3DICE coding matrix multidirectional macro-architecture modulates cell organization, shape, and co-cultures endothelization network

Natural extracellular matrix governs cells providing biomechanical and biofunctional outstanding properties, despite being porous and mostly made of soft materials. Among organs, specific tissues present specialized macro-architectures. For instance, hepatic lobules present radial organization, while vascular sinusoids are branched from vertical veins, providing specific biofunctional features. Therefore, it is imperative to mimic such structures while modeling tissues. So far, there is limited capability of coupling oriented macro-structures with interconnected micro-channels in programmable long-range vertical and radial sequential orientations. Herein, a three-directional ice crystal elongation (3DICE) system is presented to code geometries in cryogels. Using 3DICE, guided ice crystals growth templates vertical and radial pores through bulky cryogels. Translucent isotropic and anisotropic architectures of radial or vertical pores are fabricated with tunable mechanical response. Furthermore, 3D combinations of vertical and radial pore orientations are coded at the centimeter scale. Cell morphological response to macro-architectures is demonstrated. The formation of endothelial segments, CYP450 activity, and osteopontin expression, as liver fibrosis biomarkers, present direct response and specific cellular organization within radial, linear, and random architectures. These results unlock the potential of ice-templating demonstrating the relevance of macro-architectures to model tissues, and broad possibilities for drug testing, tissue engineering, and regenerative medicine.The authors are grateful for the Portuguese Foundation for Science and Technology (FCT) distinction attributed to R. F. Canadas (SFRH/ BD/92565/2013), and to J. M. Oliveira (IF/00423/2012, IF/01285/ 2015). R. F. Canadas is also thankful to FCT, Fundo Europeu de Desenvolvimento Regional (FEDER), and Programa Operacional Competitividade e Internacionalizaç˜ao (POCI) for funding the B-Liver Project (PTDC/EMD-EMD/29139/2017). The authors are also thankful to FCT for supporting the project Hierarchitech (M-ERA-NET/0001/2014) and for the funds provided under the 3 BioMeD project (JICAM/0001/2017). The authors acknowledge that this material and collaboration is based in part upon work supported by Luso-American Development Foundation (FLAD), 2016/CON15/CAN6). U. Demirci is also grateful for the Canary Center at Stanford for Cancer Early Detection Seed Award. The authors are also grateful for the support provided by Diana Bicho and Nicolas Cristini on scaffold characterization and cell culture, respectively

Potential SLA Hp-4.0 haplotype-restricted CTL epitopes identified from the membrane protein of PRRSV induce cell immune responses

Swine leukocyte antigen (SLA) class I molecule-restricted T-cell epitopes, which induce cytotoxic T lymphocyte (CTL) responses, play a critical role in the clearance of porcine reproductive and respiratory syndrome virus (PRRSV) and the development of efficient protective vaccines. The SLA-1*04:01:01, SLA-2*04:01, and SLA-3*04:01 alleles, assigned the Hp-4.0 haplotype, are highly prevalent and usually present in all pig breeds. However, the SLA Hp-4.0 haplotype-restricted CTL epitopes in the structural membrane (M) protein of PRRSV are still unknown. In this study, we predicted 27 possible 9-mer epitope peptides in M protein with high binding scores for SLA-1*04:01:01 using CTL epitope prediction tools. In total, 45 SLA class I complexes, comprising the predicted peptide, extracellular region of the SLA-I molecules, and β2-microglobulin, were constructed in vitro to detect the specific binding of these peptides to SLA-1*04:01:01 (27 complexes), SLA-2*04:01 (9 complexes), and SLA-3*04:01 (9 complexes), respectively. Our results showed that the M27 (T91WKFITSRC), M39 (N130HAFVVRRP), and M49 (G158RKAVKQGV) peptides bind specifically to SLA-1*04:01:01, SLA-2*04:01, and SLA-3*04:01, respectively. Subsequently, using peripheral blood mononuclear cells (PBMCs) isolated from the homozygous Hp-4.0 and Hp-26.0 haplotype piglets vaccinated with commercial PRRSV HuN4-F112 strain, we determined the capacities of these 27 potential peptides to stimulate their proliferation with a Cell Counting Kit-8 and their secretion and expression of interferon gamma (IFN-γ) with an ELISpot assay and real-time qPCR, respectively. The immunological activities of M27, M39, and M49 were therefore confirmed when they efficiently induced PBMC proliferation and IFN-γ secretion in PBMCs from piglets with the prevalent SLA Hp-4.0 haplotype. The amino acid sequence alignment revealed that M27, M39, and M49 are highly conserved among 248 genotype II PRRSV strains collected between 1998 and 2019. These findings contribute to the understanding of the mechanisms of cell-mediated immune responses to PRRSV. Our study also provides a novel strategy for identifying and confirming potential SLA haplotype-restricted CTL epitopes that could be used to develop novel peptide-based vaccines against swine diseases

Molecular identification and probiotic potential characterization of lactic acid bacteria isolated from the pigs with superior immune responses

Lactic acid bacteria (LAB) belong to a significant group of probiotic bacteria that provide hosts with considerable health benefits. Our previous study showed that pigs with abundant LAB had more robust immune responses in a vaccination experiment. In this study, 52 isolate strains were isolated from the pigs with superior immune responses. Out of these, 14 strains with higher antibacterial efficacy were chosen. We then assessed the probiotic features of the 14 LAB strains, including such as autoaggregation, coaggregation, acid resistance, bile salt resistance, and adhesion capability, as well as safety aspects such as antibiotic resistance, hemolytic activity, and the presence or absence of virulence factors. We also compared these properties with those of an opportunistic pathogen EB1 and two commercial probiotics (cLA and cLP). The results showed that most LAB isolates exhibited higher abilities of aggregation, acid and bile salt resistance, adhesion, and antibacterial activity than the two commercial probiotics. Out of the 14 strains, only LS1 and LS9 carried virulence genes and none had hemolytic activity. We selected three LAB strains (LA6, LR6 and LJ1) with superior probiotic properties and LS9 with a virulence gene for testing their safety in vivo. Strains EB1, cLA and cLP were also included as control bacteria. The results demonstrated that mice treated LAB did not exhibit any adverse effects on weight gain, organ index, blood immune cells, and ileum morphology, except for those treated with LS9 and EB1. Moreover, the antimicrobial effect of LR6 and LA6 strains was examined in vivo. The results indicated that these strains could mitigate the inflammatory response, reduce bacterial translocation, and alleviate liver, spleen, and ileum injury caused by Salmonella typhimurium infection. In addition, the LR6 treatment group showed better outcomes than the LA6 treatment group; treatment with LR6 substantially reduced the mortality rate in mice. The study results provide evidence of the probiotic properties of the LAB isolates, in particular LR6, and suggest that oral administration of LR6 could have valuable health-promoting benefits

Synthesis and characterization of thermotropic liquid crystalline copolyester/multi-walled carbon nanotubes composites via in situ polymerization

We present a novel approach for the preparation of thermotropic liquid crystalline copolyester (TLCP)/multi-walled carbon nanotubes (MWCNTs) composites via in situ polymerization. A two-stage polycondensation procedure was employed in our process, where carboxylic acid modified MWCNTs were dispersed in acetic anhydride via ultrasonication prior to being charged to a prepolymerization reactor together with monomers and catalysts for esterification reaction at 130 degrees C-200 degrees C. The esterified mixture was then fed into a polycondensation reactor at 280 degrees C-320 degrees C. In this way, fully exfoliated MWCNTs were dispersed in the TLCP matrix at concentrations up to 0.3 wt%. Systematic studies show that well dispersed MWCNTs acted as "pseudo nucleation sites" for the nematic ordering in the adjacent TLCP melt. Thus the extrudates show a smaller core region and higher overall orientational order. Consequently, the addition of MWCNTs is not only effective in improving the mechanical stiffness but also toughness of the composites. For example, the 0.3 wt% TLCP/MWCNT composite shows a 62%, 135% and 145% increase in Young's modulus, tensile strength and toughness, respectively, in comparison with the pure TLCP. (C) 2012 Elsevier Ltd. All rights reserved