Biology and physiology of Hanseniaspora vineae: metabolic diversity and increase flavour complexity for food fermentation.

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.

Contribution of Kazal-Like Domains of the Serine Protease Inhibitor-1 from Toxoplasma gondii in Asthma Therapeutic Vaccination Effectiveness.

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.

Anomalous self-experience, body image disturbance, and eating disorder symptomatology in first-onset anorexia nervosa.

PURPOSE: Anorexia nervosa-restrictive subtype (AN-R) is a life-threatening disorder relying on behavioural abnormalities, such as excessive food restriction or exercise. Such abnormalities may be secondary to an "objectified" attitude toward body image and self. This is the first study exploring the impact of anomalous self-experience (ASEs) on abnormal body image attitude and eating disorder (ED) symptomatology in individuals with AN-R at onset. METHODS: We recruited Italian female participants, 40 with AN-R (mean age 18.3 ± 2.3) and 45 age and educational level-matched healthy controls (HCs) (mean age 18.2 ± 2.6). ASEs, body image attitude, and ED symptom severity were assessed through the examination of anomalous self-experience (EASE), the body uneasiness test (BUT), and the eating disorder examination questionnaire (EDE-Q), respectively. We conducted multivariate analysis of variance to investigate distribution patterns of variables of interest, and mediation analysis to test the effect of ASEs and body image on ED symptomatology. RESULTS: Individuals with AN-R scored higher than HCs on the EASE (p < .0001). A direct effect of ASEs on ED severity (p = 0.009; bootstrapped LLCI = 0.067, ULCI = 0.240) was found in AN-R. After modelling the effect of abnormal body image attitude, the relationship between EASE total score and ED symptomatology was significantly mediated by BUT (p = 0.002; bootstrapped LLCI = 0.001, ULCI = 0.172). CONCLUSION: Although the exact pathways linking AN-R to self-disorder remain to be identified, a broader exploration of transdiagnostic features in AN, including explorations of different dimensions of self-experience and intersubjectivity, may shed further light on the clinical phenomenology of the disorder. LEVEL OF EVIDENCE: Level III, case-control analytic study.

Emotional dysregulation mediates the impact of childhood trauma on psychological distress: First Italian data during the early phase of COVID-19 outbreak.

BACKGROUND: The coronavirus disease 2019 pandemic represents an unprecedented traumatic stressor to mental health. Psychological distress is considered a reliable proxy for psychopathology and can be negatively influenced by childhood trauma through sensitization effects. Emotion dysregulation has been proposed as a potential mediator of this mechanism. We aimed to test this hypothesis in a national Italian sample assessed in the early phase of the coronavirus disease 2019 outbreak. METHODS: We investigated the relationship between psychological distress and childhood trauma in 500 healthy participants assessed through a survey-based study, after the coronavirus disease 2019 pandemic lockdown in Italy. Levels of psychological distress and history of childhood trauma were obtained using the Kessler-10 (K10) and the Childhood Trauma Questionnaire, respectively. We used bootstrapped mediation analysis to test the mediator role of emotional dysregulation, measured through the Difficulties in Emotion Regulation Scale, on the effect of childhood trauma on psychological distress. RESULTS: In total, 190 (38%) reported psychological distress related to the coronavirus disease 2019 outbreak. Individuals with psychological distress experienced more childhood trauma, specifically emotional abuse (F = 23.51, df = 1, p < 0.001) and neglect (F = 10.98, df = 1, p = 0.001). After modelling the effect of emotional dysregulation, the impact of childhood trauma on coronavirus disease 2019-related psychological distress resulted in indirect effects and mediated by deficits in emotion regulation mechanisms (bootstrapped lower level of confidence interval = 0.345, upper level of confidence interval = 0.598). CONCLUSION: Childhood trauma is associated with increased vulnerability to the stressful effect of the coronavirus disease 2019 outbreak. Our data suggest that emotional dysregulation may represent a dimension mediating the impact of childhood trauma on coronavirus disease 2019-related psychological distress and may be considered as specific target for interventions aiming at empowering resilience.

Affective temperament, attachment style, and the psychological impact of the COVID-19 outbreak: an early report on the Italian general population.

The outbreak of COVID-19 is severely affecting mental health worldwide, although individual response may vary. This study aims to investigate the psychological distress perceived by the Italian general population during the early phase of the COVID-19 pandemic, and to analyze affective temperament and adult attachment styles as potential mediators. Through an online survey, we collected sociodemographic and lockdown-related information and evaluated distress, temperament, and attachment using the Kessler 10 Psychological Distress Scale (K10), the Temperament Evaluation of Memphis, Pisa, Paris and San Diego-Autoquestionnaire short version (TEMPS-A) and the Attachment Style Questionnaire (ASQ). In our sample (n = 500), 62% of the individuals reported no likelihood of psychological distress, whereas 19.4% and 18.6% displayed mild and moderate-to-severe likelihood. Cyclothymic (OR: 1.24; p < 0.001), depressive (OR: 1.52; p < 0.001) and anxious (OR: 1.58; p = 0.002) temperaments, and the ASQ "Need for approval" (OR: 1.08; p = 0.01) were risk factors for moderate-to-severe psychological distress compared to no distress, while the ASQ "Confidence" (OR: 0.89; p = 0.002) and "Discomfort with closeness" were protective (OR: 0.92; p = 0.001). Cyclothymic (OR: 1.17; p = 0.008) and depressive (OR: 1.32; p = 0.003) temperaments resulted as risk factors in subjects with moderate-to-severe psychological distress compared to mild distress, while the ASQ "Confidence" (OR: 0.92; p = 0.039) and "Discomfort with closeness" (OR: 0.94; p = 0.023) were protective. Our data indicated that a relevant rate of individuals may have experienced psychological distress following the COVID-19 outbreak. Specific affective temperament and attachment features predict the extent of mental health burden. To the best of our knowledge, these are the first data available on the psychological impact of the early phase of the COVID-19 pandemic on a sizeable sample of the Italian population. Moreover, our study is the first to investigate temperament and attachment characteristics in the psychological response to the ongoing pandemic. Our results provide further insight into developing targeted intervention strategies.

Met inhibition revokes IFNγ-induction of PD-1 ligands in MET-amplified tumours.

BACKGROUND: Interferon-induced expression of programmed cell death ligands (PD-L1/PD-L2) may sustain tumour immune-evasion. Patients featuring MET amplification, a genetic lesion driving transformation, may benefit from anti-MET treatment. We explored if MET-targeted therapy interferes with Interferon-γ modulation of PD-L1/PD-L2 in MET-amplified tumours. METHODS: PD-L1/PD-L2 expression and signalling pathways downstream of MET or Interferon-γ were analysed in MET-amplified tumour cell lines and in patient-derived tumour organoids, in basal condition, upon Interferon-γ stimulation, and after anti-MET therapy. RESULTS: PD-L1 and PD-L2 were upregulated in MET-amplified tumour cells upon Interferon-γ 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-γ 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-γ 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-γ. Thus, therapeutic use of anti-MET drugs may provide additional clinical benefit over and above the intended inhibition of the target oncogene.

Genomic and Transcriptomic Basis of Hanseniaspora vineae's Impact on Flavor Diversity and Wine Quality.

Hanseniaspora is the main genus of the apiculate yeast group that represents approximately 70% of the grape-associated microflora. Hanseniaspora vineae is emerging as a promising species for quality wine production compared to other non-Saccharomyces species. Wines produced by H. vineae with Saccharomyces cerevisiae consistently exhibit more intense fruity flavors and complexity than wines produced by S. cerevisiae alone. In this work, genome sequencing, assembling, and phylogenetic analysis of two strains of H. vineae showed that it is a member of the Saccharomyces complex and it diverged before the whole-genome duplication (WGD) event from this clade. Specific flavor gene duplications and absences were identified in the H. vineae genome compared to 14 fully sequenced industrial S. cerevisiae genomes. The increased formation of 2-phenylethyl acetate and phenylpropanoids such as 2-phenylethyl and benzyl alcohols might be explained by gene duplications of H. vineae aromatic amino acid aminotransferases (ARO8 and ARO9) and phenylpyruvate decarboxylases (ARO10). Transcriptome and aroma profiles under fermentation conditions confirmed these genes were highly expressed at the beginning of stationary phase coupled to the production of their related compounds. The extremely high level of acetate esters produced by H. vineae compared to that by S. cerevisiae is consistent with the identification of six novel proteins with alcohol acetyltransferase (AATase) domains. The absence of the branched-chain amino acid transaminases (BAT2) and acyl coenzyme A (acyl-CoA)/ethanol O-acyltransferases (EEB1) genes correlates with H. vineae's reduced production of branched-chain higher alcohols, fatty acids, and ethyl esters, respectively. Our study provides sustenance for understanding and potentially utilizing genes that determine fermentation aromas.IMPORTANCE The huge diversity of non-Saccharomyces yeasts in grapes is dominated by the apiculate genus Hanseniaspora Two native strains of Hanseniaspora vineae applied to winemaking because of their high oenological potential in aroma and fermentation performance were selected to obtain high-quality genomes. Here, we present a phylogenetic analysis and the complete transcriptome and aroma metabolome of H. vineae during three fermentation steps. This species produced significantly richer flavor compound diversity than Saccharomyces, including benzenoids, phenylpropanoids, and acetate-derived compounds. The identification of six proteins, different from S. cerevisiae ATF, with diverse acetyltransferase domains in H. vineae offers a relevant source of native genetic variants for this enzymatic activity. The discovery of benzenoid synthesis capacity in H. vineae provides a new eukaryotic model to dilucidate an alternative pathway to that catalyzed by plants' phenylalanine lyases.

Vaccine potential of antigen cocktails composed of recombinant Toxoplasma gondii TgPI-1, ROP2 and GRA4 proteins against chronic toxoplasmosis in C3H mice.

The development of an effective and safe vaccine to prevent Toxoplasma gondii infection is an important aim due to the great clinical and economic impact of this parasitosis. We have previously demonstrated that immunization with the serine protease inhibitor-1 (TgPI-1) confers partial protection to C3H/HeN and C57BL/6 mice. In order to improve the level of protection, in this work, we combined this novel antigen with ROP2 and/or GRA4 recombinant proteins (rTgPI-1+rROP2, rTgPI-1+rGRA4, rTgPI-1+rROP2+rGRA4) to explore the best combination against chronic toxoplasmosis in C3H/HeN mice. All tested vaccine formulations, administered following a homologous prime-boost protocol that combines intradermal and intranasal routes, conferred partial protection as measured by the reduction of brain cyst burden following oral challenge with tissue cysts of Me49 T. gondii strain. The highest level of protection was achieved by the mixture of rTgPI-1 and rROP2 proteins with an average parasite burden reduction of 50% compared to the unvaccinated control group. The vaccine-induced protective effect was related to the elicitation of systemic cellular and humoral immune responses that included antigen-specific spleen cell proliferation, the release of Th1/Th2 cytokines, and the generation of antigen-specific antibodies in serum. Additionally, mucosal immune responses were also induced, characterized by secretion of antigen-specific IgA antibodies in intestinal lavages and specific mesenteric lymph node cell proliferation. Our results demonstrate that rTgPI-1+rROP2 antigens seem a promising mixture to be combined with other immunogenic proteins in a multiantigenic vaccine formulation against toxoplasmosis.

Effect of yeast assimilable nitrogen on the synthesis of phenolic aroma compounds by Hanseniaspora vineae strains.

In several grape varieties, the dominating aryl alkyl alcohols found are the volatile group of phenylpropanoid-related compounds, such as glycosylated benzyl and 2-phenylethyl alcohol, which contribute to wine with floral and fruity aromas after being hydrolysed during fermentation. Saccharomyces cerevisiae is largely recognized as the main agent in grape must fermentation, but yeast strains belonging to other genera, including Hanseniaspora, are known to predominate during the first stages of alcoholic fermentation. Although non-Saccharomyces yeast strains have a well-recognized genetic diversity, understanding of their impact on wine flavour richness is still emerging. In this study, 11 Hansenisapora vineae strains were used to ferment a chemically defined simil-grape fermentation medium, resembling the nutrient composition of grape juice but devoid of grape-derived secondary metabolites. GC-MS analysis was performed to determine volatile compounds in the produced wines. Our results showed that benzyl alcohol, benzyl acetate and 2-phenylethyl acetate are significantly synthesized by H. vineae strains. Levels of these compounds found in fermentations with 11 H. vineae different strains were one or two orders of magnitude higher than those measured in fermentations with a known S. cerevisiae wine strain. The implications for winemaking in response to the negative correlation of benzyl alcohol, benzyl acetate and 2-phenylethyl acetate production with yeast assimilable nitrogen concentrations are discussed. Copyright © 2016 John Wiley & Sons, Ltd.

Contribution of yeast and base wine supplementation to sparkling wine composition.

BACKGROUND: The differential characteristic of sparkling wine is the formation of foam, which is dependent, among other factors, on yeast autolysis, aging and oenological practices. In this study, we analyzed the effects of yeast strain, nutrient supplementation to the base wine and aging process on the sparkling wine composition and its foamability. RESULTS: We determined that the addition of inorganic nitrogen promoted nitrogen liberation to the extracellular medium, while the addition of inactive dry yeast to the base wine caused an increase in the polysaccharide concentration and foaming properties of the sparkling wine. The use of synthetic and natural base wines allowed us to discriminate that the differences in high-molecular-weight polysaccharides and oligosaccharides could be attributed to the yeast cells and that the higher nitrogen content in the natural wine could be due to external proteolysis. CONCLUSION: The practices of nitrogen addition and supplementation of inactive dry yeast could modulate the main characteristics of the sparkling wine and be a critical element for the design of this kind of wine. © 2016 Society of Chemical Industry.

Toxoplasma gondii infection modulate systemic allergic immune response in BALB/c mice.

The increased prevalence of allergies in developed countries has been attributed to a reduced exposure to some microbes. In agreement with epidemiological studies, we previously showed that Toxoplasma gondii infection prevents allergic airway inflammation. The mechanisms would be related to the strong Th1 response induced by the parasite and to regulatory cell induction. Herein we further characterized whether T. gondii allergy modulation extents to a systemic level or if it is limited to the lung. Parasite infection before allergic sensitization resulted in a diminished Th2 cytokine response and, when sensitized during acute infection, an increased in TGF-ß production was detected. Allergen specific T cell proliferation was also reduced. Sensitization during both acute and chronic phases of infection resulted in a decreased anaphylaxis reaction. Our results extend earlier work and show that, in addition to lung airway inflammation, T. gondii infection can suppress allergic responses at systemic level. These results open the possibility that this protozoan infection could modulate other allergic disorders such as atopic dermatitis or oral allergies. Understanding the mechanisms by which different microorganisms regulate inflammation may potentially lead to the development of strategies aimed to control atopic diseases.

Phytoextracts for Human Health from Raw and Roasted Hazelnuts and from Hazelnut Skin and Oil: A Narrative Review.

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.

Dual VEGFA/BRAF targeting boosts PD-1 blockade in melanoma through GM-CSF-mediated infiltration of M1 macrophages.

The introduction of targeted therapies represented one of the most significant advances in the treatment of BRAFV600E melanoma. However, the onset of acquired resistance remains a challenge. Previously, we showed in mouse xenografts that vascular endothelial growth factor (VEGFA) removal enhanced the antitumor effect of BRAF inhibition through the recruitment of M1 macrophages. In this work, we explored the strategy of VEGFA/BRAF inhibition in immunocompetent melanoma murine models. In BRAF mutant D4M melanoma tumors, VEGFA/BRAF targeting reshaped the tumor microenvironment, largely by stimulating infiltration of M1 macrophages and CD8+ T cells, and sensitized tumors to immune checkpoint blockade (ICB). Furthermore, we reported that the association of VEGFA/BRAF targeting with anti-PD-1 antibody (triple therapy) resulted in a durable response and enabled complete tumor eradication in 50% of the mice, establishing immunological memory. Neutralization and CRISPR-Cas-mediated editing of granulocyte-macrophage colony-stimulating factor (GM-CSF) abrogated antitumor response prompted by triple therapy and identified GM-CSF as the cytokine instrumental in M1-macrophage recruitment. Our data suggest that VEGFA/BRAF targeting in melanoma induces the activation of innate and adaptive immunity and prepares tumors for ICB. Our study contributes to understanding the tumor biology of BRAFV600E melanoma and suggests VEGFA as therapeutic target.

Maternal stress increases risk of allergic lung inflammation in adult mice.

BACKGROUND: Allergies are increasing worldwide. The presence of atopic diseases in the mother propagates the onset of allergic diseases in the offspring with a considerably stronger penetrance than atopic diseases of the father. Such observation challenges genetic predispositions as the sole cause of allergic diseases. Epidemiological studies suggest that caregiver stress in the perinatal period may predispose offspring to asthma. Only one group has studied the link between prenatal stress and neonatal asthma susceptibility in a murine model. OBJECTIVES: We aimed to study if the neonatal increased risk of developing allergic lung inflammation persists after puberty and if there are sex differences in susceptibility. METHODS: Pregnant BALB/c mice were subjected to a single restraint stress exposure at day 15 of gestation. Pups were separated by gender and subjected to a well-known sub-optimal asthma model after puberty. RESULTS: Adult mice born to stressed dams were more susceptible to developing allergic pulmonary inflammation since an increase in the number of eosinophils in bronchoalveolar lavage (BAL), a greater peribronchial and perivascular infiltrate, a higher proportion of mucus-producing cells, and increased IL-4 and IL-5 levels in BAL were detected compared to control mice. These effects were more profound in females than males. Moreover, only females from stressed dams showed an increase in IgE levels. CONCLUSIONS: Increased litter susceptibility to develop allergic lung inflammation induced by maternal stress persists after puberty and is more potent in females than in male mice.

A chloroplast-derived Toxoplasma gondii GRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice.

The parasitic protozoan Toxoplasma gondii, the causal agent of toxoplasmosis, can infect most mammals and birds. In human medicine, T. gondii can cause complications in pregnant women and immunodeficient individuals, while in veterinary medicine, T. gondii infection has economic importance due to abortion and neonatal loss in livestock. Thus, the development of an effective anti-Toxoplasma vaccine would be of great value. In this study, we analysed the expression of T. gondii GRA4 antigen by chloroplast transformation (chlGRA4) in tobacco plants and evaluated the humoral and cellular responses and the grade of protection after oral administration of chlGRA4 in a murine model. The Western blot analysis revealed a specific 34-kDa band mainly present in the insoluble fractions. The chlGRA4 accumulation levels were approximately 6 µg/g of fresh weight (equivalent to 0.2% of total protein). Oral immunization with chlGRA4 resulted in a decrease of 59% in the brain cyst load of mice compared to control mice. ChlGRA4 immunization elicited both a mucosal immune response characterized by the production of specific IgA, and IFN-γ, IL-4 and IL-10 secretion by mesenteric lymph node cells, and a systemic response in terms of GRA4-specific serum antibodies and secretion of IFN-γ, IL-4 and IL-10 by splenocytes. Our results indicate that oral administration of chlGRA4 promotes the elicitation of both mucosal and systemic balanced Th1/Th2 responses that control Toxoplasma infection, reducing parasite loads.

Tandem repeat-tRNA (TRtRNA) PCR method for the molecular typing of non-Saccharomyces subspecies.

There is a worldwide trend to understand the impact of non-Saccharomyces yeast species on the process of winemaking. Although the predominant species at the end of the fermentation is Saccharomyces cerevisiae, several non-Saccharomyces species present during the first days of the process can produce and/or release aromas that improve the bouquet and complexity of the final wine. Since no genomic sequences are available for the predominant non-Saccharomyces species selected from grapes or musts (Hanseniaspora uvarum, Hanseniaspora vineae, Hanseniaspora opuntiae, Metschnikowia pulcherrima, Candida zemplinina), a reproducible PCR method was devised to discriminate strains at the subspecies level. The method combines different oligonucleotides based on tandem repeats with a second oligonucleotide based on a conserved tRNA region, specific for ascomycetes. Tandem repeats are randomly dispersed in all eukaryotic genomes and tRNA genes are conserved and present in several copies in different chromosomes. As an example, the method was applied to discriminate native M. pulcherrima strains but it could be extended to differentiate strains from other non-Saccharomyces species. The biodiversity of species and strains found in the grape ecosystem is a potential source of new enzymes, fungicides and/or novel sustainable methods for biological control of phytopathogens.

Combination of CpG-oligodeoxynucleotides with recombinant ROP2 or GRA4 proteins induces protective immunity against Toxoplasma gondii infection.

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) have been characterized as Th1-promoting immunopotentiators, an adjuvant activity desirable for vaccination against intracellular parasites like Toxoplasma gondii. In an attempt to find new antigen-adjuvant combinations that enhance the immunogenicity of antigen candidates for toxoplasma vaccines, we analyzed the extent of protection in mice immunized with ROP2 and GRA4 recombinant proteins when co-administered with CpG-ODN. Both GRA4+CpG-ODN and ROP2+CpG-ODN formulations were shown to induce a strong humoral Th1-biased response characterized by a high IgG(2a) to IgG(1) antibody ratio. Both vaccination regimens led to increased secretion of IFN-γ and IL-10, and negligible amounts of IL-4, upon specific re-stimulation of spleen cells from these groups of mice. After a non-lethal challenge with tissue cysts of a moderately virulent strain, only the brains from mice vaccinated with ROP2 or GRA4 in combination with CpG-ODN showed a significant reduction (63% and 62%, respectively) in their parasite load compared to the controls. The rate of protection obtained with GRA4+ROP2+CpG-ODN resulted equivalent (66%) to those achieved with the single antigens plus CpG-ODN. Taken together, these results indicate that CpG-ODN is an important candidate adjuvant for use in potential multicomponent anti-T. gondii vaccines for animals and humans.

Synergistic effect of GRA7 and profilin proteins in vaccination against chronic Toxoplasma gondii infection.

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.

Heart rate variability and interoceptive accuracy predict impaired decision-making in Gambling Disorder.

BACKGROUND AND AIMS: Gambling Disorder (GD) entails maladaptive patterns of decision-making. Neurophysiological research points out the effect of parasympathetic arousal, including phasic changes in heart rate variability (HRV), and interoceptive accuracy (IA, i.e., the ability to track changes in bodily signals), on decision-making. Nevertheless, scarce evidence is available on their role in GD. This is the first study exploring the impact in GD of respiratory sinus arrhythmia (RSA), an index of HRV, and IA on decision-making, as measured by the Iowa Gambling Task (IGT). METHODS: Twenty-two patients experiencing problems with slot-machines or video lottery terminals gambling and 22 gender- and age-matched healthy controls (HC) were recruited. A resting ECG was performed before and after the completion of the IGT. IA was assessed throughout the heartbeat detection task. We conducted a MANCOVA to detect the presence of significant differences between groups in RSA reactivity and IA. A linear regression model was adopted to test the effect of factors of interest on IGT scores. RESULTS: Patients with GD displayed significantly decreased RSA reactivity (P = 0.002) and IA (P = 0.024) compared to HCs, even after controlling for affective symptoms, age, smoking status, and BMI. According to the linear regression model, cardiac vagal reactivity and IA significantly predict decision-making impairments on the IGT (P = 0.008; P = 0.019). DISCUSSION AND CONCLUSIONS: Although the exact pathways linking HRV and IA to impaired decision-making in GD remain to be identified, a broader exploration relying upon an embodiment-informed framework may contribute to shed further light on the clinical phenomenology of the disorder.

Effect of codon optimization and subcellular targeting on Toxoplasma gondii antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice.

BACKGROUND: Codon optimization and subcellular targeting were studied with the aim to increase the expression levels of the SAG178-322 antigen of Toxoplasma gondii in tobacco leaves. The expression of the tobacco-optimized and native versions of the SAG1 gene was explored by transient expression from the Agrobacterium tumefaciens binary expression vector, which allows targeting the recombinant protein to the endoplasmic reticulum (ER) and the apoplast. Finally, mice were subcutaneously and orally immunized with leaf extracts-SAG1 and the strategy of prime boost with rSAG1 expressed in Escherichia coli was used to optimize the oral immunization with leaf extracts-SAG1. RESULTS: Leaves agroinfiltrated with an unmodified SAG1 gene accumulated 5- to 10-fold more than leaves agroinfiltrated with a codon-optimized SAG1 gene. ER localization allowed the accumulation of higher levels of native SAG1. However, no significant differences were observed between the mRNA accumulations of the different versions of SAG1. Subcutaneous immunization with leaf extracts-SAG1 (SAG1) protected mice against an oral challenge with a non-lethal cyst dose, and this effect could be associated with the secretion of significant levels of IFN-gamma. The protection was increased when mice were ID boosted with rSAG1 (SAG1+boost). This group elicited a significant Th1 humoral and cellular immune response characterized by high levels of IFN-gamma. In an oral immunization assay, the SAG1+boost group showed a significantly lower brain cyst burden compared to the rest of the groups. CONCLUSION: Transient agroinfiltration was useful for the expression of all of the recombinant proteins tested. Our results support the usefulness of endoplasmic reticulum signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The results showed that this plant-produced protein has potential for use as vaccine and provides a potential means for protecting humans and animals against toxoplasmosis.