A new approach to obtain pure and active proteins from Lactococcus lactis protein aggregates

The production of pure and soluble proteins is a complex, protein-dependent and time-consuming process, in particular for those prone-to-aggregate and/or difficult-to-purify. Although Escherichia coli is widely used for protein production, recombinant products must be co-purified through costly processes to remove lipopolysaccharide (LPS) and minimize adverse effects in the target organism. Interestingly, Lactococcus lactis, which does not contain LPS, could be a promising alternative for the production of relevant proteins. However, to date, there is no universal strategy to produce and purify any recombinant protein, being still a protein-specific process. In this context and considering that L. lactis is also able to form functional protein aggregates under overproduction conditions, we explored the use of these aggregates as an alternative source of soluble proteins. In this study, we developed a widely applicable and economically affordable protocol to extract functional proteins from these nanoclusters. For that, two model proteins were used: mammary serum amyloid A3 (M-SAA3) and metalloproteinase 9 (MMP-9), a difficult-to-purify and a prone-to-aggregate protein, respectively. The results show that it is possible to obtain highly pure, soluble, LPS-free and active recombinant proteins from L. lactis aggregates through a cost-effective and simple protocol with special relevance for difficult-to-purify or highly aggregated proteins

Phosphatidylserine-Liposomes promote tolerogenic features on dendritic cells in human type 1 diabetes by apoptotic mimicry

Type 1 diabetes (T1D) is a metabolic disease caused by the autoimmune destruction of insulin-producing β-cells. With its incidence increasing worldwide, to find a safe approach to permanently cease autoimmunity and allow β-cell recovery has become vital. Relying on the inherent ability of apoptotic cells to induce immunological tolerance, we demonstrated that liposomes mimicking apoptotic β-cells arrested autoimmunity to β-cells and prevented experimental T1D through tolerogenic dendritic cell (DC) generation. These liposomes contained phosphatidylserine (PS)-the main signal of the apoptotic cell membrane-and β-cell autoantigens. To move toward a clinical application, PS-liposomes with optimum size and composition for phagocytosis were loaded with human insulin peptides and tested on DCs from patients with T1D and control age-related subjects. PS accelerated phagocytosis of liposomes with a dynamic typical of apoptotic cell clearance, preserving DCs viability. After PS-liposomes phagocytosis, the expression pattern of molecules involved in efferocytosis, antigen presentation, immunoregulation, and activation in DCs concurred with a tolerogenic functionality, both in patients and control subjects. Furthermore, DCs exposed to PS-liposomes displayed decreased ability to stimulate autologous T cell proliferation. Moreover, transcriptional changes in DCs from patients with T1D after PS-liposomes phagocytosis pointed to an immunoregulatory prolife. Bioinformatics analysis showed 233 differentially expressed genes. Genes involved in antigen presentation were downregulated, whereas genes pertaining to tolerogenic/anti-inflammatory pathways were mostly upregulated. In conclusion, PS-liposomes phagocytosis mimics efferocytosis and leads to phenotypic and functional changes in human DCs, which are accountable for tolerance induction. The herein reported results reinforce the potential of this novel immunotherapy to re-establish immunological tolerance, opening the door to new therapeutic approaches in the field of autoimmunity

Use of autoantigen-loaded phosphatidylserine-liposomes to arrest autoimmunity in type 1 diabetes

INTRODUCTION: The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow β-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes. OBJECTIVE: To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to β-cells in type 1 diabetes. METHODS: A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides. RESULTS: We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4+ T cells in vivo. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4+ T cell expansion. CONCLUSIONS: We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases

Functional protein-based nanomaterial produced in GRAS microorganism : a new platform for biotechnology

Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest

Use of Autoantigen-Loaded Phosphatidylserine-Liposomes to Arrest Autoimmunity in Type 1 Diabetes

Introduction: The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow a-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes. Objective: To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to a-cells in type 1 diabetes. Methods: A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides. Results: We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4+ T cells in vivo. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4+ T cell expansion. Conclusions: We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases.This work was supported by a grant from Spanish Government (FIS PI12/00195). IPA was supported by AGAUR, Generalitat de Catalunya. MVP and RA are supported by the Health Dept. of the Catalan Government, Generalitat de Catalunya. Special thanks to Ms. M.A. Cardus and her family for their generous donatio

Rubrica d’avaluació de l’activitat Demostracions Psicosocials

La rúbrica que es presenta és l’instrument per avaluar el seminari de Demostracions Psicosocials que es realitza com activitat d’avaluació continuada en l’assignatura de Ciències Psicosocials Aplicades a la Salut, en el primer curs del Grau d’Infermeria

Use of autoantigen-loaded phosphatidylserine-liposomes to arrest autoimmunity in type 1 diabetes

Introduction The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow β -cell regeneration. Based on the immuno- modulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes. Objective To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to β -cells in type 1 diabetes. Methods A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by anti- gen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the re- establishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides.Results We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tol- erogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4 + T cells in vivo . The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4 + T cell expansion. Conclusions We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune disease

Non-twist invariant circles in conformally symplectic systems

Dissipative mechanical systems on the torus with a friction that is proportional to the velocity are modeled by conformally symplectic maps on the annulus, which are maps that transport the symplectic form into a multiple of itself (with a conformal factor smaller than 1). It is important to understand the structure and the dynamics on the attractors. It is well-known that, with the aid of parameters, and under suitable non-degeneracy conditions, one can obtain that there is an attractor that is an invariant torus whose internal dynamics is conjugate to a rotation. By analogy with symplectic dynamics, a natural question is establishing appropriate definitions for twist and non-twist invariant tori in conformally symplectic systems. The main goals of this paper are: (a) to establish proper definitions of twist and non-twist invariant tori in families of conformally symplectic systems; (b) to interpret these definitions in terms of dynamical properties; (c) to derive algorithms to compute twist and non-twist invariant tori; (d) to implement these algorithms in examples; (e) to explore the mechanisms of breakdown of twist and non-twist invariant tori. Hence, the last part of the paper is devoted to implementations of the algorithms, illustrating the definitions presented in this paper, and studying robustness properties of invariant tori

Procedimiento e intermedios para la preparación de L-carbidopa

La presente invención se refiere a un procedimiento para la preparación del compuesto L-carbidopa, así como a intermedios utilizados en el mismo

Spectral approach to the modeling of the singing voice

Comunicació presentada a la 111th Audio Engineering Society Convention, que va tenir lloc del 30 de novembre al 3 de desembre de 2001 a Nova York, Estats Units.Comunicació presentada a la 111th Audio Engineering Society Convention, que va tenir lloc del 30 de novembre al 3 de desembre de 2001 a Nova York, Estats Units.In this paper we present two different approaches to the modeling of the singing voice. Each of these approaches has been thought to fit in the specific requirements of two applications. These are an automatic voice impersonator for karaoke systems and a singing voice synthesizer