Limbal stem cells (LSCs) are of paramount importance in corneal epithelial tissue repair. The cornea becomes opaque in case of limbal stem cell deficiency (LSCD), which may cause serious damage to ...the ocular visual function. There are many techniques to restore damaged epithelium, one of which is the transplantation of healthy cultured LSCs, usually onto a human amniotic membrane or onto bio-based engineered scaffolds in recent years. In this study, melt electrospun polylactic acid (PLA) was modified by silk fibroin or gelatin and further cultured with LSCs originating from three different donors. In terms of physicochemical properties, both modifications slightly increased PLA scaffold porosity (with a significantly larger pore area for the PLA/gelatin) and improved the scaffolds' swelling percentage, as well as their biodegradation rate. In terms of the scaffold application function, the aim was to detect/visualize whether LSCs adhered to the scaffolds and to further determine cell viability (total number), as well as to observe p63 and CK3 expressions in the LSCs. LSCs were attached to the surface of microfibers, showing flattened conformations or 3D spheres in the formation of colonies or agglomerations, respectively. All scaffolds showed the ability to bind the cells onto the surface of individual microfibers (PLA and PLA/gelatin), or in between the microfibers (PLA/silk fibroin), with the latter showing the most intense red fluorescence of the stained cells. All scaffolds proved to be biocompatible, while the PLA/silk fibroin scaffolds showed the highest 98% viability of 2.9 × 10
LSCs, with more than 98% of p63 and less than 20% of CK3 expressions in the LSCs, thus confirming the support of their growth, proliferation and corneal epithelial differentiation. The results show the potential of these bio-engineered scaffolds to be used as an alternative clinical approach.
Canine babesiosis caused by the intraerythrocytic protozoan parasite Babesia canis is a tick-borne disease characterized by a host response that involves both cellular and humoral immunity. This ...study focuses on the secretion of cytokines Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Keratinocyte Chemotactic-like (KC-like), Interleukins (IL)-2, IL-7, IL-8, IL-10, IL-15, IL-18 and Monocyte Chemotactic Protein-1 (MCP-1) in babesiosis caused by Babesia canis upon treatment with Imizol®. We assessed time dependent changes in cytokine levels and tested whether these changes correlate with pathogenesis of the disease. Sixteen healthy dogs and 31 dogs infected with Babesia canis, of which 18 showed complications, were treated with Imizol®. One dog died during the study (3.2%). Longitudinal study was perfomed by monitoring dogs at the first day of presentation (day 1) and 6 days later (day 7). Our results show that higher MCP-1 levels on day 1 are positively associated with the occurrence of complications, (complicated vs. uncomplicated; p = 0.00016). A similar pattern was observed for KC-like on day 1 (p = 0.0326) and day 7 (p = 0.044). Moreover, babesiosis caused by B. canis produced a steady increase in IL-8 levels with a moderate to strong negative correlation with erythrocyte counts and hematocrit in uncomplicated diseased dogs only (Spearman's rank correlation coefficient rs = -0.582 and rs = -0.598 respectively). Like for MCP-1, KC-like levels also differed in complicated and uncomplicated diseased dogs on day 1 (p = 0.03236) and day 7 (p = 0.044). Furthermore, KC-like levels were strongly correlated with IL-8 levels (rs = 0.663-0.7) and non-segmented neutrophil counts (rs = 0.572-0.732) in both diseased groups. Analysis of ROC suggests the use of serum levels of MCP-1 and IL-7 as predictors of the occurrence of complications with an AUC of 0.906 and 0.896 respectively and linear combinations of MCP-1, KC-Like, IL-7 and GM-CSF with values up to AUC = 0.983. Cytokine cluster analysis presented in this study can contribute to a better understanding of the pathogenesis of babesiosis and serve as a prognostic tool for the early detection of cases with highest likelihood of developing complications. Overall, our studies show that infection by B. canis elicits a cytokine pattern that is distinct from that observed with B. rossi, and that some of the inflammatory mediators can be useful to predict complications. Our results also suggest targets for the development of novel therapeutic strategies in babesiosis caused by B. canis.
Newcastle disease (ND) is one of the classic viral infections of poultry which resists all the efforts of eradication. Newcastle disease virus (NDV) strain ZG1999HDS was isolated during the outbreak ...in 1,999 at a broiler farm in Croatia. Previous trials in chickens confirmed it to be a lentogenic pathotype and immunogenic by stimulating humoral and cell mediated immunity. Further characterization by deduced amino acid sequence at the cleavage site of fusion protein confirmed its lentogenic nature, and in vitro tests its oncolytic capacity. Owing to its immunogenicity, strain ZG1999HDS is considered for vaccine development. In this study, 1-day-old chicks were vaccinated using strain ZG1999HDS oculonasally or by nebulization. Strain ZG1999HDS induced humoral immune response in both immunized groups The cell-mediated immune response occurred earlier in the group immunized by nebulization, as shown by a higher frequency rate of T and B lymphocytes, and significantly higher expression of IFN-α in respiratory organs and IFN-γ expression in the spleen. Viral genomic RNA was not detected in investigated organs. Thus, NDV strain ZG1999HDS is immunogenic when administered by means of nebulization or oculonasally without any adverse effects and is therefore suitable for further research and vaccine development. Further research is needed regarding its tropism.
The interaction of silver nanoparticles (AgNPs) with the immune system has not yet been sufficiently elucidated even though they belong to the most investigated and exploited group of nanomaterials. ...This study aimed to evaluate immunomodulatory effect of four different AgNPs on human peripheral blood mononuclear cells (hPBMCs). Fresh hPBMCs were exposed to the small sized (~ 10 nm) AgNPs immediately after isolation from the whole blood of healthy volunteers. The study considered coating-, time- and dose-dependent response of hPBMSc and stimulation of both early and intermediate activation of lymphocytes and monocytes using flow cytometry. The AgNPs differed in surface charge and were stabilised with polyvinyl pyrrolidone (PVP), poly-L-lysine (PLL), bis(2-ethylhexyl) sulfosuccinate sodium (AOT) or blood serum albumin (BSA). Response of hPBMCs to coating agents and ionic Ag form was evaluated to distinguish their effect from the AgNPs action as they may be released from the nanosurface. There was no significant effect of any tested AgNPs on relative count of hPBMCs subpopulations. The T-cells and monocytes were not activated after treatment with AgNPs, but the highest concentration of PLL- and BSA-AgNPs decreased density of CD4 and CD8 markers on T-helper and T-cytotoxic cells, respectively. The same AgNPs activated B- and NK-cells. Ionic Ag activated T-, B- and NK-cells, but at very higher concentration, whereas only PLL exhibited immunomodulatory activity. This study evidenced immunomodulatory activity of AgNPs that may be fine-tuned by the design of their surface functionalization.
Graphic abstract
This study aimed to define a consortium of lactic acid bacteria (LAB) that will bring added value to dried fresh cheese through specific probiotic properties and the synthesis of bioactive peptides ...(biopeptides). The designed LAB consortium consisted of three
strains: S-layer carrying
D6, exopolysaccharides producing
D12 and plantaricin expressing
D13, and one
strain,
ZGZA7-10. Chosen autochthonous LAB strains exhibited efficient adherence to the Caco-2 cell line and impacted faecal microbiota biodiversity. The cheese produced by the LAB consortium showed better physicochemical, textural and sensory properties than the cheese produced by a commercial starter culture. Liquid chromatography coupled with matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (LC-MALDI-TOF/TOF) showed the presence of 18 specific biopeptides in dried fresh cheeses. Their identification and relative quantification was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM). The results also showed that their synthesis resulted mainly from β-casein and also α-S1 casein degradation by proteolytic activities of the LAB consortium. The designed LAB consortium enhanced the functional value of the final product through impact on biopeptide concentrations and specific probiotic properties.
Despite increasing use of silver nanoparticles (AgNPs) in different medicinal products, knowledge about their effects on hemostasis and platelets functionality is still scarce. Published scientific ...reports provide neither data on oxidative stress response of platelets to AgNPs nor information about the effects of AgNPs physicochemical properties on functionality and activation of platelets. This study aimed to explore the role of AgNPs surface functionalization on cell viability, particle uptake, oxidative stress response, and activation of platelets. Small sized, spherical AgNPs were surface functionalized by negatively charged sodium bis(2-ethylhexyl) sulphosuccinate (AOT), neutral polymer polyvinylpyrrolidone (PVP), positively charged polymer poly-l-lysine (PLL) and bovine serum albumin (BSA). Platelet viability, activation and particle uptake were evaluated by flow cytometry. Oxidative stress response was evaluated by measuring the levels of intracellular glutathione (GSH), peroxy and superoxide radicals using assays based on fluorescence dies. Cytotoxicity and uptake of AgNPs to platelets were found to be dose-dependent in a following order PLL-AgNP >> > BSA-AgNP > AOT-AgNP > PVP-AgNP. Particle internalization was further confirmed by transmission electron microscopy. Treatment of platelets with AgNPs induced superoxide radical formation, depletion of GSH and hyperpolarization of the mitochondrial membrane. Small, but statistically significant increase of P-selectin expression in cells treated with all AgNPs compared to non-treated controls evidenced AgNPs-induced activation of platelets. Increased PAC-1 expression was found only in platelets treated with PLL-AgNPs. Obtained results demonstrate that different surface decoration of AgNPs determines their biological effects on platelets highlighting the importance of careful design of AgNPs-based medicinal products regarding their biocompatibility and functionality.
Synopsis: Surface functionalization of silver nanoparticles (AgNPs) determines their interaction with platelets affecting cell viability, particle uptake, oxidative stress response, and platelets activation. Display omitted
•Response of platelets to silver nanoparticles (AgNPs) was evaluated.•Surface functionalization of AgNPs determines their effects on platelets.•Cytotoxicity and uptake of AgNPs in platelets is dose-dependent.•Mild activation of platelets by AgNPs was observed by P-selectin expression.•PAC-1 expression was found only for platelets treated by positively charged AgNPs.
Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly ...homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications.
After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined.
Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype.
This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.
Respiratory syncytial virus (RSV) stimulates the production of specific immunoglobulin (Ig) E and IgG4 antibodies as a hallmark of the Th2 immune response. In this paper, we evaluated the occurrence ...of atopic diseases in 10-year-old children who were positive for RSV-specific IgG antibodies during infancy.
The prospective follow-up of 72 children included a physical examination, an International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire and the determination of RSV-specific antibodies and total and allergen-specific IgE.
Children with asthma had their first wheezing episode at a younger age (χ2 8.097, df = 1,
= 0.004). RSV-specific IgG4 levels at year one were positively correlated with atopic dermatitis (AD) (tau_b = 0.211,
= 0.049) and current AD (tau_b = 0.269,
= 0.012); and RSV-specific IgE levels were positively correlated with allergic rhinitis (AR) (tau_b = 0.290,
= 0.012) and current AR (tau_b = 0.260,
= 0.025). Positive RSV-specific IgE at the age of one increased the chances of asthma occurrence by 5.94 (OR = 5.94, 95% CI = 1.05-33.64;
= 0.044) and the chances of AR by more than 15 times (OR = 15.03, 95% CI = 2.08-108.72;
= 0.007). A positive family history of atopy increased the chances of asthma occurrence by 5.49 times (OR = 5.49, 95% CI = 1.01-30.07;
= 0.049), and a longer duration of exclusive breastfeeding lowered that chance (OR = 0.63, 95% CI = 0.45-0.89;
= 0.008). Prenatal smoking increased the chances of AR occurrence by 7.63 times (OR = 7.63, 95% CI = 1.59-36.53;
= 0.011).
RSV-specific IgE and RSV-specific IgG4 antibodies could be risk markers for the development of atopic diseases in children.
Newcastle disease (ND) is a highly contagious avian disease. Global control of ND is mainly based on vaccination of poultry; however, reported outbreaks of ND in vaccinated flocks indicate a constant ...need to re-evaluate the existing vaccines and a development of the new ones. In this study, 4-week-old male chickens of the layer commercial hybrid were immunized oculonasally with a commercial NDV live La Sota vaccine (LS group), a suspension of lyophilized NDV strain ZG1999HDS (ZG group), or saline (Control (K) group). Antibody response was determined by haemagglutination inhibition (HI) assay. Cell-mediated immunity (CMI) was characterized by immunophenotyping of leukocyte's and T-lymphocyte's subpopulations (flow cytometry). Applied NDV strains did not cause any adverse reaction in treated chickens. Both strains induced the significantly higher HI antibody response in comparison to the control group, and overall antibody titer was higher in ZG group than in LS group. CMI, manifested as a higher proliferation of B- and T-helper cells, yielded better results in the ZG groups than in the LS group. Based on the obtained results, we conclude that the strain ZG1999HDS is immunogenic and is a suitable candidate for further research and development of poultry vaccines.
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