Astrocytes and human artificial blood-brain barrier models

The blood-brain barrier (BBB) functions as a highly selective border of endothelial cells, protecting the central nervous system from potentially harmful substances by selectively controlling the entry of cells and molecules, including components of the immune system. To study the BBB properties, find suitable therapies, and identify new drug targets, there is a need to develop representative in vitro BBB models. In this article, we describe the astrocyte roles in the BBB functioning and human in vitro BBB models

Royal Jelly and Human Interferon-Alpha (HuIFN-αN3) Affect Proliferation, Glutathione Level, and Lipid Peroxidation in Human Colorectal Adenocarcinoma Cells In Vitro

The purpose was to investigate the influence of RJ-F(M), 10-hydroxy-2-decenoic acid and HuIFN-αN3 on the proliferation of CaCo-2 cells and ascertain their effects on intracellular glutathione level and lipid peroxidation. The antiproliferative (AP) activity of RJ-F (M) (0.1 g/10 mL PBS), HuIFN-αN3 (1000 IU mL−1), 10-HDA (100.0 μmol L−1) and their combinations, in the ratios 1:1, 1:2, and 2:1 on CaCo-2 cells were measured. Single RJ-F (M) had a low AP activity: 2.0 (0.5 mg mL−1). HuIFN-αN3 had an AP activity of 2.5 (208.33 IU mL−1), while 10-HDA had an AP activity of 1.5 (37.5 μmol mL−1). AP activity of 3.8 was obtained when RJ-F(M) and HuIFN-αN3 were in the ratio 2:1. In it, the level of GSH was 24.9 ± 2.4 nmol g−3 of proteins (vs. 70.2 ± 3.2 nmol g−3 in the control), and level of MDA was 72.3 ± 3.1 nmol g−3 (vs. 23.6 ± 9.1 nmol g−3 in the control). 10-HDA, together with HuIFN-αN3, is responsible for the inhibition of CaCo-2 cell proliferation in vitro. RJ-F (M) and HuIFN-αN3 applied at 2:1 decreased level of GSH and increased lipid peroxidation via MDA in CaCo-2 cells. Future studies are needed whether these GSH- and MDA-related activities of RJ-F (M), HuIFN-αN3, 10-HDA, and their combinations may decrease the tumorigenicity index and tumorigenic potential of tumor cells in vitr

Platelet-rich plasma, especially when combined with a TGF-ß inhibitor promotes proliferation, viability and myogenic differentiation of myoblasts in vitro

Regeneration of skeletal muscle after injury is limited by scar formation, slow healing time and a high recurrence rate. A therapy based on platelet-rich plasma (PRP) has become a promising lead for tendon and ligament injuries in recent years, however concerns have been raised that PRP-derived TGF-β could contribute to fibrotic remodelling in skeletal muscle after injury. Due to the lack of scientific grounds for a PRP -based muscle regeneration therapy, we have designed a study using human myogenic progenitors and evaluated the potential of PRP alone and in combination with decorin (a TGF-β inhibitor), to alter myoblast proliferation, metabolic activity, cytokine profile and expression of myogenic regulatory factors (MRFs). Advanced imaging multicolor single-cell analysis enabled us to create a valuable picture on the ratio of quiescent, activated and terminally committed myoblasts in treated versus control cell populations. Finally high-resolution confocal microscopy validated the potential of PRP and decorin to stimulate the formation of polynucleated myotubules. PRP was shown to down-regulate fibrotic cytokines, increase cell viability and proliferation, enhance the expression of MRFs, and contribute to a significant myogenic shift during differentiation. When combined with decorin further synergistc effects were identified. These results suggest that PRP could not only prevent fibrosis but could also stimulate muscle commitment, especially when combined with a TGF-β inhibitor

Single-cell analysis reveals IGF-1 potentiation of inhibition of the TGF-ß/Smad pathway of fibrosis in human keratocytes in vitro

Corneal wound healing is often affected by TGF-β–mediated fibrosis and scar formation. Guided fibrosis with IGF-1 and antifibrotic substances might maintain corneal transparency. Primary human corneal keratocytes under serum-free conditions were used as a model of corneal stromal wounding, with markers of corneal fibrosis and opacity studied under TGF-β2 stimulation. Single-cell imaging flow cytometry was used to determine nuclearization of Smad3, and intracellular fluorescence intensity of Smad7 and the corneal crystallin aldehyde dehydrogenase 3A1. Extracellular matrix proteoglycans keratocan and biglycan were quantified using ELISAs. On the TGF-β2 background, the keratocytes were treated with IGF-1, and suberoylanilidehydroxamic acid (SAHA) or halofuginone ± IGF-1. IGF-1 alone decreased Smad3 nuclearization and increased aldehyde dehydrogenase 3A1 expression, with favorable extracellular matrix proteoglycan composition. SAHA induced higher Smad7 levels and inhibited translocation of Smad3 to the nucleus, also when combined with IGF-1. Immunofluorescence showed that myofibroblast transdifferentiation is attenuated and appearance of fibroblasts is favored by IGF-1 alone and in combination with the antifibrotic substances. The TGF-β/Smad pathway of fibrosis and opacity was inhibited by IGF-1, and further with SAHA in particular, and with halofuginone. IGF-1 is thus a valid aid to antifibrotic treatment, with potential for effective and transparent corneal wound healing

Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced by PGen-1 Impulse Generator Device, Affect the Proliferation Patterns of Different Animal Cells

The influence of the medium-strength electric forces (MSE) on the proliferation of adherent chicken embryo fibroblasts (CEF), VERO, MDBK, MRC-5, and HeLa; lymphoblast cells, FB1 and K562; and cell multiplications were analyzed by growth index (GI). Impulse generator device PGen-1 provided 100 V/cm square-wave impulses of 10 ms. Treatment: Samples were subjected to one or three MSE. GIs were compared with controls after 72 hours and one or three treatments: Monolayers: CEF: GI in the control is 16.76, and after one and three MSE, it is 15.81 and 7.09. Vero cells: GI in the controls is 8.39, and after one and three MSE, it is 5.39 and 5.69. MDBK cells: GI in controls is 8.39, and after one and three MSE, it is 5.39 and 5.69. MRC-5 cells: GI in controls is 5.58, and after one and three MSE, it is 4.18 and 2.60. HeLa cells: GI in controls is 13.69, and after one and three MSE, it is 10.16 and 5.37. Suspension cells: Lymphoblast FB1: GI in controls is 6.55, and after one and three MSE, it is 13.48 and 12.25. Lymphoblast K562: GI in controls is 9.07, and after one or three MSE, it is 12.37 and 13.55. To conclude: MSE in monolayer cells inhibits the GI, depending on the nature of cells. MSE enhances the multiplication of lymphoblast FB1 or K562

Vpliv matičnega mlečka in humanega interferona-alfa (HuIFN-αN3) na proliferacijo, nivo glutationa in na preoksidacijo lipidov v humanih kolorektalnih adenokarcinomskih celicah in vitro

Among royal jelly’s (RJ) various biological activities, its possible antitumour activity deserves particular attention. The purpose of this study was to investigate the influence of RJ, its bioactive component 10-hydroxy-2-decenoic acid (10- HDA), and human interferon-alpha (HuIFN-αN3) on the proliferation of human colorectal adenocarcinoma cells (CaCo- 2), and ascertain their effect on intracellular glutathione (GSH) level and lipid peroxidation. We studied the antiproliferative (AP) activity of RJ [(0.1 g/10 mL phosphate buffer saline (PBS)], HuIFN-αN3 (1000 I.U. mL-1), 10-HDA at 100.0 μmol L-1, and their different combinations, in the ratio 1:1, 1:2, and 2:1 on CaCo-2 cells. The GSH level was measured by glutathione assay. The lipid peroxidation was measured by malondialdehyde (MDA) assay. Single RJ had a low AP activity: 2.0 (0.5 mg mL-1). HuIFN-αN3 had an AP activity of 2.5 (208.33 I.U. mL-1), while 10-HDA had an AP activity of 1.5 (37.5 μmol mL-1). The highest AP activity of 3.8 was obtained when RJ and HuIFN-αN3 were applied at the ratio 2:1. In that combination the level of GSH was 24.9±2.4 nmol g-3 of proteins (vs. 70.2±3.2 nmol g-3 in the control) and the level of MDA was 72.3±3.1 nmol g-3 (vs. 23.6±9.1 nmol g-3 in the control). It is generally assumed that 10-HDA, an important constituent of RJ, together with HuIFN-αN3, is responsible for the inhibition of CaCo-2 cells proliferation in vitro. In our study, however, RJ and HuIFN-αN3 applied at 2:1 decreased the level of GSH the most and significantly increased lipid peroxidation via MDA in CaCo-2 cells. Future studies should show whether these GSH- and MDA-related activities of RJ, HuIFN-αN3, 10-HDA, and their combinations may decrease the tumorigenicity index and tumorigenic potential of various tumour cells in vitro.Kot del biološke aktivnosti MM (Matičnega mlečka) so avtorji preučevali njegovo protitumorsko delovanje kot tudi možno interakcijo s humanim interferonom alfa (HuIFN-αN3). Cilj opravljenih poskusov je bil preučiti vpliv kombinacije med MM in HuIFN-αN3 na proliferacijo celic Humanega kolorektalnega adenokarcinoma (CaCo-2) in njun vpliv na znotrajcelični nivo glutationa (GSH) in peroksidacijo lipidov. Avtorji so preučevali AP (Antiproliferativno) delovanje MM (0.1 g/10 mL fosfatnega pufra) (PBS), HuIFN-αN3, (1000 I.U. mL-1), 10-hidroxy-2-decenoične kisline (10-HDA) (100.0 μmol L-1) in različne kombinacije med njimi (1:1, 1:2 in 2:1) na celice CaCo-2 in vitro. Njihov vpliv na znotrajcelični nivo GSH so merili s pomočjo komercialnega kita. Peroksidacijo lipidov so merili s pomočjo meritve vrednosti malondialdehida (MDA). MM sam kaže AP aktivnost 2.0 (0.5 mg mL-1 ). HuIFN-αN3 ima AP aktivnost 2.5 (208.33 I.U. mL-1) medtem ko ima 10-HDA AP aktivnost 1.5 (37.5 μmol mL-1). AP aktivnost kombinacije MM:HuIFN-αN3 (2:1) je bila 3.8. Pri tej kombinaciji je bil viden vpliv na nivo GSH: 24.9±2.4 nmol g-3 proteinov (70.2±3.2 nmol g-3 pri kontroli). Nivo MDA je bil 72.3±3.1 nmol g-3 pri kontroli). 10-HDA je glavna sestavina MM, ki v kombinaciji s HuIFN-αN3 deluje antiproliferativno na CaCo-2 celice. MM in HuIFN-αN3 v kombinaciji 2:1 pospešujeta peroksidacijo lipidov (MDA) in zmanjšujeta nivo glutationa (GSH). Nadaljni poskusi bodo pokazali ali z GSH- in MDA- povezane aktivnosti MM, HuIFN-αN3, 10-HDA in kombinacij med njimi, zmanjšujejo indeks tumorigenosti in s tem tumorigeni potencijal različnih tumorskih celic in vitro

Additive Effects of Water-Soluble Propolis (Greit 120) and Human Interferon Alfa (HuIFN-αN3) against Influenza Viruses in Vitro

Abstract: Influenza virus affects the respiratory tract in humans causing a range of distinct manifestations including fever, nasal secretions, cough, headaches, muscle pain and pneumonia, which could become violent and severe. It was found that influenza A viruses remain resistant to amantadine and rimantadin with high level of oseltamvir resistance. Therefore, there is a need for constant improvement of drugs active against resistant influenza viruses. Propolis has anti-influenza activity both in vitro and in vivo. Human leukocyte interferon (HuIFN-αN3) is a multi-subtype protein that displays an antiviral activity against influenza virus. In this study we elucidated the anti-influenza activity of the mixes of water-soluble propolis (WSP) (Greit 120) and HuIFN-αN3 at different ratios. Greit 120 polyphenols, total phenol acids and bioflavonoid were characterized by HPLC-UV-ESI-MS504971 and HuIFN-αN3 by reverse-phase high-performance liquid chromatography (RP-HPLC). Influenza A and B viruses were separately added to the LLC-MK2 cells treated with WSP (Greit 120) and HuIFN-αN3 alone or in proportions 1:1, 1:2 and 2:1. Plates were incubated and cytopathic effect was determined. The best results (ID50) were obtained with the mix of 10% WSP and HuIFN-αN3 in proportion 1:2, showing ID50 at 12 ± 0.2 μg/mL and 19 ± 0.6 μg/mL for influenza A and B viruses, respectively. When comparing anti-influenza activity of WSP (Greit 120)/HuIFN-αN3 with that of ribavirin, it was found that 1:2 was the optimal ratio for WSP (Greit 120)/HuIFN-αN3 (0.5 and 0.6 for influenza A and B, respectively). This new formulation of WSP (Greit 120) and HuIFN-αN3, showing better anti-Influenza activity, will definitely improve its application in flu infections

Polysaccharide Thin Solid Films for Analgesic Drug Delivery and Growth of Human Skin Cells

Chronic wounds not only lower the quality of patient's life significantly, but also present a huge financial burden for the healthcare systems around the world. Treatment of larger wounds often requires the use of more complex materials, which can ensure a successful renewal or replacement of damaged or destroyed tissues. Despite a range of advanced wound dressings that can facilitate wound healing, there are still no clinically used dressings for effective local pain management. Herein, alginate (ALG) and carboxymethyl cellulose (CMC), two of the most commonly used materials in the field of chronic wound care, and combination of ALG-CMC were used to create a model wound dressing system in the form of multi-layered thin solid films using the spin-assisted layer-by-layer (LBL) coating technique. The latter multi-layer system was used to incorporate and study the release kinetics of analgesic drugs such as diclofenac and lidocaine at physiological conditions. The wettability, morphology, physicochemical and surface properties of the coated films were evaluated using different surface sensitive analytical tools. The influence of in situ incorporated drug molecules on the surface properties (e.g., roughness) and on the proliferation of human skin cells (keratinocytes and skin fibroblasts) was further evaluated. The results obtained from this preliminary study should be considered as the basis for the development “real” wound dressing materials and for 3D bio-printing applications