How dietary advanced glycation end products could facilitate the occurrence of food allergy.

BACKGROUND: Food allergy (FA) is one of the most common chronic conditions in children with an increasing prevalence facilitated by the exposure to environmental factors in predisposed individuals. It has been hypothesized that the increased consumption of ultra-processed foods, containing high levels of dietary advanced glycation end products (AGEs), could facilitate the occurrence of FA. OBJECTIVE: We sought to provide preclinical and clinical evidence on the potential role of AGEs in facilitating the occurrence of FA. METHODS: Human enterocytes, human small intestine organ culture, and PBMCs from children at risk for allergy were used to investigate the direct effect of AGEs on gut barrier, inflammation, TH2 cytokine response, and mitochondrial function. Intake of the 3 most common glycation products in Western diet foods, Nε-(carboxymethyl) lysine, Nε-(1-carboxyethyl) lysin, and Nδ-(5-hydro-5- methyl-4-imidazolone-2-yl)-ornithine (MG-H1), and the accumulation of AGEs in the skin were comparatively investigated in children with FA and in age-matched healthy controls. RESULTS: Human enterocytes exposed to AGEs showed alteration in gut barrier, AGE receptor expression, reactive oxygen species production, and autophagy, with increased transepithelial passage of food antigens. Small intestine organ cultures exposed to AGEs showed an increase of CD25+ cells and proliferating crypt enterocytes. PBMCs exposed to AGEs showed alteration in proliferation rate, AGE receptor activation, release of inflammatory and TH2 cytokines, and mitochondrial metabolism. Significant higher dietary AGE intake and skin accumulation were observed children with FA (n = 42) compared with age-matched healthy controls (n = 66). CONCLUSIONS: These data, supporting a potential role for dietary AGEs in facilitating the occurrence of FA, suggest the importance of limiting exposure to AGEs children as a potential preventive strategy against this common condition.

O protagonismo da criança em cuidados paliativos para a efetivação da sua segurança / Protagonism of the child in palliative care and the impact on its safety / El protagonismo de los niños en los cuidados paliativos para la realización de su seguridad

Objetivo: contribuir com o debate sobre o protagonismo da criança no seu processo de cuidado em saúde, alertando sobre o papel importante dessa participação no Cuidado Paliativo Pediátrico (CPP) e consequentemente, na sua própria segurança. Metodologia: revisão narrativa, a partir do levantamento bibliográfico nas bases de dados MEDLINE (via PubMed) e SciELO, nos últimos 10 anos, acrescida da experiência das autoras e captação secundária. Resultados: foram incluídos 14 artigos na síntese narrativa. A análise desses artigos em relação a segurança do paciente em CPP revelou a dificuldade da abordagem dos CPP na prática, além de lacunas na formação dos profissionais, a importância do cuidado centrado no paciente, das decisões compartilhadas, da comunicação apropriada e do uso seguro de opioides. Em relação a participação da criança como protagonista do seu cuidado, foi observado o desejo da criança em participar efetivamente da tomada de decisão, o reconhecimento da sua autonomia e a avaliação objetiva da sua competência para tal. Conclusão: muitos aspectos do CPP coadunam com a segurança do paciente, como a comunicação apropriada e a participação da criança como protagonista do seu processo de cuidado. A autonomia da criança enquanto paciente encontra-se num processo crescente de reconhecimento. Não obstante haja um arcabouço normativo acerca deste tema, a perspectiva observada em muitos artigos ainda é do cuidado centrado na família e para impulsionar a mudança necessária, é fundamental que se invista em todos os níveis de educação em saúde e em pesquisa.

Objective: to contribute to the debate on the role of the child in his or her own health process and to highlight the important role of this involvement in pediatric palliative care (PPC) and, thus, in his or her own safety. Methods: this was a narrative review based on a literature search in MEDLINE and SciELO over the past 10 years, with additional input from the authors' experiences and secondary literature. Results: Fourteen articles were included in the narrative synthesis. Analysis of the articles on patient safety in PPC revealed difficulty in practicing PPC, there are gaps in professional education, importance of putting the patient first, shared decision-making, appropriate communication, and safe opioid use. Regarding the child's participation as a protagonist in PPC, concerns were raised about the child's wishes, effective participation in decision making, and objective assessment of the child's competence in this area. Conclusion: many aspects of PPC are consistent with patient safety, such as appropriate communication and the child's participation in his or her own care process. There was greater recognition of the child's autonomy as a patient. Although this topic is normative, many articles are about family-centered care. To reshape this approach, it is important that health care education and research be strengthened.

Objetivo: contribuir con el debate sobre el papel de los niños en su proceso de cuidado y salud, alertando sobre el papel importante de esta participación en los Cuidados Paliativos Pediátricos (CPP) y, consecuentemente, en su propia seguridad. Metodología: revisión narrativa, a partir del levantamiento bibliográfico en las bases de datos MEDLINE (vía PubMed) y SciELO, en los últimos 10 años, más la experiencia de los autores y captura secundaria. Resultados: fueron incluidos 14 artículos en la síntesis narrativa. El análisis de estos artículos en relación con la seguridad del paciente en PPC reveló la dificultad de abordar la PPC en la práctica, además de las lagunas en la formación de los profesionales, la importancia del cuidado centrado en el paciente, las decisiones compartidas, la comunicación adecuada y el uso de opioides seguro. En cuanto a la participación del niño como protagonista de su CPP, se observó el deseo del niño de participar de manera efectiva en la toma de decisiones, el reconocimiento de su autonomía y la evaluación objetiva de su competencia para hacerlo. Conclusión: muchos aspectos de la CPP son consistentes con la seguridad del paciente, así como la comunicación adecuada y participación del niño como protagonista de su proceso de atención. A autonomía da crianza se encuentra en unproceso creciente de reconocimiento. No obstante, hay un marco de referencia normativo acerca de este tema, una perspectiva observada en muchos artículos y un cuidado centrado en la familia y para impulsar una mudanza necesaria, es fundamental que se informe a todos los niños de educación en salud e en investigación.

Protective effects of the postbiotic deriving from cow's milk fermentation with L. paracasei CBA L74 against Rotavirus infection in human enterocytes.

Rotavirus (RV) is the leading cause of acute gastroenteritis-associated mortality in early childhood. Emerging clinical evidence suggest the efficacy of the postbiotic approach based on cow's milk fermentation with the probiotic Lacticaseibacillus paracasei CBAL74 (FM-CBAL74) in preventing pediatric acute gastroenteritis, but the mechanisms of action are still poorly characterized. We evaluated the protective action of FM-CBAL74 in an in vitro model of RV infection in human enterocytes. The number of infected cells together with the relevant aspects of RV infection were assessed: epithelial barrier damage (tight-junction proteins and transepithelial electrical resistance evaluation), and inflammation (reactive oxygen species, pro-inflammatory cytokines IL-6, IL-8 and TNF-α, and mitogen-activated protein kinase pathway activation). Pre-incubation with FM-CBA L74 resulted in an inhibition of epithelial barrier damage and inflammation mediated by mitogen-activated protein kinase pathway activation induced by RV infection. Modulating several protective mechanisms, the postbiotic FM-CBAL74 exerted a preventive action against RV infection. This approach could be a disrupting nutritional strategy against one of the most common killers for the pediatric age.

A New Butyrate Releaser Exerts a Protective Action against SARS-CoV-2 Infection in Human Intestine.

Butyrate is a major gut microbiome metabolite that regulates several defense mechanisms against infectious diseases. Alterations in the gut microbiome, leading to reduced butyrate production, have been reported in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. A new butyrate releaser, useful for all the known applications of butyrate, presenting physiochemical characteristics suitable for easy oral administration, (N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA), has been recently developed. We investigated the protective action of FBA against SARS-CoV-2 infection in the human small intestine and enterocytes. Relevant aspects of SARS-CoV-2 infection were assessed: infectivity, host functional receptor angiotensin-converting enzyme-2 (ACE2), transmembrane protease serine 2 (TMPRSS2), neuropilin-1 (NRP1), pro-inflammatory cytokines expression, genes involved in the antiviral response and the activation of Nf-kB nuclear factor (erythroid-derived 2-like) 2 (Nfr2) pathways. We found that FBA positively modulates the crucial aspects of the infection in small intestinal biopsies and human enterocytes, reducing the expression of ACE2, TMPRSS2 and NRP1, pro-inflammatory cytokines interleukin (IL)-15, monocyte chemoattractant protein-1 (MCP-1) and TNF-α, and regulating several genes involved in antiviral pathways. FBA was also able to reduce the number of SARS-CoV-2-infected cells, and ACE2, TMPRSS2 and NRP1 expression. Lastly, through the inhibition of Nf-kB and the up-regulation of Nfr2, it was also able to reduce the expression of pro-inflammatory cytokines IL-15, MCP-1 and TNF-α in human enterocytes. The new butyrate releaser, FBA, exerts a preventive action against SARS-CoV-2 infection. It could be considered as an innovative strategy to limit COVID-19.

Evidence on the Preventive Effects of the Postbiotic Derived from Cow's Milk Fermentation with Lacticaseibacillus paracasei CBA L74 against Pediatric Gastrointestinal Infections.

Postbiotics are commonly defined as preparations of inanimate probiotics and/or their cellular components and/or their metabolites/end products that confer health benefits on the host. They have been suggested as a promising strategy to limit infectious diseases. Emerging evidence support the efficacy of the postbiotic derived from cow's milk fermentation with the probiotic Lacticaseibacillus paracasei CBAL74 (FM-CBAL74) in preventing pediatric infectious diseases. We aimed at reviewing the evidence available.

Corrigendum: Age-Related Differences in the Expression of Most Relevant Mediators of SARS-CoV-2 Infection in Human Respiratory and Gastrointestinal Tract.

[This corrects the article DOI: 10.3389/fped.2021.697390.].

Phenylalanine Butyramide Is a New Cosmetic Ingredient with Soothing and Anti-Reddening Potential.

Human skin is colonized by diverse commensal microbes, making up the skin microbiota (SM), contributing to skin integrity and homeostasis. Many of the beneficial effects aroused by the SM are exerted by microbial metabolites such as short-chain fatty acids (SCFAs), including butyric acid. The SCFAs can be used in cosmetic formulations against skin diseases to protect SM by preserving and/or restoring their natural balance. Unpleasant sensorial properties and unfavorable physico-chemical properties of butyrate strongly limit its cosmetic use. In contrast, some butyrate derivatives, including phenylalanine butyramide (C13H18N2O2, FBA), a solid form of butyric acid, are odorless while retaining the pharmacokinetic properties and safety profile of butyric acid. This study assessed the FBA's permeation across the skin and its soothing and anti-reddening potential to estimate its cosmetic application. The dosage method used to estimate FBA's levels was validated to be sure of analytical results. The FBA diffusion tests were estimated in vitro using a Franz-type vertical diffusion cell. The soothing action was evaluated in vivo by Colorimeter CL400, measuring the erythema index. The results suggest that the FBA represents an innovative way to exploit the benefits of butyric acid in the cosmetic fields since it cannot reach the bloodstream, is odorless, and has a significative soothing action (decrease the erythema index -15.7% after 30', and -17.8% after 60').

Protective effects elicited by cow milk fermented with L. Paracasei CBAL74 against SARS-CoV-2 infection in human enterocytes.

Fermented foods have been proposed in limiting SARS-CoV-2 infection. Emerging evidence suggest the efficacy of cow's milk fermented with the probiotic L. paracasei CBAL74 (FM-CBAL74) in preventing infectious diseases. We evaluated the protective action of FM-CBAL74 against SARS-CoV-2 infection in human enterocytes. Relevant aspects of SARS-CoV-2 infection were assessed: infectivity, host functional receptor angiotensin-converting enzyme-2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and pro-inflammatory cytokines expression (IL-6, IL-15, IL-1ß, VEGFß, TNF-α, MCP-1, CXCL1). Pre-incubation with FM-CBA L74 reduced the number of infected cells. The expression of ACE2 and the pro-inflammatory cytokines IL-6, VEGFß, IL-15, IL-1ß was downregulated by the pre-treatment with this fermented food. No effect on TMPRSS2, MCP-1, TNF-α and CXCL1 expression was observed. Modulating the crucial aspects of the infection, the fermented food FM-CBAL74 exerts a preventive action against SARS-CoV-2. These evidence could pave the way to innovative nutritional strategy to mitigate the COVID-19.

Age-Related Differences in the Expression of Most Relevant Mediators of SARS-CoV-2 Infection in Human Respiratory and Gastrointestinal Tract.

Background: Clinical features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection seem to differ in children compared to that in adults. It has been hypothesized that the lower clinical severity in children could be influenced by differential expression of the main host functional receptor to SARS-CoV-2, the angiotensin-converting enzyme 2 (ACE2), but data are still conflicting. To explore the origin of age-dependent clinical features of coronavirus disease 2019 (COVID-19), we comparatively evaluated the expression in children and adult subjects of the most relevant mediators of the SARS-CoV-2 infection: ACE2, angiotensin-converting enzyme 1 (ACE1), transmembrane serine protease-2 (TMPRSS2), and neuropilin-1 (NRP1), at upper respiratory tract and small intestine level. Methods: The expression of ACE2, ACE1, TMPRSS2, and NRP1 in nasal epithelium and in small intestine epithelium was investigated by quantitative real-time PCR analysis. Results: We found no differences in ACE2, ACE1, and TMPRSS2 expression in the nasal epithelium comparing children and adult subjects. In contrast, nasal epithelium NRP1 expression was lower in children compared to that in adults. Intestinal ACE2 expression was higher in children compared to that in adults, whereas intestinal ACE1 expression was higher in adults. Intestinal TMPRSS2 and NRP1 expression was similar comparing children and adult subjects. Conclusions: The lower severity of SARS-CoV-2 infection observed in children may be due to a different expression of nasal NRP1, that promotes the virus interaction with ACE2. However, the common findings of intestinal symptoms in children could be due to a higher expression of ACE2 at this level. The insights from these data will be useful in determining the treatment policies and preventive measures for COVID-19.

Butyrate as a bioactive human milk protective component against food allergy.

BACKGROUND: Food allergy (FA) is a growing health problem worldwide. Effective strategies are advocated to limit the disease burden. Human milk (HM) could be considered as a protective factor against FA, but its mechanisms remain unclear. Butyrate is a gut microbiota-derived metabolite able to exert several immunomodulatory functions. We aimed to define the butyrate concentration in HM, and to see whether the butyrate concentration detected in HM is able to modulate the mechanisms of immune tolerance. METHODS: HM butyrate concentration from 109 healthy women was assessed by GS-MS. The effect of HM butyrate on tolerogenic mechanisms was assessed in in vivo and in vitro models. RESULTS: The median butyrate concentration in mature HM was 0.75 mM. This butyrate concentration was responsible for the maximum modulatory effects observed in all experimental models evaluated in this study. Data from mouse model show that in basal condition, butyrate up-regulated the expression of several biomarkers of gut barrier integrity, and of tolerogenic cytokines. Pretreatment with butyrate significantly reduced allergic response in three animal models of FA, with a stimulation of tolerogenic cytokines, inhibition of Th2 cytokines production and a modulation of oxidative stress. Data from human cell models show that butyrate stimulated human beta defensin-3, mucus components and tight junctions expression in human enterocytes, and IL-10, IFN-γ and FoxP3 expression through epigenetic mechanisms in PBMCs from FA children. Furthermore, it promoted the precursors of M2 macrophages, DCs and regulatory T cells. CONCLUSION: The study's findings suggest the importance of butyrate as a pivotal HM compound able to protect against FA.

Protective action of Bacillus clausii probiotic strains in an in vitro model of Rotavirus infection.

Rotavirus is the most common cause of acute gastroenteritis (AGE) in young children. Bacillus clausii (B. clausii) is a spore-forming probiotic that is able to colonize the gut. A mixture of four B. clausii strains (O/C, T, SIN and N/R) is commonly used for the treatment of AGE, and it has been demonstrated that it can reduce the duration and severity of diarrhea in children with AGE. Few studies have sought to characterize the mechanisms responsible for such beneficial effects. Intestinal effects of probiotics are likely to be strain-specific. We conducted a series of in vitro experiments investigating the activities of this mixture of B. clausii strains on biomarkers of mucosal barrier integrity and immune function in a cellular model of Rotavirus infection. B. clausii protected enterocytes against Rotavirus-induced decrease in trans-epithelial electrical resistance, and up-regulated expression of mucin 5AC and tight junction proteins (occludin and zonula occludens-1), all of which are important for effective mucosal barrier function. B. clausii also inhibited reactive oxygen species production and release of pro-inflammatory cytokines (interleukin-8 and interferon-ß) in Rotavirus-infected cells, and down-regulated pro-inflammatory Toll-like receptor 3 pathway gene expression. Such mechanisms likely contributed to the observed protective effects of B. clausii against reduced cell proliferation and increased apoptosis in Rotavirus-infected enterocytes.

Tolerogenic Effect Elicited by Protein Fraction Derived From Different Formulas for Dietary Treatment of Cow's Milk Allergy in Human Cells.

Several formulas are available for the dietary treatment of cow's milk allergy (CMA). Clinical data suggest potentially different effect on immune tolerance elicited by these formulas. We aimed to comparatively evaluate the tolerogenic effect elicited by the protein fraction of different formulas available for the dietary treatment of CMA. Five formulas were compared: extensively hydrolyzed whey formula (EHWF), extensively hydrolyzed casein formula (EHCF), hydrolyzed rice formula (HRF), soy formula (SF), and amino acid-based formula (AAF). The formulas were reconstituted in water according to the manufacturer's instructions and subjected to an in vitro infant gut simulated digestion using a sequential gastric and duodenal static model. Protein fraction was then purified and used for the experiments on non-immune and immune components of tolerance network in human enterocytes and in peripheral mononuclear blood cells (PBMCs). We assessed epithelial layer permeability and tight junction proteins (occludin and zonula occludens-1, ZO-1), mucin 5AC, IL-33, and thymic stromal lymphopoietin (TSLP) in human enterocytes. In addition, Th1/Th2 cytokine response and Tregs activation were investigated in PBMCs from IgE-mediated CMA infants. EHCF-derived protein fraction positively modulated the expression of gut barrier components (mucin 5AC, occludin and ZO-1) in human enterocytes, while SF was able to stimulate the expression of occludin only. EHWF and HRF protein fractions elicited a significant increase in TSLP production, while IL-33 release was significantly increased by HRF and SF protein fractions in human enterocytes. Only EHCF-derived protein fraction elicited an increase of the tolerogenic cytokines production (IL-10, IFN-γ) and of activated CD4+FoxP3+ Treg number, through NFAT, AP1, and Nf-Kb1 pathway. The effect paralleled with an up-regulation of FoxP3 demethylation rate. Protein fraction from all the study formulas was unable to induce Th2 cytokines production. The results suggest a different regulatory action on tolerogenic mechanisms elicited by protein fraction from different formulas commonly used for CMA management. EHCF-derived protein fraction was able to elicit tolerogenic effect through at least in part an epigenetic modulation of FoxP3 gene. These results could explain the different clinical effects observed on immune tolerance acquisition in CMA patients and on allergy prevention in children at risk for atopy observed using EHCF.

The therapeutic efficacy of Bifidobacterium animalis subsp. lactis BB-12® in infant colic: A randomised, double blind, placebo-controlled trial.

BACKGROUND: The pathogenesis of infant colic is poorly defined. Gut microbiota seems to be involved, supporting the potential therapeutic role of probiotics. AIMS: To assess the rate of infants with a reduction of ≥50% of mean daily crying duration after 28 days of intervention with the probiotic Bifidobacterium animalis subsp. lactis BB-12® (BB-12). Secondary outcomes were daily number of crying episodes, sleeping time, number of bowel movements and stool consistency. METHODS: Randomized controlled trial (RCT) on otherwise healthy exclusively breastfed infants with infant colic randomly allocated to receive BB-12 (1 × 109  CFU/day) or placebo for 28 days. Gut microbiota structure and butyrate, beta-defensin-2 (HBD-2), cathelicidin (LL-37), secretory IgA (sIgA) and faecal calprotectin levels were assessed. RESULTS: Eighty infants were randomised, 40/group. The rate of infants with reduction of ≥50% of mean daily crying duration was higher in infants treated with BB-12, starting from the end of 2nd week. No infant relapsed when treatment was stopped. The mean number of crying episodes decreased in both groups, but with a higher effect in BB-12 group (-4.7 ± 3.4 vs -2.3 ± 2.2, P < 0.05). Mean daily stool frequency decreased in both groups but the effect was significantly higher in the BB-12 group; stool consistency was similar between the two groups. An increase in Bifidobacterium abundance (with significant correlation with crying time reduction), butyrate and HBD-2, LL-37, sIgA levels associated with a decrease in faecal calprotectin level were observed in the BB-12 group. CONCLUSIONS: Supplementation with BB-12 is effective in managing infant colic. The effect could derive from immune and non-immune mechanisms associated with a modulation of gut microbiota structure and function.

Publisher Correction: Randomized controlled trial on the influence of dietary intervention on epigenetic mechanisms in children with cow's milk allergy: the EPICMA study.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Gut Microbiome as Target for Innovative Strategies Against Food Allergy.

The dramatic increase in food allergy prevalence and severity globally requires effective strategies. Food allergy derives from a defect in immune tolerance mechanisms. Immune tolerance is modulated by gut microbiota function and structure, and microbiome alterations (dysbiosis) have a pivotal role in the development of food allergy. Environmental factors, including a low-fiber/high-fat diet, cesarean delivery, antiseptic agents, lack of breastfeeding, and drugs can induce gut microbiome dysbiosis, and have been associated with food allergy. New experimental tools and technologies have provided information regarding the role of metabolites generated from dietary nutrients and selected probiotic strains that could act on immune tolerance mechanisms. The mechanisms are multiple and still not completely defined. Increasing evidence has provided useful information on optimal bacterial species/strains, dosage, and timing for intervention. The increased knowledge of the crucial role played by nutrients and gut microbiota-derived metabolites is opening the way to a post-biotic approach in the stimulation of immune tolerance through epigenetic regulation. This review focused on the potential role of gut microbiome as the target for innovative strategies against food allergy.

Randomized controlled trial on the influence of dietary intervention on epigenetic mechanisms in children with cow's milk allergy: the EPICMA study.

Epigenetic mechanisms could drive the disease course of cow's milk allergy (CMA) and formula choice could modulate these pathways. We compared the effect of two different dietary approaches on epigenetic mechanisms in CMA children. Randomized controlled trial on IgE-mediated CMA children receiving a 12-month treatment with extensively hydrolyzed casein formula containing the probiotic L.rhamnosus GG (EHCF + LGG) or with soy formula (SF). At the baseline, after 6 and 12 months of treatment FoxP3 methylation rate and its expression in CD4+ T cells were assessed. At same study points IL-4, IL-5, IL-10, and IFN-γ methylation rate, expression and serum concentration, miRNAs expression were also investigated. 20 children (10/group) were evaluated. Baseline demographic, clinical and epigenetic features were similar in the two study groups. At 6 and 12 months, EHCF + LGG group showed a significant increase in FoxP3 demethylation rate compared to SF group. At the same study points, EHCF + LGG group presented a higher increase in IL-4 and IL-5 and a higher reduction in IL-10 and IFN-γ DNA methylation rate compared to SF group. A different modulation of miR-155, -146a, -128 and -193a expression was observed in EHCF + LGG vs. SF. Dietary intervention could exert a different epigenetic modulation on the immune system in CMA children.

Targeting Food Allergy with Probiotics.

The dramatic increase in food allergy prevalence and severity globally is demanding effective strategies. Food allergy derives from a defect in immune tolerance mechanisms. Immune tolerance is modulated by gut microbiota composition and function, and gut microbiota dysbiosis has been associated with the development of food allergy. Selected probiotic strains could act on immune tolerance mechanisms. The mechanisms are multiple and still not completely defined. Increasing evidence is providing useful information on the choice of optimal bacterial species/strains, dosage, and timing for intervention. The increased knowledge on the crucial role played by gut microbiota-derived metabolites, such as butyrate, is also opening the way to a post-biotic approach in the stimulation of immune tolerance.

The preparation and characterization of PLG nanoparticles with an entrapped synthetic TLR7 agonist and their preclinical evaluation as adjuvant for an adsorbed DTaP vaccine.

The design of safe and potent adjuvants able to enhance and modulate antigen-specific immunity is of great interest for vaccine research and development. In the present study, negatively charged poly(lactide-co-glycolide) (PLG) nanoparticles have been combined with a synthetic immunepotentiator molecule targeting the Toll-like receptor 7. The selection of appropriate preparation and freeze-drying conditions resulted in a PLG-based adjuvant with well-defined and stable physico-chemical properties. The adjuvanticity of such nanosystem has later been evaluated in the mouse model with a diphtheria-tetanus-pertussis (DTaP) vaccine, on the basis of the current need to improve the efficacy of acellular pertussis (aP) vaccines. DTaP antigens were adsorbed onto PLG nanoparticles surface, allowing the co-delivery of TLR7a and multiple antigens through a single formulation. The entrapment of TLR7a into PLG nanoparticles resulted in enhanced IgG and IgG2a antibody titers. Notably, the immune potentiator effect of TLR7a was less evident when it was used in not-entrapped form, indicating that co-localization of TLR7a and antigens is required to adequately stimulate immune responses. In conclusion, the rational selection of adjuvants and formulation here described resulted as a highly valuable approach to potentiate and better tailor DTaP vaccine immunogenicity.

Multiplex immunoassay for in vitro characterization of acellular pertussis antigens in combination vaccines.

Vaccines characterization is required to ensure physical, chemical, and biological integrity of antigens and adjuvants. Current analytical methods mostly require complete antigen desorption from aluminum-based adjuvants and are not always suitable to distinguish individual antigens in multivalent formulations. Here, Luminex technology is proposed to improve the analytics of vaccine characterization. As proof of concept, TdaP (tetanus, diphtheria and acellular pertussis) combination, adjuvanted with aluminum hydroxide, was chosen as model formulation to quantify and determine the level of adsorption of acellular pertussis (aP) antigens onto adjuvant surface at the same time. The assay used specific antibodies bound to magnetic microspheres presenting unique digital signatures for each pertussis antigen, allowing the simultaneous recognition of respective antigens in the whole vaccine, avoiding laborious procedures for adjuvant separation. Accurate and reproducible quantification of aP antigens in TdaP vaccine has been achieved in the range 0.78-50 ng/mL, providing simultaneously information on antigen identity, quantity, and degree of adsorption to aluminum hydroxide. The current study could further be considered as a model to set up in vitro potency assays thus supporting the replacement of animal tests accordingly to the 3Rs concept.