A new generation of recombinant polypeptides combines multiple protein domains for effective antimicrobial activity

Background Although most of antimicrobial peptides (AMPs), being relatively short, are produced by chemical synthesis, several AMPs have been produced using recombinant technology. However, AMPs could be cytotoxic to the producer cell, and if small they can be easily degraded. The objective of this study was to produce a multidomain antimicrobial protein based on recombinant protein nanoclusters to increase the yield, stability and effectivity. Results A single antimicrobial polypeptide JAMF1 that combines three functional domains based on human α-defensin-5, human XII-A secreted phospholipase A2 (sPLA2), and a gelsolin-based bacterial-binding domain along with two aggregation-seeding domains based on leucine zippers was successfully produced with no toxic effects for the producer cell and mainly in a nanocluster structure. Both, the nanocluster and solubilized format of the protein showed a clear antimicrobial effect against a broad spectrum of Gram-negative and Gram-positive bacteria, including multi-resistant strains, with an optimal concentration between 1 and 10 µM. Conclusions Our findings demonstrated that multidomain antimicrobial proteins forming nanoclusters can be efficiently produced in recombinant bacteria, being a novel and valuable strategy to create a versatile, highly stable and easily editable multidomain constructs with a broad-spectrum antimicrobial activity in both soluble and nanostructured format.info:eu-repo/semantics/publishedVersio

Exploring the impact of the recombinant Escherichia coli strain on defensins antimicrobial activity: BL21 versus Origami strain

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a β-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality.info:eu-repo/semantics/publishedVersio

Lactiplantibacillus plantarum: a new example of inclusion body producing bacteria

Background Lactic Acid Bacteria such as Lactococcus lactis, Latilactobacillus sakei (basonym: Lactobacillus sakei) and Lactiplantibacillus plantarum (basonym: Lactobacillus plantarum) have gained importance as recombinant cell factories. Although it was believed that proteins produced in these lipopolysaccharides (LPS)-free microorganisms do not aggregate, it has been shown that L. lactis produce inclusion bodies (IBs) during the recombinant production process. These protein aggregates contain biologically active protein, which is slowly released, being a biomaterial with a broad range of applications including the obtainment of soluble protein. However, the aggregation phenomenon has not been characterized so far in L. plantarum. Thus, the current study aims to determine the formation of protein aggregates in L. plantarum and evaluate their possible applications. Results To evaluate the formation of IBs in L. plantarum, the catalytic domain of bovine metalloproteinase 9 (MMP-9cat) protein has been used as model protein, being a prone-to-aggregate (PTA) protein. The electron microscopy micrographs showed the presence of electron-dense structures in L. plantarum cytoplasm, which were further purified and analyzed. The ultrastructure of the isolated protein aggregates, which were smooth, round and with an average size of 250–300 nm, proved that L. plantarum also forms IBs under recombinant production processes of PTA proteins. Besides, the protein embedded in these aggregates was fully active and had the potential to be used as a source of soluble protein or as active nanoparticles. The activity determination of the soluble protein solubilized from these IBs using non-denaturing protocols proved that fully active protein could be obtained from these protein aggregates. Conclusions These results proved that L. plantarum forms aggregates under recombinant production conditions. These aggregates showed the same properties as IBs formed in other expression systems such as Escherichia coli or L. lactis. Thus, this places this LPS-free microorganism as an interesting alternative to produce proteins of interest for the biopharmaceutical industry, which are obtained from the IBs in an important number of cases.info:eu-repo/semantics/publishedVersio

Potential of Oral Nanoparticles Containing Cytokines as Intestinal Mucosal Immunostimulants in Pigs : A Pilot Study

Antibiotics are essential compounds to cope with bacterial infections. However, their inadequate and excessive use has triggered the rapid arising of antimicrobial-resistant bacteria. In this scenario, immunostimulants, which are molecules that boost the immune system, open up a new approach to face this problem, enhancing treatment efficacy and preventing infections by immune system response. Cytokines are central effector molecules of the immune system, and their recombinant production and administration in animals could be an interesting immune modulation strategy. The aim of this study was the development of a highly stable nanoparticle of porcine cytokines to achieve the immunostimulation of intestinal mucosa in piglets. The outcomes of the present study prove this approach is able to stimulate swine intestinal cells and macrophages in vitro and tends to modulate inflammatory responses in vivo, although further studies are required to definitively evaluate their potential in animals. Antimicrobial resistance is a global threat that is worryingly rising in the livestock sector. Among the proposed strategies, immunostimulant development appears an interesting approach to increase animal resilience at critical production points. The use of nanoparticles based on cytokine aggregates, called inclusion bodies (IBs), has been demonstrated as a new source of immunostimulants in aquaculture. Aiming to go a step further, the objective of this study was to produce cytokine nanoparticles using a food-grade microorganism and to test their applicability to stimulate intestinal mucosa in swine. Four cytokines (IL-1β, IL-6, IL-8, and TNF-α) involved in inflammatory response were produced recombinantly in Lactococcus lactis in the form of protein nanoparticles (IBs). They were able to stimulate inflammatory responses in a porcine enterocyte cell line (IPEC-J2) and alveolar macrophages, maintaining high stability at low pH and high temperature. In addition, an in vivo assay was conducted involving 20 piglets housed individually as a preliminary exploration of the potential effects of IL-1β nanoparticles in piglet intestinal mucosa after a 7 d oral administration. The treated animals tended to have greater levels of TNF-α in the blood, indicating that the tested dose of nanoparticles tended to generate an inflammatory response in the animals. Whether this response is sufficient to increase animal resilience needs further evaluation

A Novel Generation of Tailored Antimicrobial Drugs Based on Recombinant Multidomain Proteins

Antibiotic resistance has exponentially increased during the last years. It is necessary to develop new antimicrobial drugs to prevent and treat infectious diseases caused by multidrug- or extensively-drug resistant (MDR/XDR)-bacteria. Host Defense Peptides (HDPs) have a versatile role, acting as antimicrobial peptides and regulators of several innate immunity functions. The results shown by previous studies using synthetic HDPs are only the tip of the iceberg, since the synergistic potential of HDPs and their production as recombinant proteins are fields practically unexplored. The present study aims to move a step forward through the development of a new generation of tailored antimicrobials, using a rational design of recombinant multidomain proteins based on HDPs. This strategy is based on a two-phase process, starting with the construction of the first generation molecules using single HDPs and further selecting those HDPs with higher bactericidal efficiencies to be combined in the second generation of broad-spectrum antimicrobials. As a proof of concept, we have designed three new antimicrobials, named D5L37βD3, D5L37D5L37 and D5LAL37βD3. After an in-depth exploration, we found D5L37D5L37 to be the most promising one, since it was equally effective against four relevant pathogens in healthcare-associated infections, such as methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis (MRSE) and MDR Pseudomonas aeruginosa, being MRSA, MRSE and P. aeruginosa MDR strains. The low MIC values and versatile activity against planktonic and biofilm forms reinforce the use of this platform to isolate and produce unlimited HDP combinations as new antimicrobial drugs by effective means.info:eu-repo/semantics/publishedVersio

Angiotensin II type 1/adenosine A2A receptor oligomers: a novel target for tardive dyskinesia

Tardive dyskinesia (TD) is a serious motor side effect that may appear after long-term treatment with neuroleptics and mostly mediated by dopamine D2 receptors (D2Rs). Striatal D2R functioning may be finely regulated by either adenosine A2A receptor (A2AR) or angiotensin receptor type 1 (AT1R) through putative receptor heteromers. Here, we examined whether A2AR and AT1R may oligomerize in the striatum to synergistically modulate dopaminergic transmission. First, by using bioluminescence resonance energy transfer, we demonstrated a physical AT1R-A2AR interaction in cultured cells. Interestingly, by protein-protein docking and molecular dynamics simulations, we described that a stable heterotetrameric interaction may exist between AT1R and A2AR bound to antagonists (i.e. losartan and istradefylline, respectively). Accordingly, we subsequently ascertained the existence of AT1R/A2AR heteromers in the striatum by proximity ligation in situ assay. Finally, we took advantage of a TD animal model, namely the reserpine-induced vacuous chewing movement (VCM), to evaluate a novel multimodal pharmacological TD treatment approach based on targeting the AT1R/A2AR complex. Thus, reserpinized mice were co-treated with sub-effective losartan and istradefylline doses, which prompted a synergistic reduction in VCM. Overall, our results demonstrated the existence of striatal AT1R/A2AR oligomers with potential usefulness for the therapeutic management of TD

Development of new immunostimulant and antimicrobial protein-based molecules from a One Health perspective

El descobriment dels antibiòtics és considerat una de les principals fites de la medicina moderna. No obstant això, l'abús i l'ús excessiu d'antibiòtics ha desencadenat una ràpida expansió de la resistència antimicrobiana (AMR), comprometent tant la salut humana com l'animal. En aquest escenari, l'Organització Mundial de la Salut va demanar que s'abordés la crisis de AMR mitjançant una estratègia multifacética, transdisciplinària i integradora, anomenada One Health Approach. El desenvolupament de tractaments alternatius als antibiòtics és un aspecte crític en la lluita contra la resistència antimicrobiana. Dins d'aquest marc, els pèptids de defensa de l'hoste (HDPs) presenten una dualitat destacada com antimicrobians d'ampli espectre -fins i tot contra els bacteris AMR-, i molècules de regulació immunitària de l'hoste, despertant l'interès de la comunitat científica. Tot i així, generalment els HDPs es sintetitzen químicament, provocant un alt cost associat que implica un gran inconvenient per la seva implementació a gran escala. En aquest context, la producció de proteïnes recombinats emergeix com una alternativa assequible per compostos antimicrobians basats en proteïnes amb uns rendiments notables i fàcil escalat. No obstant, la producció recombinant de HDPs és complexa degut a les seves característiques intrínseques, on la seva reduïda mida condiciona una primerenca degradació proteolítica en l'hoste bacterià recombinant, juntament amb una toxicitat no desitjada, esdevenen en conjunt els principals obstacles per la seva producció recombinant. Per tant, amb l'objectiu de trobar el potencial dels HDPs com a els agents antimicrobians de pròxima generació, aquesta tesi s'ha focalitzat en el desenvolupament de medicaments antimicrobians modificables basats en HDPs utilitzant i millorant l'aproximació de la seva producció recombinant. Com a primer pas, hem explorat quines fàbriques cel·lulars microbianes són més adients per la seva producció, sempre tenint en compte la presència de ponts de disulfur conservats en les defensines, una de les famílies més rellevants dels HDPs. A més, per fer front als problemes relacionats amb la proteòlisis i potencial toxicitat envers el productor bacterià, hem desenvolupat una primera generació de pèptids antimicrobians en els quals els HDPs s'han fusionat amb la Green Fluorescence Protein (GFP). Addicionalment, també hem considerat fonts alternatives per purificar els HDPs tenint en compte la seva alta ratio d'agregació i formació de cossos d'inclusió (IBs). Els nostres primers resultats van indicar que Escherichia coli BL21 és un bon hoste per la seva producció, aconseguint molècules antimicrobianes amb gran activitat i puresa. També hem demostrat que IBs són una font natural de HDPs d'alta qualitat, desenvolupant específicament un protocol no desnaturalitzant lliure de detergents per evitar pèrdues d'activitat inesperades. Aquests resultats ens han encoratjat a construir una segona generació de proteïnes antimicrobianes basades en HDPs, combinant els HDPs més prometedors en un sol polipèptid i eliminant la GFP. En general, vam provar com aquestes construccions multidomini tenen un activitat antimicrobiana millorada d'ampli aspecte i una MIC més baixa que els seus anàlegs monodomini, demostrant l'efecte sinèrgic de combinar diferents HDPs. Al mateix temps, també hem abordat la problemàtica de AMR mitjançant un enfocament complementari, basat en el desenvolupament de nous IBs formats per citocines amb capacitat immunoestimulants produït en Lactococcus lactis. La nostra hipòtesis era augmentar la resiliència de l'animal a les infeccions mitjançant l'activació del seu sistema immunitari de forma prèvia a un moment de producció crític, reduint la necessitat de tractaments amb antibiòtic. Interessantment, les primeres troballes in vitro van demostrar les propietats immunoestimulants de les citocines nanoparticulades de porcí, encara que només és va observar una tendència en els experiments preliminars in vivo amb garrins.El descubrimiento de los antibióticos es considerado uno de los principales hitos de la medicina moderna. Sin embargo, el abuso y el uso excesivo de antibióticos ha desencadenado una rápida expansión de la resistencia antimicrobiana (AMR), comprometiendo tanto la salud humana como la de los animales. En este escenario, la Organización Mundial de la Salud pidió que se abordara la crisis de AMR mediante una estrategia multifacética, transdisciplinaria e integradora, denominada One Health Approach. El desarrollo de tratamientos alternativos a los antibióticos es un aspecto crítico en la lucha contra la resistencia antimicrobiana. Dentro de esta conjetura, los péptidos de defensa del huésped (HDPs) presentan una dualidad notoria como antimicrobianos de amplio espectro -incluso contra las bacterias AMR-, y moléculas de regulación inmunitaria del huésped, despertando el interés de la comunidad científica. Aun así, generalmente los HDPs se sintetizan químicamente, provocando un alto coste asociado, que implica un gran inconveniente en su implementación a gran escala. En este contexto, la producción de proteínas recombinantes emerge como una alternativa asequible para compuestos antimicrobianos basados en proteínas con unos rendimientos notables y fácil cambio de escala. No obstante, la producción recombinante de los HDPs es compleja debido a sus características intrínsecas, donde su reducido tamaño condiciona una temprana degradación proteolítica en el huésped bacteriano recombinante, junto con una toxicidad no deseada, acontecen en conjunto los principales obstáculos para su producción recombinante. Por lo tanto, con el objetivo de desenmascarar el potencial de los HDPs como agentes antimicrobianos de vanguardia, esta tesis se ha focalizado en el desarrollo de medicamentos antimicrobianos modificables basados en HDPs utilizando y mejorando la aproximación de su producción recombinante. Como primer paso, hemos explorado qué fábricas celulares microbianas son más adecuadas para su producción, siempre teniendo en cuenta la presencia de puentes de disulfuro conservados en las defensinas, una de las familias más relevantes de los HDPs. Además, para hacer frente a los problemas relacionados con la proteólisis y potencial toxicidad hacia el huésped bacteriano, hemos desarrollado una primera generación de péptidos antimicrobianos en los cuales los HDPs se han fusionado con la Green Fluorescence Protein (GFP). Adicionalmente, también hemos considerado fuentes alternativas para purificar los HDPs teniendo en cuenta su alto ratio de agregación y formación de cuerpos de inclusión (IBs). Nuestros primeros resultados indicaron que Escherichia coli BL21 es un buen huésped para su producción, consiguiendo moléculas antimicrobianas con gran actividad y pureza. También hemos demostrado que IBs son una fuente natural de HDPs de alta calidad, desarrollando específicamente un protocolo no desnaturalizante libre de detergentes para evitar así pérdidas de actividad inesperadas. Estos resultados nos han alentado a construir una segunda generación de proteínas antimicrobianas basadas en HDPs, combinando los HDPs más prometedores en un solo polipéptido y eliminando la GFP. En general, probamos como estas construcciones multidominio tienen una actividad antimicrobiana amplio aspecto mejorada y una MIC más baja que sus análogos monodominio, demostrando el efecto sinérgico de combinar diferentes HDPs. Al mismo tiempo, también hemos abordado la problemática de AMR mediante un enfoque complementario, basado en el desarrollo de nuevos IBs formados por citocinas con capacidad inmunoestimuladora producidas en Lactococcus lactis. Nuestra hipótesis era aumentar la resiliencia del animal a las infecciones mediante la activación de su sistema inmunitario de forma previa a un momento de producción crítico, reduciendo la necesidad de tratamientos con antibiótico. Interesantemente, los primeros hallazgos in vitro demostraron las propiedades inmunoestimuladoras de las citocinas nanoparticuladas de porcino, aunque solo es observó una tendencia en los experimentos preliminares in vivo con lechones.Antibiotics breakthrough is considered among the most remarkable hallmarks of modern medicine. However, antibiotic misuse and overuse have triggered the swift expansion of antimicrobial resistance (AMR), compromising both human and animal health. In this scenario, the World Health Organization called for addressing the AMR crisis using a multifaceted, transdisciplinary and integrative strategy, named One Health Approach. The development of alternative treatments to antibiotics is a critical aspect in the AMR fight. Within this framework, the Host Defense Peptides (HDPs) hold an outstanding duality as broad-spectrum antimicrobials -even against AMR bacteria-, and host immune regulation molecules, arousing the scientific community interest. Still, HDPs are generally chemically synthesized, but the high associated cost entails a major drawback for broader implementation. In this context, recombinant protein production emerges as an inexpensive source of antimicrobial protein-based compounds with noteworthy yields and straightforward scale-up. Although promising, recombinant HDP production is challenging due to the HDPs characteristics, being their small size, an early proteolytic degradation in the recombinant bacterial host, coupled with non-desirable recombinant host toxicity, the major obstacles for their recombinant production. With the aim of pursuing HDPs potential as the next generation antimicrobials, this thesis has been focused on the development of tunable HDPs-based antimicrobial drugs using and improving a recombinant production approach. As a first step, we have explored the most appropriate microbial cell factory for their production, taking into account the presence of conserved disulfide bridges in defensins, one of the most relevant HDPs family. In addition, to overcome proteolytic-related issues and potential toxicity for the bacterial producer, we have developed first-generation antimicrobial peptides in which HDPs have been fused to the Green Fluorescence Protein (GFP) carrier. Besides, we have also assessed alternative sources to purify the HDPs, considering their high aggregation ratio and inclusion bodies (IBs) formation. Our first findings pointed out that Escherichia coli BL21 is a suitable host for their production, achieving highly pure and active antimicrobial molecules. We have also demonstrated that IBs are a natural source of high-quality HDPs, developing also a free-detergent non-denaturing protocol to avoid unexpected activity losses. These results have encouraged us to construct a second-generation of HDP-based antimicrobial proteins, combining the most promising HDPs in a single polypeptide and removing the GFP carrier. Overall, we showed how these multidomain constructs hold an enhanced broad-spectrum antimicrobial activity and lower minimal inhibitory concentration (MIC) than their monodomain analogs, proving the synergic effect of combining different HDPs. Concurrently, we also tackled the AMR problems by using a complementary approach based on the development of novel cytokine-based IBs immunostimulants produced in Lactococcus lactis. The hypothesis was to increase the animal resilience to infections by activation of their immune system previous to critical productive moments, which will decrease the need of using antibiotic treatments. Interestingly, the first in vitro findings demonstrated the immunostimulant properties of the nanoparticulated porcine cytokines, although only a tendency was observed in preliminary in vivo experiments with piglets

Quality comparison of recombinant soluble proteins and proteins solubilized from bacterial inclusion bodies

Recombinant protein production in bacteria is often accompanied by the formation of aggregates, known as inclusion bodies (IBs). Although several strategies have been developed to minimize protein aggregation, many heterologous proteins are produced in aggregated form. For these proteins, purification necessarily requires processes of solubilization and refolding, often involving denaturing agents. However, the presence of biologically active recombinant proteins forming IBs has driven a redefinition of the protocols used to obtain soluble protein avoiding the protein denaturation step. Among the different strategies described, the detergent n-lauroylsarcosine (NLS) has proved to be effective. However, the impact of the NLS on final protein quality has not been evaluated so far. Here, the activity of three antimicrobial proteins (all as GFP fusions) obtained from the soluble fraction was compared with those solubilized from IBs. Results showed that NLS solubilized proteins from IBs efficiently, but that protein activity was impaired. Thus, a solubilization protocol without detergents was evaluated, demonstrating that this strategy efficiently solubilized proteins embedded in IBs while retaining their biological activity. These results showed that the protocol used for IB solubilization has an impact on final protein quality and that IBs can be solubilized through a very simple step, obtaining fully active proteins.info:eu-repo/semantics/publishedVersio

Exploring the impact of the recombinant Escherichia coli strain on defensins antimicrobial activity: BL21 versus Origami strain

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a β-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality.This work was funded by Ministerio de Ciencia, Innovación y Universidades Grants (PID2019-107298RB-C21) to AA and EG-F, PID2019-105622RB-I00 to IR and through the ‘‘Severo Ochoa’’ Programme for Centers of Excellence in R&D (FUNFUTURE CEX2019-000917-S and SEV-2017-0706). The authors are also grateful to Marató de TV3 foundation for the Grant 201812-30-31-32-33 to EG-F and IR, and the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). AL-C received a pre-doctoral fellowship from Generalitat de Catalunya (FI-AGAUR), EG-F a post-doctoral fellowship from INIA and JG a Ramón y Cajal fellowship from MINN (RyC-2017-22614).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Potential of Oral Nanoparticles Containing Cytokines as Intestinal Mucosal Immunostimulants in Pigs: A Pilot Study

Antimicrobial resistance is a global threat that is worryingly rising in the livestock sector. Among the proposed strategies, immunostimulant development appears an interesting approach to increase animal resilience at critical production points. The use of nanoparticles based on cytokine aggregates, called inclusion bodies (IBs), has been demonstrated as a new source of immunostimulants in aquaculture. Aiming to go a step further, the objective of this study was to produce cytokine nanoparticles using a food-grade microorganism and to test their applicability to stimulate intestinal mucosa in swine. Four cytokines (IL-1β, IL-6, IL-8, and TNF-α) involved in inflammatory response were produced recombinantly in Lactococcus lactis in the form of protein nanoparticles (IBs). They were able to stimulate inflammatory responses in a porcine enterocyte cell line (IPEC-J2) and alveolar macrophages, maintaining high stability at low pH and high temperature. In addition, an in vivo assay was conducted involving 20 piglets housed individually as a preliminary exploration of the potential effects of IL-1β nanoparticles in piglet intestinal mucosa after a 7 d oral administration. The treated animals tended to have greater levels of TNF-α in the blood, indicating that the tested dose of nanoparticles tended to generate an inflammatory response in the animals. Whether this response is sufficient to increase animal resilience needs further evaluation.info:eu-repo/semantics/publishedVersio