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Supplementary Figure S6: Sequential chemo-immunotherapy associates with improved survival

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DNA extraction and Sanger Sequencing In vivo Limiting Dilution Assay Cell growth inhibition assay Analysis of LV.Oct4.eGFP integration in eGFP positive and negative cell fractions

Supplementary Table 1: Patients Characteristics Supplementary Table 2: Comparative transduction of melanoma cell cultures

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Supplementary Figure S5:Tumor infiltration by CIK cells

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Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses.Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4. Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP+ mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro. First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models.Results: We visualized eGFP+ mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP+ MCs (1/42) compared with the eGFP− counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP+ mCSCs (P = 0.001). Sequential CHT–immunotherapy treatment retained antitumor activity (n = 12, P = 0.001) reducing mCSC rates (P = 0.01).Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277–88. ©2016 AACR.

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Purpose: MET, the tyrosine kinase receptor for hepatocyte growth factor, is frequently overexpressed in colon cancers with high metastatic tendency. We aimed to evaluate the role of its negative regulators, miR-1 and miR-199a*, and its transcriptional activator, the metastasis-associated in colon cancer 1 (MACC1), in controlling MET expression in human colon cancer samples.Experimental Design: The expression of MET, miR-1, miR-199a*, and MACC1 was evaluated by real-time PCR in 52 matched pairs of colorectal cancers and nontumoral surrounding tissues. The biological role of miR-1 in controlling MET expression and biological activity was assessed in colon cancer cells either by its forced expression or by AntagomiR-mediated inhibition.Results: MiR-1 was downregulated in 84.6% of the tumors and its decrease significantly correlated with MET overexpression, particularly in metastatic tumors. We found that concurrent MACC1 upregulation and miR-1 downregulation are required to elicit the highest increase of MET expression. Consistent with a suppressive role of miR-1, its forced in vitro expression in colon cancer cells reduced MET levels and impaired MET-induced invasive growth. Finally, we identified a feedback loop between miR-1 and MET, resulting in their mutual regulation.Conclusions: This study identifies an oncosuppressive role of miR-1 in colorectal cancer in which it acts by controlling MET expression through a feedback loop. Concomitant downregulation of miR-1 and increase of MACC1 can thus contribute to MET overexpression and to the metastatic behavior of colon cancer cells. Clin Cancer Res; 18(3); 737–47. ©2011 AACR.

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Background: We have previously showed rTgPI-1 tolerogenic adjuvant properties in asthma treatment, turning it a promising candidate for allergen-specific immunotherapy. This therapy is an alternative treatment to control asthma that still presents several concerns related to its formulation. rTgPI-1 contains independent inhibitory domains able to inhibit trypsin and neutrophil elastase, both involved in asthma pathology. Objectives: In view of the need to design rational therapies, herein we investigated the contribution of the different inhibitory domains in rTgPI-1 therapeutic effectiveness. Methods: BALB/c mice were rendered allergic by intraperitoneal OVA-alum sensitization and airway challenged. Once the asthmatic phenotype was achieved, mice were intranasally treated with OVA combined with the full-length recombinant protein rTgPI-1 or its truncated versions, Nt (containing trypsin-inhibitory domains) or Ct (containing neutrophil elastase-inhibitory domains). Afterward, mice were aerosol re-challenged. Results: Asthmatic mice treated with the neutrophil elastase- or the trypsin-inhibitory domains separately failed to improve allergic lung inflammation. Only when all inhibitory domains were simultaneously administered, an improvement was achieved. Still, a better outcome was obtained when mice were treated with the full-length rTgPI-1. Conclusions: Adjuvant ability depends on the presence of all its inhibitory domains in a single entity, so it should be included in potential asthma treatment formulations as a full-length protein.

Apiculate yeasts belonging to the genus Hanseniaspora are predominant on grapes and other fruits. While some species, such as Hanseniaspora uvarum, are well known for their abundant presence in fruits, they are generally characterized by their detrimental effect on fermentation quality because the excessive production of acetic acid. However, the species Hanseniaspora vineae is adapted to fermentation and currently is considered as an enhancer of positive flavour and sensory complexity in foods. Since 2002, we have been isolating strains from this species and conducting winemaking processes with them. In parallel, we also characterized this species from genes to metabolites. In 2013, we sequenced the genomes of two H. vineae strains, being these the first apiculate yeast genomes determined. In the last 10 years, it has become possible to understand its biology, discovering very peculiar features compared to the conventional Saccharomyces yeasts, such as a natural and unique G2 cell cycle arrest or the elucidation of the mandelate pathway for benzenoids synthesis. All these characteristics contribute to phenotypes with proved interest from the biotechnological point of view for winemaking and the production of other foods.

Toxoplasmosis is a zoonotic disease with worldwide prevalence in humans and warm-blooded animal populations. In livestock Toxoplasma gondii is the causal agent of significant economic losses since it can cause abortions in goats and sheep. It is estimated that one third of the world population is infected. Although there are effective therapies for acute infection, these are sometimes poorly tolerated, teratogenic, and have a long administration time. Considering the deficiencies that exist related to the prevention and treatment of toxoplasmosis, the development of a safe and effective vaccine would be extremely valuable in fighting against this infection. In the present work, we characterize for the first time the adjuvant and immunogenic potential of a recombinant profilin protein (rTgPF), in a vaccine formulation alone or in combination with the well-known GRA7 antigen candidate in a murine toxoplasmosis model. Since TgPF acts as a ligand for TLR11 and 12 inducing innate immune responses that promote type 1 adaptive responses, we first study the capacity of the mix rGRA7 + rTgPF to initiate an immune response by evaluating dendritic cell activation. Both rTgPF and rGRA7 induces activation of mouse BMDCs more efficiently than the single proteins, evidenced by increased expression of CD80 and CD86 co-stimulatory proteins and secretion of IL-6, IL-10 and IL-12 cytokines after in vitro stimulation. The sum of the effects of rGRA7 and rTgPF on BMDCs maturation led us to assay them in a vaccination protocol. BALB/c mice vaccinated with this mix elicited a Th1-biased immunity via the induction of lymphocyte proliferation, activation of CD4+T cells and increased IFN-γ production that resulted in enhanced protection against chronic Toxoplama gondii infection. Profilin per se induce only cellular immunity but augments the effect of rGRA7 immune responses when used together, thus allowing us to postulate rTgPF as a potential adjuvant in a protein vaccine.

The objectives of this narrative review are as follows: an evaluation of the bromatological composition of hazelnuts and a comparison of the nutritional properties of raw versus roasted hazelnuts, taking into account potential differences among varieties from different production territories such as Turkey, Italy, Chile, and New Zealand; an evaluation of nutrients contained in hazelnut skin; and an evaluation of nutrients contained in hazelnut oil. This review incorporates 27 scientific articles that measured and reported the concentrations of macro- and micro-nutrients in hazelnuts. These hazelnuts were subjected to different processing methods, originated from various geographical areas, or belonged to different varieties. Our results showed that the different varieties and territories where the hazelnuts were cultivated influence their bromatological composition, and we found that different processing steps can largely influence the concentration of specific nutrients. The removal of the skin, which contains a very high concentration of compounds with antioxidant action, is particularly critical. We should give greater attention to the skin, considering it not as a waste product, but as an important part of the hazelnut due to its nutritional properties of primary relevance in the Mediterranean diet. We provide a detailed assessment of the nutritional properties of the hazelnut kernel, skin, and oil, evaluating nutrient compositions and possible modifications (increases or reductions) that occur during the roasting process or that depend on the production territory and origin.

Vitis vinifera Tannat was introduced in Uruguay in 1870 from the Basque Pyrenees, and within several grapevines it became the variety best adapted to our viticultural conditions. Recently, through genetic analysis it was demonstrated that Manseng Noir, in addition to originate from the same region of Tannat, is the only natural sister identified within 2500 Vitis varieties surveyed [1]. Given the small commercial vineyard of this variety in France, after several years we have managed to plant in 2019 the first vineyard outside the Pyrenees in Uruguay. In 2021 and 2022 harvests, its elaboration is achieved with the aim of comparing its wine with Tannat. Results obtained show that its agronomic phenotype, as well as aspects of sanity, acidity, and color intensity are similar to Tannat, but with the particularity that grapes are of moderate ripening, 12.5% of alc., total polyphenol index was 12% lower than the control Tannat at 14% of alcohol. Interestingly, Manseng Noir shows an early smoothness in its tannins that allow to achieve lower alcohol wines, still powerful color and structure but less astringency in the mouth. Its complete genome was sequenced by Illumina technology and comparative genome analysis with Tannat was carried on. Genetic, metabolomic and sensory analyzes comparison with Tannat are discussed in this work.

Apiculate yeasts from Hanseniaspora genus are predominant on the native flora of grapevines, while Hanseniaspora uvarum is well known for its abundant presence in grapes, it results generally, in detrimental quality effects on wine due to high production of acetic acid. By contrast, Hanseniaspora vineae is better adapted to fermentation, increasing flavor complexity in wines as it has been demonstrated in winemaking practices during the last decade. We obtained a collection of more than 22 different Hanseniaspora vineae strains from which we could detect, by a plating screening method, high and moderate protease activity. In this study, we tested these strains under real winemaking conditions on a Sauvignon blanc white wine must. Results obtained demonstrated that there is an interesting protease active diversity in all H. vineae strains when compared to conventional fermentations conducted by Saccharomyces cerevisiae. Four H. vineae strains showed a decrease in wine protein turbidity, up to three times lower than that observed in S. cerevisiae, which imply a significant decrease in the demand for bentonite before bottling. This attribute of some H. vineaestrains should be very attractive at the commercial level to reduce manipulations and flavor removal in the production of delicate white wines such as Sauvignon blanc.

L’analyse video dans l’observation de l’interaction parent-enfant permet d’observer les sequences interactives a la base du developpement sociocognitif du jeune enfant. Son utilisation, en consultation de pediatrie, en video-feedback, apparait tres interessante pour l’intervention precoce. A travers l’etude d’une serie de cas, on montre ici la faisabilite et acceptabilite d’une intervention precoce de video-feedback, adaptation de la Video Intervention Therapy (PC-VIT), dans un centre de soins primaires pediatrique public italien.L’intervention vise a soutenir la sensibilite et la capacite de mentalisation du caregiving, au cours de la premiere annee de vie du bebe. L’etude de la serie de cas presentee indique que le PC-VIT se presente comme un nouvel outil capable de repondre aux besoins de prevention en sante mentale du jeune enfant.

Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses. Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4. Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP+ mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro. First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models. Results: We visualized eGFP+ mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP+ MCs (1/42) compared with the eGFP− counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP+ mCSCs (P = 0.001). Sequential CHT–immunotherapy treatment retained antitumor activity (n = 12, P = 0.001) reducing mCSC rates (P = 0.01). Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277–88. ©2016 AACR.

Background Supporting the hypothesis thatT. gondii infection protects against allergy in humans we previously demonstrated that this infection can modulate not only the susceptibility to develop respiratory allergies in mice but also suppresses allergic responses at systemic level. This latter finding suggests that T. gondii infection could prevent the onset of other allergic diseases, such as atopic dermatitis. At present, few studies have investigated the modulation of atopic dermatitis by parasite infections. Objective Here, we sought to investigate whether chronic infection with T. gondii is capable of modulating the development of atopic dermatitis. Methods Chronically infected mice were sensitized by repeated epicutaneous ovalbumin administration. Skin histopathology, humoral response, cytokine production and innate type-II lymphoid cells (ILC2) were assessed. Results A marked reduction in epidermal thickness and dermal inflammatory infiltrate along with a reduction in mast cell count was observed in infected mice compared to non-infected mice. These results correlated with a diminished TH2 and TH1 allergen specific response. Reduced type-II IL-4 and IL-5 cytokines were already detected during the first 24 h of allergen sensitization in splenocytes and draining lymph nodes from infected mice. Moreover, this reduced type-II profile in chronically infected animals correlated with diminished ILC2 number in draining lymph nodes. Conclusion Chronic infection withT. gondii prevents the development of atopic dermatitis. The diminished susceptibility seems to result from changes in type-II innate immune response that may lead to the induction of a deficient TH2 response and consequently to a lower susceptibility to develop atopic dermatitis.

The introduction of BRAF inhibitors (BRAFi) has improved response rate and overall survival of metastatic melanoma patients compared to standard chemotherapy. However, acquired drug resistance occurs in nearly all patients. The comprehension of cellular and molecular mechanisms underlying BRAFi resistance could help to identify novel actionable pathways in the treatment of BRAF-dependent tumors. VEGFA is an attractive target for combinatorial cancer therapy, and we have recently demonstrated in melanoma and CRC xenografts that targeting VEGFA enhanced the antitumor effect of BRAFi by normalizing the tumor vasculature, recruiting M1 macrophages, and inducing a remodeling of the extracellular matrix characterized by a reduction in collagen I and in cancer-associated fibroblasts (Comunanza et al., EMBO Mol Med 2017). Based on our previous proof of concept, obtained within an immunodeficient model, here we investigated the therapeutic effect of VEGFA targeting in association with PLX4720 (BRAFi) in a dedicated immune-competent model. D4M cells, a BRAFV600E-mutant melanoma murine cell line, were subcutaneously injected in syngeneic C57BL/6J mice. We demonstrated that the association of BRAFi with DC101 (antibody anti-VEGFR2) had a weak activity while we observed a synergistic antitumor effect when combined with B20 (murine anti-VEGFA neutralizing antibody). Although targeted inhibition of either BRAF or VEGFA delayed the tumor growth, only combined inhibition of both pathways resulted in the regression of initial tumor size, with an evident apoptotic effect, and delayed the onset of acquired resistance to the BRAF inhibition. Since the immune-suppressive role of VEGFA in tumors has been well characterized, we further investigated whether contrasting the VEGF effect along with simultaneous BRAF inhibition can turn into a promotion of both innate and adaptive immunity. Immune phenotype analysis revealed that the combinatorial regimen activated the host immune system, inducing the tumor infiltration of macrophages with tumor-suppressive features, NKs, CD4+ and CD8+ lymphocytes. Most of the infiltrating CD8+ lymphocytes in D4M tumors expressed high levels of PD-1 and none of the treatments significantly modulated PD-1 expression on T cells, suggesting a sustained T-cell exhaustion. We then postulated that the therapeutic effect obtained by the combinatorial VEGF blockade and BRAFi could be further enhanced by the association with an immune-checkpoint inhibitor targeting PD-1. Interestingly, we observed that the addition of anti-PD-1 blocking antibody boosted the antitumor effect and induced striking tumor volume regression in mice receiving the triplet therapy (BRAFi+anti-VEGFA+anti-PD1-1). Our findings provide biologic rationale to explore the association of immunotherapy in novel combinatorial approaches that could improve the clinical outcome exerted by oncogene-targeted therapy, and further investigation is warranted. Citation Format: Valentina Comunanza, Chiara Gigliotti, Gabriella Doronzo, Valentina Martin, Anna Gattuso, Dario Sangiolo, Federica Di Nicolantonio, Federico Bussolino. VEGF removal delays the onset of acquired resistance to target therapy and increases the efficacy of immune checkpoint inhibitors in BRAF-mutated melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B17.

Receptor tyrosine kinases (RTKs) inhibitors&rsquo, activity in advanced osteosarcoma is significant but short-lived. To prevent or at least delay drug resistance, we explored a vertical inhibition by combining drugs acting at different levels of the RTK pathways (pazopanib + trametinib). We studied pazopanib + trametinib antitumor activity both in vitro and in vivo (MNNG-HOS and KHOS xenografts in NOD/SCID mice) investigating the molecular mechanisms and potential escapes. The involvement of MAPK-PI3K pathways was validated by Nanostring technology, western blot and by silencing/overexpression experiments. Pazopanib targets were expressed on seven osteosarcoma cell lines and their pathways were activated. Pazopanib + trametinib exhibited synergistic antitumor activity by inducing apoptosis and inhibiting ERK1/2 and Akt. In vivo antitumor activity was shown in osteosarcoma-bearing mice. The drug combination significantly down-modulated RTK Ephrin Type-A Receptor 2 (EphA2) and Interleukin-7 Receptor (IL-7R), whereas induced mitogen-activated protein-kinase kinase (MAPKK) MEK6. EphA2 silencing significantly reduced osteosarcoma cell proliferation and migration, while impeding MEK6 up-regulation in the treated cells significantly increased the antitumor effect of the studied drugs. Moreover, the up-regulation of MEK6 reduced combination activity. Pazopanib + trametinib demonstrated synergistic antitumor effects in osteosarcoma models through ERK and Akt inhibition and EphA2 and IL-7R down-modulation. MEK6 up-regulation might evoke escaping mechanism.

BACKGROUND: Interferon-induced expression of programmed cell death ligands (PD-L1/PD-L2) may sustain tumour immuneevasion. Patients featuring MET amplification, a genetic lesion driving transformation, may benefit from anti-MET treatment. We explored if MET-targeted therapy interferes with Interferon-gamma modulation of PD-L1/PD-L2 in MET-amplified tumours.METHODS: PD-L1/PD-L2 expression and signalling pathways downstream of MET or Interferon-gamma were analysed in MET-amplified tumour cell lines and in patient-derived tumour organoids, in basal condition, upon Interferon-gamma stimulation, and after anti-MET therapy.RESULTS: PD-L1 and PD-L2 were upregulated in MET-amplified tumour cells upon Interferon-gamma treatment. This induction was impaired by JNJ-605, a selective inhibitor of MET kinase activity, and MvDN30, an antibody inducing MET proteolytic cleavage. We found that activation of JAKs/STAT1, signal transducers downstream of the Interferon-gamma receptor, was neutralised by MET inhibitors. Moreover, JAK2 and MET associated in the same signalling complex depending on MET phosphorylation. Results were confirmed in MET-amplified organoids derived from human colorectal tumours, where JNJ-605 treatment revoked Interferon-gamma induced PD-L1 expression.CONCLUSIONS: These data show that in MET-amplified cancers, treatment with MET inhibitors counteracts the induction of PD-1 ligands by Interferon-gamma. Thus, therapeutic use of anti-MET drugs may provide additional clinical benefit over and above the intended inhibition of the target oncogene.

Apiculate yeasts of the genus Hanseniaspora/Kloeckera are the main species present on mature grapes and play a significant role at the beginning of fermentation, producing enzymes and aroma compounds that expand the diversity of wine color and flavor. Ten species of the genus Hanseniaspora have been recovered from grapes and are associated in two groups: H. valbyensis, H. guilliermondii, H. uvarum, H. opuntiae, H. thailandica, H. meyeri, and H. clermontiae; and H. vineae, H. osmophila, and H. occidentalis. This review focuses on the application of some strains belonging to this genus in co-fermentation with Saccharomyces cerevisiae that demonstrates their positive contribution to winemaking. Some consistent results have shown more intense flavors and complex, full-bodied wines, compared with wines produced by the use of S. cerevisiae alone. Recent genetic and physiologic studies have improved the knowledge of the Hanseniaspora/Kloeckera species. Significant increases in acetyl esters, benzenoids, and sesquiterpene flavor compounds, and relative decreases in alcohols and acids have been reported, due to different fermentation pathways compared to conventional wine yeasts.

Toxoplasma gondii is an intracellular protozoan which is widely distributed. Infection occurs as a result of ingestion of uncooked meat and exposure to cat feces. Immunocompetent individuals are generally asymptomatic, while severe disease may occur in immunocompromised subjects and in congenital toxoplasmosis, which is caused by transplacental acquisition of T. gondii. Genetic diversity of T. gondii has often been studied using a PCR-RFLP scheme based on nine molecular markers. These studies led to the description of a clonal population structure with three main lineages (I, II and III) in North America and Europe and higher genetic diversity in South America. The aim of this study was to develop molecular markers that could allow the discrimination of genetic variants within each clonal lineage. We analyzed the genome of T. gondii to identify genes containing variable number tandem repeats (VNTRs). The coding sequences of T. gondii ME49 genome were processed with Tandem Repeat Finder software. A panel of candidate markers was selected based on the following parameters: the repeat unit size (>9 bp) and composition (to avoid single and dinucleotide runs), the number of copies (