soluble α-amylases were 9.6 and 3.8 mg starch ml−1, respectively. In conclusion, this method shows that the immobilized α-amylase proved to be more efficient than its soluble form, and hence could be used during saccharification of starch.

Fatty acid amides (FAAs) are of great interest due to their broad industrial applications. They can be synthesized enzymatically with many advantages over chemical synthesis. In this study, the fatty acid moieties of lipids of Cunninghamella echinulata ATHUM 4411, Umbelopsis isabellina ATHUM 2935, Nannochloropsis gaditana CCAP 849/5, Olive oil and an eicosapentaenoic acid (EPA) concentrate were converted into their fatty acid methyl esters and used in the FAA (i.e. ethylene diamine amides) enzymatic synthesis, using lipases as biocatalysts. The FAA synthesis, monitored using in situ NMR, FT-IR and thin-layer chromatography, was catalyzed efficiently by the immobilized Candida rugosa lipase. The synthesized FAAs exhibited a significant antimicrobial activity, especially those containing oleic acid in high proportions (i.e. derived from Olive oil and U. isabellina oil), against several human pathogenic microorganisms, insecticidal activity against yellow fever mosquito, especially those of C. echinulata containing gamma linolenic acid, and anti-cancer properties against SKOV-3 ovarian cancer cell line, especially those containing EPA in their structures (i.e. EPA concentrate and N. gaditana oil). We conclude that FAAs can be efficiently synthesized using microbial oils of different fatty acid composition and used in specific biological applications.

Oral squamous cell carcinoma (OSCC) is a malignant tumour of Head and Neck Cancer (HNC). The recent therapeutic approaches used to treat cancer have adverse side effects. The natural agents exhibiting anticancer activities are generally considered to have a robust therapeutic potential. Curcuminoids, one of the major active compounds of the turmeric herb, are used as a therapeutic agent for several diseases including cancer. In this study, the cytotoxicity of curcuminoids was investigated against OSCC cell line HNO97. Our data showed that curcuminoids significantly inhibits the proliferation of HNO97 in a time and dose-dependent manner (IC50=35 μM). Cell cycle analysis demonstrated that curcuminoids increased the percentage of G2/M phase cell populations in the treated groups. Treating HNO97 cells with curcuminoids led to cell shrinking and increased detached cells, which are the typical appearance of apoptotic cells. Moreover, flow cytometry analysis revealed that curcuminoids significantly induced apoptosis in a time-dependent manner. Furthermore, as a response to curcuminoids treatment, comet tails were formed in cell nuclei due to the induction of DNA damage. Curcuminoids treatment reduced the colony formation capacity of HNO97 cells and induced morphological changes. Overall, these findings demonstrate that curcuminoids can in vitro inhibit HNC proliferation and metastasis and induce apoptosis.