Differential Activation of Splenic cDC1 and cDC2 Cell Subsets following Poxvirus Infection of BALB/c and C57BL/6 Mice.

Conventional dendritic cells (cDCs) are innate immune cells that play a pivotal role in inducing antiviral adaptive immune responses due to their extraordinary ability to prime and polarize naïve T cells into different effector T helper (Th) subsets. The two major subpopulations of cDCs, cDC1 (CD8α+ in mice and CD141+ in human) and cDC2 (CD11b+ in mice and CD1c+ in human), can preferentially polarize T cells toward a Th1 and Th2 phenotype, respectively. During infection with ectromelia virus (ECTV), an orthopoxvirus from the Poxviridae family, the timing and activation of an appropriate Th immune response contributes to the resistance (Th1) or susceptibility (Th2) of inbred mouse strains to the lethal form of mousepox. Due to the high plasticity and diverse properties of cDC subpopulations in regulating the quality of a specific immune response, in the present study we compared the ability of splenic cDC1 and cDC2 originating from different ECTV-infected mouse strains to mature, activate, and polarize the Th immune response during mousepox. Our results demonstrated that during early stages of mousepox, both cDC subsets from resistant C57BL/6 and susceptible BALB/c mice were activated upon in vivo ECTV infection. These cells exhibited elevated levels of surface MHC class I and II, and co-stimulatory molecules and showed enhanced potential to produce cytokines. However, both cDC subsets from BALB/c mice displayed a higher maturation status than that of their counterparts from C57BL/6 mice. Despite their higher activation status, cDC1 and cDC2 from susceptible mice produced low amounts of Th1-polarizing cytokines, including IL-12 and IFN-γ, and the ability of these cells to stimulate the proliferation and Th1 polarization of allogeneic CD4+ T cells was severely compromised. In contrast, both cDC subsets from resistant mice produced significant amounts of Th1-polarizing cytokines and demonstrated greater capability in differentiating allogeneic T cells into Th1 cells compared to cDCs from BALB/c mice. Collectively, our results indicate that in the early stages of mousepox, splenic cDC subpopulations from the resistant mouse strain can better elicit a Th1 cell-mediated response than the susceptible strain can, probably contributing to the induction of the protective immune responses necessary for the control of virus dissemination and for survival from ECTV challenge.

Ectromelia Virus Affects the Formation and Spatial Organization of Adhesive Structures in Murine Dendritic Cells In Vitro.

Ectromelia virus (ECTV) is a causative agent of mousepox. It provides a suitable model for studying the immunobiology of orthopoxviruses, including their interaction with the host cell cytoskeleton. As professional antigen-presenting cells, dendritic cells (DCs) control the pericellular environment, capture antigens, and present them to T lymphocytes after migration to secondary lymphoid organs. Migration of immature DCs is possible due to the presence of specialized adhesion structures, such as podosomes or focal adhesions (FAs). Since assembly and disassembly of adhesive structures are highly associated with DCs' immunoregulatory and migratory functions, we evaluated how ECTV infection targets podosomes and FAs' organization and formation in natural-host bone marrow-derived DCs (BMDC). We found that ECTV induces a rapid dissolution of podosomes at the early stages of infection, accompanied by the development of larger and wider FAs than in uninfected control cells. At later stages of infection, FAs were predominantly observed in long cellular extensions, formed extensively by infected cells. Dissolution of podosomes in ECTV-infected BMDCs was not associated with maturation and increased 2D cell migration in a wound healing assay; however, accelerated transwell migration of ECTV-infected cells towards supernatants derived from LPS-conditioned BMDCs was observed. We suggest that ECTV-induced changes in the spatial organization of adhesive structures in DCs may alter the adhesiveness/migration of DCs during some conditions, e.g., inflammation.

Graphene 2D platform is safe and cytocompatibile for HaCaT cells growing under static and dynamic conditions.

The study concerns the influence of graphene monolayer, as a 2 D platform, on cell viability, cytoskeleton, adhesions sites andmorphology of mitochondria of keratinocytes (HaCaT) under static conditions. Based on quantitative and immunofluorescent analysis, it could be stated that graphene substrate does not cause any damage to membrane or disruption of other monitored parameters. Spindle poles and cytokinesis bridges indicating proliferation of cells on this graphene substrate were detected. Moreover, the keratinocyte migration rate on the graphene substrate was comparable to control glass substrate when the created wound was completely closed after 38 hours. HaCaT morphology and viability were also assessed under dynamic conditions (lab on a chip - micro scale). For this purpose, microfluidic graphene system was designed and constructed. No differences as well as no anomalies were observed during cultivation of these cells on the graphene or glass substrates in relation to cultivation conditions: static (macro scale) and dynamic (micro scale). Only natural percentage of dead cells was determined using different methods, which proved that the graphene as the 2 D platform is cytocompatible with keratinocytes. The obtained results encourage the use of the designed lab on a chip system in toxicity testing of graphene also on other cells and further research on the use of graphene monolayers to produce bio-bandages for skin wounds in animal tests.

Bioavailability of Macro- and Microelements in Rats Fed Hypercholesterolemic Diets Containing Actinidia arguta Fruits.

The aim of this study was to estimate the influence of different cultivars of Actinidia arguta (kiwiberry) on the bioavailability of mineral elements and to examine the mineral profile of rats fed atherogenic diets enriched with kiwiberries. The following cultivars of Actinidia arguta were used: Bingo, M1, Anna, Weiki, Jumbo, and Geneva. Kiwiberry has recently become popular in the market. It is a precious source of biologically active components, vitamins, and minerals. The livers, spleens, and kidneys were examined for mineral contents using the flame atomic absorption spectroscopy method. The bioavailability of Ca, Mg, Fe, Mn, Zn, and Cu was evaluated. The addition of kiwiberries in atherogenic diets increased the contents of Fe in the rat liver. The bioavailability of Mn, Zn, and Cu, calculated on the basis of the contents in the livers, was significantly decreased in rats fed diets with 5% additional kiwiberries. We supposed that the effect of kiwiberry on the bioavailability of the studied minerals may be related to the diet components of bioactive substances present in fruits (polyphenols, vitamins, dietary fiber, and tannins).

Cytocompatibility of Graphene Monolayer and Its Impact on Focal Cell Adhesion, Mitochondrial Morphology and Activity in BALB/3T3 Fibroblasts.

This study investigates the effect of graphene scaffold on morphology, viability, cytoskeleton, focal contacts, mitochondrial network morphology and activity in BALB/3T3 fibroblasts and provides new data on biocompatibility of the "graphene-family nanomaterials". We used graphene monolayer applied onto glass cover slide by electrochemical delamination method and regular glass cover slide, as a reference. The morphology of fibroblasts growing on graphene was unaltered, and the cell viability was 95% compared to control cells on non-coated glass slide. There was no significant difference in the cell size (spreading) between both groups studied. Graphene platform significantly increased BALB/3T3 cell mitochondrial activity (WST-8 test) compared to glass substrate. To demonstrate the variability in focal contacts pattern, the effect of graphene on vinculin was examined, which revealed a significant increase in focal contact size comparing to control-glass slide. There was no disruption in mitochondrial network morphology, which was branched and well connected in relation to the control group. Evaluation of the JC-1 red/green fluorescence intensity ratio revealed similar levels of mitochondrial membrane potential in cells growing on graphene-coated and uncoated slides. These results indicate that graphene monolayer scaffold is cytocompatible with connective tissue cells examined and could be beneficial for tissue engineering therapy.

Human mesenchymal stem cell (hMSC) differentiation towards cardiac cells using a new microbioanalytical method.

Stem cells (SCs) are more and more often applied in tissue engineering and cell therapies, e.g. in regenerative medicine. Standard methods of SC differentiation are time consuming and ineffective. Therefore, new bioanalytical methods (i.e. Lab-on-a-Chip systems) are develop to improve such type of studies. Although, microtechnology is a rapidly growing research area, there are so far not too many works which present SC differentiation into cardiomyocytes in the microsystems. Therefore, we present new microbioanalytical method of SC differentiation towards cardiac cells using a newly developed digitally controlled microdispenser integrated with a Heart-on-a-chip system. Seven-day culture of human mesenchymal stem cells (hMSCs) and their differentiation using biochemical factors such as 5-AZA (2 µM, 24 h) and VEGF (20 ng ml-1, 72 h) were investigated in the microsystem which was automatically operated using smartphone software. hMSC differentiation into the cardiac cells was confirmed using immunostaining of cardiac markers (α-actinin and troponin T). The usage of the microsystem allowed shortening the time of hMSC differentiation in comparison to macroscale method. We showed that the microsystem, in which the in vivo microenvironment is mimicked and dynamic conditions are provided by a microdispenser, favorably affect hMSC differentiation towards cardiac cells. Based on the presented research we can conclude that the developed digitally controlled microsystem could be successfully utilized as a new microbioanalytical method for stem cells differentiation and analysis of their function under dynamic conditions. In the future, this could be a helpful tool for scientists working on regenerative medicine.

The effects of graphene and mesenchymal stem cells in cutaneous wound healing and their putative action mechanism.

This study provides a review of the therapeutic potential of graphene dressing scaffolds and mesenchymal stem cells (MSCs) and their synergistic effects with respect to cutaneous wound healing. This study also considers their putative action mechanism based on the antibacterial, immunomodulating, angiogenic, matrix remodeling effects of materials belonging to the graphene family and MSCs during the wound healing process. In addition, this study discusses the cytocompatibility of graphene, its uses as a platform for skin substitutes, the properties it possesses with respect to providing protection against microbial invasion as well as strategies aimed at minimizing the chance of the occurrence of sepsis. MSCs are capable of secreting several factors that exert a therapeutic impact on reparative processes and tissue regeneration. In light of experiments conducted to date, graphene combined with MSCs appears to have the potential to enhance both the wound healing process and infection control at the injury site.

Biocompatibility of pristine graphene monolayer: Scaffold for fibroblasts.

The aim of the present study was to evaluate the cytotoxicity of pristine graphene monolayer and its utility as a scaffold for murine fibroblast L929 cell line. Cell viability, morphology, cytoskeleton architecture (microfilaments and microtubules), cell adhesion and migration into the scratch-wound area were determined using pristine graphene-coated microscopic slides. We found that fibroblasts cultured on pristine graphene monolayer exhibited changes in cell attachment, motility and cytoskeleton organization. Graphene was found to have no cytotoxicity on L929 fibroblasts and increased cell adhesion and proliferation within 24 h of culture. The area of cells growing on graphene was comparable to the area of fibroblasts cultured on glass. Migration of cells on the surface of graphene substrate appeared to be more regular in comparison to uncoated glass surface, however in both control (glass) and experimental (graphene) groups the scratch wound was closed after 48 h of culture. Taken together, our results indicate that pristine graphene monolayer is non-toxic for murine subcutaneous connective tissue fibroblasts and could be beneficial for recovery of damaged tissues after injury. These studies could be helpful in evaluating biocompatibility of graphene, which still remains ambiguous.