Lactic acid bacteria are components of the gastrointestinal tract microbiota in both humans and animals and are widely used as probiotics. Lactobacillus is the most closely related genus to probiotic activity. It is capable of releasing membrane microvesicles (MVs), whose primary functions include carrying and transmitting antigens to host tissues and modulating host defense responses. In the present study, MVs were isolated from Lactobacillus acidophilus resident in the ileum of free-living rats, and their immunostimulant effect was evaluated in two biological models. MVs were characterized using SDS-PAGE electrophoresis, electron microscopy, and nanoparticle tracking analysis. In the first model, the immunostimulatory effect of MVs was evaluated on ovine abomasal explants, which had been previously stimulated with MVs and then challenged with third-stage larvae of Haemonchus contortus. This resulted in a decrease in the percentage of larval association and favored the migration of inflammatory cells to the infection site. In the second model, the macrophage cell line RAW 264.7 was stimulated with MVs to evaluate the expression of transcripts encoding IL-1β and TNF-α. MVs isolated from L. acidophilus demonstrate immunostimulatory and probiotic effects in the two biological models assessed. This suggested that the MVs possess similar immunostimulatory effects as those reported for the parent bacteria.

A strategy to increase the transfection efficiency of chitosan-based nanoparticles for gene therapy is by adding nuclear localization signals through karyophilic peptides. Here, the effect of the length and sequence of these peptides and their interaction with different plasmids on the physical characteristics and biological functionality of nanoparticles is reported. The karyophilic peptides (P1 or P2) were used to assemble nanoparticles by complex coacervation with pEGFP-N1, pQBI25 or pSelect-Zeo-HSV1-tk plasmids, and chitosan. Size, polydispersity index, zeta potential, and morphology, as well as in vitro nucleus internalization and transfection capability of nanoparticles were determined. The P2 nanoparticles resulted smaller compared to the ones without peptides or P1 for the three plasmids. In general, the addition of either P1 or P2 did not have a significant impact on the polydispersity index and the zeta potential. P1 and P2 nanoparticles were localized in the nucleus after 30 min of exposure to HeLa cells. Nevertheless, the presence of P2 in pEGFP-N1 and pQBI25 nanoparticles raised their capability to transfect and express the green fluorescent protein. Thus, karyophilic peptides are an efficient tool for the optimization of nonviral vectors for gene delivery; however, the sequence and length of peptides have an impact on characteristics and functionality of nanoparticles.

The interaction between probiotic bacteria and the innate immune system is of increasing interest due to its capacity to modulate inflammatory and antimicrobial responses. The murine macrophage cell line RAW 264.7 is widely used to investigate the immunomodulatory effects of probiotic bacteria and their cell-free derivatives, such as membrane vesicles (MVs). In this study, we evaluated whether MVs derived from Lactobacillus acidophilus promote superior modulation of cytokine production and antimicrobial signaling in RAW 264.7 macrophages compared with whole cells (WCs). Our results show that L. acidophilus MVs exhibited direct bactericidal activity against Escherichia coli and induced a more selective and balanced cytokine profile than whole cells. These findings highlight the potential of probiotic-derived membrane vesicles as acellular immunomodulatory effectors for the development of novel cell-free biotherapeutic strategies.

Lactic acid bacteria are components of the gastrointestinal tract microbiota in both humans and animals and are widely used as probiotics. Lactobacillus is the most related genus to probiotic activity and is able to release membrane microvesicles (MVs), which primary functions include to carry and transmit antigens to host tissues and modulate host defense responses. In the present study, MVs were isolated from Lactobacillus acidophilus resident in the ileum of free-living rats, and its immunostimulant effect was evaluated in two biological models. MVs were characterized using SDS-PAGE electrophoresis, electron microscopy, and nanoparticle tracking analysis. In the first model, the immunostimulant effect of MVs was evaluated on ovine abomasal explants, previously stimulated with MVs and challenged with third-stage larvae of Haemonchus contortus, demonstrating a decrease in the percentage of larvae association, and favoring the migration of inflammatory cells to the infection site. In the second model, macrophage cell line (RAW 264.7) was stimulated with MVs to evaluate the expression of transcripts coding for IL-1β and TNF-α. In both cases, the immunostimulatory effect of MVs was verified, demonstrating that MVs can carry important antigens that could be considered in a formulation to promote the intestinal health in livestock production.

The interaction between probiotic bacteria and the innate immune system is of in-creasing interest due to its capacity to modulate inflammatory and antimicrobial re-sponses. The murine macrophage cell line RAW 264.7 is widely used to investigate the immunomodulatory effects of probiotic bacteria and their cell-free derivatives, such as membrane vesicles (MVs). In this study, we evaluated whether MVs derived from Lactobacillus acidophilus promote superior modulation of cytokine production and antimicrobial signaling in RAW 264.7 macrophages compared with whole cells (W.C). Our results show that L. acidophilus MVs exhibited direct bactericidal activity against Escherichia coli and induced a more selective and balanced cytokine profile than whole cells. These findings highlight the potential of probiotic-derived membrane vesicles as acellular immunomodulatory effectors for the development of novel cell-free biotherapeutic strategies.