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1.
Nanotechnol Sci Appl ; 17: 77-94, 2024.
Article in English | MEDLINE | ID: mdl-38523648

ABSTRACT

Purpose: Mastitis in dairy cows is a worldwide problem faced by dairy producers. Treatment mainly involves antibiotic therapy, however, due to widespread antibiotic resistance among bacteria, such treatments are no longer effective. For this reason, scientists are searching for new solutions to combat mastitis, which is caused by bacteria, fungi, and algae. One of the most promising solutions, nanotechnology, is attracting research due to its biocidal properties. The purpose of this research was to determine the biocidal properties of nanocomposites as a potential alternative to antibiotics in the control of mastitis, as well as to determine whether the use of nanoparticles and what concentration is safe for the breeder and the animal. Patients and Methods: In this study, the effects of Ag, Au, Cu, Fe, and Pt nanoparticles and their complexes were evaluated in relation to the survival of bacteria and fungi isolated from cattle diagnosed with mastitis, their physicochemical properties, and their toxicity to bovine and human mammary epithelial cells BME-UV1 and HMEC (human microvascular endothelial cells). Moreover, E. coli, S. aureus, C. albicans, and Prototheca sp. invasion was assessed using the alginate bead (bioprinted) model. The NPs were tested at concentrations of 25, 12.5, 6.25, 3.125, 1.56 mg/l for Au, Ag, Cu and Fe NPs, and 10, 5, 2.5, 1.25, 0.625 mg/l for Pt. Results: With the exception of Fe and Pt, all exhibited biocidal properties against isolates, while the AgCu complex had the best effect. In addition, nanoparticles showed synergistic effects, while the low concentrations had no toxic effect on BME-UV1 and HMEC cells. Conclusion: Synergistic effects of nanoparticles and no toxicity to bovine and human cells might, in the future, be an effective alternative in the fight against microorganisms responsible for mastitis, and the implementation of research results in practice would reduce the percentage of dairy cows suffering from mastitis. The problem of increasing antibiotic resistance is posing a global threat to human's and animal's health, and requires comprehensive research to evaluate the potential use of nanoparticles - especially their complexes - as well as to determine whether nanoparticles are safe for the breeders and the animals. The conducted series of studies allows further consideration of the use of the obtained results in practice, creating a potentially new alternative to antibiotics in the treatment and prevention of mastitis in dairy cattle.

2.
J Cell Mol Med ; 27(19): 2995-3008, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37679999

ABSTRACT

Mucosal melanoma (MM) is a very rare and aggressive type of cancer for which immunotherapy or targeted therapy such as BRAF/MEK inhibitors, used in cutaneous melanoma, usually fail. Due to our earlier experience showing the high effectiveness of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (MET) inhibitors in reducing the activation of the MAPK and PI3K/AKT signalling pathways, we aim to test whether these drugs would also be effective for mucosal melanoma. Cells representing two commercially available mucosal melanoma cell lines (GAK and HMVII) and one cell line obtained from a patient's vaginal melanoma were treated with MET or EGFR inhibitors, or combinations of these agents. The dual-inhibitor treatment strategy resulted in a decrease of cell proliferation, migration and invasion. Moreover, combinations of inhibitors led to reduction of pEGFR/EGFR and pMET/MET ratio and downregulation of PI3K/AKT and MEK/ERK1/2-based signalling pathways. Our findings indicate a potential therapeutic strategy based on EGFR and MET inhibitors in mucosal melanoma, which should be further evaluated in vivo and in clinical experiments. They also suggest that targeting multiple receptor tyrosine kinases may block signalling crosstalk and possibly delay the appearance of resistance to kinase inhibitors in mucosal melanoma cells.

3.
Biomedicines ; 11(8)2023 Aug 17.
Article in English | MEDLINE | ID: mdl-37626787

ABSTRACT

Mastitis prevention and treatment in dry cows are complex issues with limited solutions. The most common is intramammary antibiotic treatment. However, the effectiveness of this treatment varies among countries and even within herds in the same region. Therefore, it is necessary to develop new strategies for dry cow therapy. Metal nanoparticles (NPs), which have strong biocidal properties for treating diseases caused by bacteria, fungi, and algae, are increasingly used to reduce antibiotic use. In this study, AuNPs, CuNPs, AgNPs, PtNPs, NP-FeCs, and their triple complexes were used at different concentrations to evaluate their practical use in treating cows during their dry period. The nanoparticles were in hydrocolloid form and were added separately to form a mixture with beeswax, a mixture with oil, or a mixture based on vegetable glycerin and propylene glycol. The NPs' concentrations were 0.5, 1, 2, and 5 ppm. Gram-positive and Gram-negative bacteria, and fungi isolated from cows diagnosed with mastitis were used to determine pathogen viability. The results indicated that AuNPs, CuNPs, AgNPs, and their complexes show biocidal properties against mastitis pathogens. AgNPs at 5 ppm had the strongest biocidal properties and reduced Streptococcus agalactiae's survival rate by 50%; however, the nanoparticle complexes showed poor synergism. The strongest biocidal properties of NPs in wax and in glycerin mixed with glycol were shown against Escherichia coli. Additionally, low nanoparticle concentrations showed no cytotoxicity for BME-UV1 bovine cells, suggesting that these mixtures might be used for further in vivo testing.

4.
Cell Mol Biol Lett ; 28(1): 58, 2023 Jul 22.
Article in English | MEDLINE | ID: mdl-37481560

ABSTRACT

BACKGROUND: One of the factors that affect the progression of melanoma is the tumor microenvironment, which consists of cellular elements, extracellular matrix, acidification, and a hypoxic state. Adipocytes are one of the types of cell present in the niche and are localized in the deepest layer of the skin. However, the relationship between fat cells and melanoma remains unclear. METHODS: We assessed the influence of melanoma cells on adipocytes using an indirect coculture system. We estimated the level of cancer-associated adipocyte (CAA) markers through quantitative PCR analysis. The fibroblastic phenotype of CAAs was confirmed by cell staining and western blotting analysis. The lipid content was estimated by lipid detection in CAAs using LipidSpot and by quantitative analysis using Oil Red O. The expression of proteins involved in lipid synthesis, delipidation, and metabolic processes were assessed through quantitative PCR or western blotting analysis. Lactate secretion was established using a Lactate-Glo™ assay. Proteins secreted by CAAs were identified in cytokine and angiogenesis arrays. The proliferation of melanoma cells cocultured with CAAs was assessed using an XTT proliferation assay. Statistical analysis was performed using a one-way ANOVA followed by Tukey's test in GraphPad Prism 7 software. RESULTS: Obtained CAAs were identified by decreased levels of leptin, adiponectin, resistin, and FABP4. Adipocytes cocultured with melanoma presented fibroblastic features, such as a similar proteolytic pattern to that of 3T3L1 fibroblasts and increased levels of vimentin and TGFßRIII. Melanoma cells led to a reduction of lipid content in CAAs, possibly by downregulation of lipid synthesis pathways (lower FADS, SC4MOL, FASN) or enhancement of lipolysis (higher level of phosphorylation of ERK and STAT3). Adipocytes cocultured with melanoma cells secreted higher IL6 and SerpinE1 levels and produced less CCL2, CXCL1, and angiogenic molecules. CAAs also showed metabolic changes comprising the increased secretion of lactate and enhanced production of glucose, lactate, and ion transporters. In addition, changes in adipocytes observed following melanoma coculture resulted in a higher proliferation rate of cancer cells. CONCLUSIONS: Melanoma cells led to decreased lipid content in adipocytes, which might be related to enhanced delipidation or reduction of lipid synthesis. Fibroblast-like CAAs showed metabolic changes that may be the reason for accelerated proliferation of melanoma cells.


Subject(s)
Adipocytes , Melanoma , Humans , Adipocytes/metabolism , Coculture Techniques , Lactates/metabolism , Lipids , Tumor Microenvironment
5.
Biochim Biophys Acta Mol Cell Res ; 1870(7): 119549, 2023 10.
Article in English | MEDLINE | ID: mdl-37506884

ABSTRACT

Microenvironment of the melanoma consists of cellular elements like fibroblasts, adipocytes, and keratinocytes as well as extracellular matrix and physicochemical conditions. In our previous research, we have established that melanoma influences strongly above mentioned cells present in the tumor niche and recruits them to support cancer progression. In this work, we evaluated the impact of cancer-associated cells, namely fibroblasts (CAFs), adipocytes (CAAs), and keratinocytes (CAKs) on melanoma proliferation, signaling pathways activation, metabolism as well as the effectiveness of used anti-cancer therapy. Obtained results indicated elevated phosphorylation of STAT3, upregulated GLUT1 and GLUT3 as well as downregulated of MCT-1 expression level in melanoma cells under the influence of all examined cells present in the tumor niche. The proliferation of melanoma cells was increased after co-culture with CAFs and CAKs, while epithelial-mesenchymal transition markers' expression level was raised in the presence of CAFs and CAAs. The level of perilipin 2 and lipid content was elevated in melanoma cells under the influence of CAAs. Moreover, increased expression of CYP1A1, gene encoding drug metabolizing protein, in melanoma cells co-cultured with CAFs and CAKs prompted us to verify the effectiveness of the previously proposed by us anti-melanoma therapy based on combination of EGFR and MET inhibitors. Obtained results indicate that the designed therapy is still efficient, even if the fibroblasts, adipocytes, and keratinocytes, are present in the melanoma vicinity.


Subject(s)
Melanoma , Humans , Melanoma/drug therapy , Melanoma/genetics , Melanoma/metabolism , Fibroblasts/metabolism , Signal Transduction , ErbB Receptors/genetics , ErbB Receptors/metabolism , Biology , Tumor Microenvironment
6.
Int J Mol Sci ; 24(7)2023 Mar 24.
Article in English | MEDLINE | ID: mdl-37047119

ABSTRACT

Digital dermatitis (DD) is the second most prevalent disease in dairy cattle. It causes significant losses for dairy breeders and negatively impacts cows' welfare and milk yield. Despite this, its etiology has not been entirely identified, and available data are limited. Antibiotic therapy is a practical method for managing animal health, but overuse has caused the evolution of antibiotic-resistant bacteria, leading to a loss in antimicrobial efficacy. The antimicrobial properties of metal nanoparticles (NPs) may be a potential alternative to antibiotics. The aim of this study was to determine the biocidal properties of AgNPs, CuNPs, AuNPs, PtNPs, FeNPs, and their nanocomposites against pathogens isolated from cows suffering from hoof diseases, especially DD. The isolated pathogens included Sphingomonas paucimobilis, Ochrobactrum intermedium I, Ochrobactrum intermedium II, Ochrobactrum gallinifaecis, and Actinomyces odontolyticus. Cultures were prepared in aerobic and anaerobic environments. The viability of the pathogens was then determined after applying nanoparticles at various concentrations. The in vitro experiment showed that AgNPs and CuNPs, and their complexes, had the highest biocidal effect on pathogens. The NPs' biocidal properties and their synergistic effects were confirmed, which may forecast their use in the future treatment and the prevention of lameness in cows, especially DD.


Subject(s)
Anti-Infective Agents , Cattle Diseases , Digital Dermatitis , Metal Nanoparticles , Female , Cattle , Animals , Digital Dermatitis/drug therapy , Digital Dermatitis/prevention & control , Lameness, Animal , Gold , Metal Nanoparticles/therapeutic use , Anti-Infective Agents/pharmacology , Anti-Infective Agents/therapeutic use , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Cattle Diseases/drug therapy , Cattle Diseases/prevention & control , Dairying
7.
Int J Mol Sci ; 24(2)2023 Jan 14.
Article in English | MEDLINE | ID: mdl-36675172

ABSTRACT

Mastitis is one of the most common issues for milk producers around the world. Antibiotic therapy is often ineffective, and therefore, scientists must find a new solution. The aim of this paper is to estimate the influence of common and well-known cosmetic substrates and mixtures of nanoparticles (NPs) and cosmetic substrates on the viability of frequently occurring mastitis pathogens, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The obtained results suggest that only collagen + elastin and glycerine influenced and increased bacteria viability. In case of the rest of the cosmetic substrates, the viability of E. coli and S. aureus was decreased, and the results were statistically significant (p ≤ 0.01). Prepared pre-dipping and dipping mixtures decrease (p ≤ 0.01) the viability of the mentioned pathogens. The obtained results of the in vitro analysis are very promising. In the next step, prepared mixtures should be tested in different herd conditions if they can be used in mastitis prevention or decrease the number of subclinical mastitis cases. Furthermore, these mixtures could become an interesting alternative for organic milk production where conventional preparations and antibiotics are forbidden. However, further analysis, especially on the influence of prepared mixtures on other bacteria species and, algae, fungi, are necessary.


Subject(s)
Disinfectants , Mastitis, Bovine , Nanoparticles , Staphylococcal Infections , Animals , Cattle , Female , Humans , Milk/microbiology , Disinfectants/pharmacology , Staphylococcus aureus , Copper/pharmacology , Silver/pharmacology , Escherichia coli , Mastitis, Bovine/drug therapy , Mastitis, Bovine/microbiology , Anti-Bacterial Agents/pharmacology
8.
Cell Commun Signal ; 20(1): 146, 2022 09 19.
Article in English | MEDLINE | ID: mdl-36123693

ABSTRACT

BACKGROUND: Keratinocytes constitute a major part of the melanoma microenvironment, considering their protective role towards melanocytes in physiological conditions. However, their interactions with tumor cells following melanomagenesis are still unclear. METHODS: We used two in vitro models (melanoma-conditioned media and indirect co-culture of keratinocytes with melanoma cells on Transwell inserts) to activate immortalized keratinocytes towards cancer-associated ones. Western Blotting and qPCR were used to evaluate keratinocyte markers and mediators of cell invasiveness on protein and mRNA expression level respectively. The levels and activity of proteases and cytokines were analysed using gelatin-FITC staining, gelatin zymography, chemiluminescent enzymatic test, as well as protein arrays. Finally, to further study the functional changes influenced by melanoma we assessed the rate of proliferation of keratinocytes and their invasive abilities by employing wound healing assay and the Transwell filter invasion method. RESULTS: HaCaT keratinocytes activated through incubation with melanoma-conditioned medium or indirect co-culture exhibit properties of less differentiated cells (downregulation of cytokeratin 10), which also prefer to form connections with cancer cells rather than adjacent keratinocytes (decreased level of E-cadherin). While they express only a small number of cytokines, the variety of secreted proteases is quite prominent especially considering that several of them were never reported as a part of secretome of activated keratinocytes' (e.g., matrix metalloproteinase 3 (MMP3), ADAM metallopeptidase with thrombospondin type 1 motif 1). Activated keratinocytes also seem to exhibit a high level of proteolytic activity mediated by MMP9 and MMP14, reduced expression of TIMPs (tissue inhibitor of metalloproteinases), upregulation of ERK activity and increased levels of MMP expression regulators-RUNX2 and galectin 3. Moreover, cancer-associated keratinocytes show slightly elevated migratory and invasive abilities, however only following co-culture with melanoma cells on Transwell inserts. CONCLUSIONS: Our study offers a more in-depth view of keratinocytes residing in the melanoma niche, drawing attention to their unique secretome and mediators of invasive abilities, factors which could be used by cancer cells to support their invasion of surrounding tissues. Video abstract.


Subject(s)
Matrix Metalloproteinase 3 , Melanoma , Cadherins/metabolism , Core Binding Factor Alpha 1 Subunit , Culture Media, Conditioned/pharmacology , Cytokines , Fluorescein-5-isothiocyanate , Galectin 3 , Gelatin , Humans , Keratinocytes/pathology , Keratins , Matrix Metalloproteinase 14 , Matrix Metalloproteinase 9/metabolism , Melanoma/pathology , RNA, Messenger/metabolism , Thrombospondins , Tissue Inhibitor of Metalloproteinases
9.
Cells ; 11(15)2022 07 23.
Article in English | MEDLINE | ID: mdl-35892570

ABSTRACT

In mammalian cells, SLC35A2 delivers UDP-galactose for galactosylation reactions that take place predominantly in the Golgi lumen. Mutations in the corresponding gene cause a subtype of a congenital disorder of glycosylation (SLC35A2-CDG). Although more and more patients are diagnosed with SLC35A2-CDG, the link between defective galactosylation and disease symptoms is not fully understood. According to a number of reports, impaired glycosylation may trigger the process of epithelial-to-mesenchymal transition (EMT). We therefore examined whether the loss of SLC35A2 activity would promote EMT in a non-malignant epithelial cell line. For this purpose, we knocked out the SLC35A2 gene in Madin-Darby canine kidney (MDCK) cells. The resulting clones adopted an elongated, spindle-shaped morphology and showed impaired cell-cell adhesion. Using qPCR and western blotting, we revealed down-regulation of E-cadherin in the knockouts, while the fibronectin and vimentin levels were elevated. Moreover, the knockout cells displayed reorganization of vimentin intermediate filaments and altered subcellular distribution of a vimentin-binding protein, formiminotransferase cyclodeaminase (FTCD). Furthermore, depletion of SLC35A2 triggered Golgi compaction. Finally, the SLC35A2 knockouts displayed increased motility and invasiveness. In conclusion, SLC35A2-deficient MDCK cells showed several hallmarks of EMT. Our findings point to a novel role for SLC35A2 as a gatekeeper of the epithelial phenotype.


Subject(s)
Epithelial-Mesenchymal Transition , Kidney , Animals , Dogs , Kidney/metabolism , Madin Darby Canine Kidney Cells , Mammals , Phenotype , Vimentin/metabolism
10.
Cell Commun Signal ; 20(1): 63, 2022 05 10.
Article in English | MEDLINE | ID: mdl-35538545

ABSTRACT

BACKGROUND: The tumor microenvironment consists of stromal cells, extracellular matrix, and physicochemical properties (e.g., oxygenation, acidification). An important element of the tumor niche are cancer-associated fibroblasts (CAFs). They may constitute up to 80% of the tumor mass and share some features with myofibroblasts involved in the process of wound healing. CAFs can facilitate cancer progression. However, their interaction with melanoma cells is still poorly understood. METHODS: We obtained CAFs using conditioned media derived from primary and metastatic melanoma cells, and via co-culture with melanoma cells on Transwell inserts. Using 2D and 3D wound healing assays and Transwell invasion method we evaluated CAFs' motile activities, while coverslips with FITC-labeled gelatin, gelatin zymography, and fluorescence-based activity assay were employed to determine the proteolytic activity of the examined cells. Western Blotting method was used for the identification of CAFs' markers as well as estimation of the mediators of MMPs' (matrix metalloproteinases) expression levels. Lastly, CAFs' secretome was evaluated with cytokine and angiogenesis proteomic arrays, and lactate chemiluminescence-based assay. RESULTS: Acquired FAP-α/IL6-positive CAFs exhibited elevated motility expressed as increased migration and invasion ratio, as well as higher proteolytic activity (area of digestion, MMP2, MMP14). Furthermore, fibroblasts activated by melanoma cells showed upregulation of the MMPs' expression mediators' levels (pERK, p-p38, CD44, RUNX), enhanced secretion of lactate, several cytokines (IL8, IL6, CXCL1, CCL2, ICAM1), and proteins related to angiogenesis (GM-CSF, DPPIV, VEGFA, PIGF). CONCLUSIONS: Observed changes in CAFs' biology were mainly driven by highly aggressive melanoma cells (A375, WM9, Hs294T) compared to the less aggressive WM1341D cells and could promote melanoma invasion, as well as impact inflammation, angiogenesis, and acidification of the tumor niche. Interestingly, different approaches to CAFs acquisition seem to complement each other showing interactions between studied cells. Video Abstract.


Subject(s)
Interleukin-6 , Melanoma , Cell Line, Tumor , Cell Movement , Cell Proliferation , Female , Fibroblasts/metabolism , Gelatin/metabolism , Humans , Interleukin-6/metabolism , Lactates/metabolism , Melanoma/pathology , Placenta Growth Factor/metabolism , Proteomics , Tumor Microenvironment
11.
Animals (Basel) ; 10(9)2020 Sep 07.
Article in English | MEDLINE | ID: mdl-32906645

ABSTRACT

Mastitis is one of the most common diseases of high-yielding dairy cows, and over 90% of cases are caused by Streptococcus spp., Enterobacteriaceae, or Staphylococcus spp. Certain groups of proteins are very significant in terms of the cow's antioxidant, bacteriostatic, and germicidal properties: lysozyme (Lz), lactoferrin (Lf), and ß-lactoglobulin (BLG). This study aimed to determine the influence of Staphylococcus spp., Streptococcus spp., and Enterobacteriaceae on the secretion of bioactive whey proteins and oxidative stress markers. From the herd, 60 multiparous cows with diagnosed mastitis were selected. Samples were taken individually from each quarter and pooled, which gave 60 samples. Enterobacteriaceae did not affect the BLG synthesis, whereas lysozyme and lactoferrin responded to a high concentration of these bacterial strains. In the case of Staphylococcus spp. infection, the BLG level increased. These strains did not affect the levels of di-malonic aldehyde (MDA), lactoferrin, and lysozyme. In contrast, they were significantly influenced by Streptococcus spp. In summary, the levels of whey proteins and oxidative stress markers changed depending on the bacterial strain inducing inflammation. Lysozyme and lactoferrin may be markers of udder inflammation caused by Enterobacteriaceae and Streptococcus spp., whereas ß-lactoglobulin may prove useful in diagnosing Staphylococcus spp. induced mastitis.

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