Journal of Cellular Biotechnology - Volume 2, issue 1

Authors: Trapani, Adriana | Tripodo, Giuseppe | Mandracchia, Delia | Cioffi, Nicola | Ditaranto, Nicoletta | Cerezuela, Rebeca | Esteban, Maria Angeles

Article Type: Research Article

Abstract: BACKGROUND: Solid lipid nanoparticles (SLN) have not been used for peptide supply to fish cells. OBJECTIVE: To evaluate the potential of SLN to deliver the antioxidant glutathione (GSH) to the primary cultures of head-kidney (HK) leucocytes of Sparus aurata L . METHODS: SLN were produced using the amphiphilic lipid Gelucire® 50/13 according to the melt-emulsification method. In vitro stability and colorimetric studies for the antioxidant activity were carried out prior to biological assays on HK leucocytes. RESULTS: SLN sonicated in acidic medium were stable up to 3 months at 4°C. A …strong in vitro antioxidant activity effect for SLN was shown. The incubation of gilthead seabream HK leucocytes with GSH, GSSG or the sonicated and non-sonicated GSH-SLN (HAc )  for 4 h had very few effects on HK cell activities. The phagocytic ability of HK leucocytes previously incubated with GSH was significantly increased in respect to the control cells. CONCLUSIONS: The technique of melt-emulsification provided particles with high association efficiency in GSH and even below 100 nm in size. However, no stimulant properties were observed after incubation of HK leucocytes with SLN and possible hypotheses explaining the observed findings are discussed. Show more

Keywords: Glutathione, antioxidant activity, solid lipid nanoparticles, fish leucocytes, teleosts

DOI: 10.3233/JCB-15022

Citation: Journal of Cellular Biotechnology, vol. 2, no. 1, pp. 1-14, 2016

Authors: Sokolova, Irina | Gafarova, Marina | Khokhlova, Maria | Muravyev, Alexei | Lyubin, Evgeny | Skryabina, Maria | Fedyanin, Andrey | Krasnova, Tatiana | Shahnazarov, Alexandr

Article Type: Research Article

Abstract: It is commonly known that red blood cell (RBC) tendency to reversibly aggregate contribute to the unique flow properties of blood. The fibrinogen-induced aggregation is primarily based on non-specific processes. Nevertheless, recent investigations disclosed a possibility of the specific fibrinogen-RBC binding, however, its role in RBC aggregation remains unclear. We conducted a study to determine whether this specific interaction is essential for natural RBC aggregation. The influence of glycoprotein (GP) IIb-IIIa inhibitor, monafram, on RBC aggregation was compared in RBC suspensions and in whole blood samples from healthy volunteers and, in addition, from patients with systemic lupus erythematosus (SLE) using …laser-light backscattering detection and optical tweezers. The effect of monafram on normal RBC aggregation was detectable only in the early phase of intercellular interactions and it involved a deceleration of the aggregation, which was equally expressed in native blood samples and RBC suspensions based on platelet-free plasma. Additionally, in SLE patients, monafram weakened the strength of paired RBC aggregates in platelet-free suspensions. In healthy subjects, strengthened aggregates were found mainly in platelet-containing bulk RBC suspensions. These findings suggest that normal specific fibrinogen-RBC binding could lead to early accelerated formation of RBC aggregates, which could increase possible beneficial effects of reversible RBC aggregation. The strength of RBC aggregates could be modified by platelets in normal subjects and by specific fibrinogen-RBC binding in SLE patients. Show more

Keywords: Red blood cell aggregation, specific fibrinogen binding, monafram

DOI: 10.3233/JCB-15023

Citation: Journal of Cellular Biotechnology, vol. 2, no. 1, pp. 15-22, 2016

Authors: Cicha, Iwona | Goppelt-Struebe, Margarete

Article Type: Research Article

Abstract: BACKGROUND: Tissue injuries and pathological changes are often associated with changes in cell adhesion and reorganization of cytoskeletal structures. This is of particular relevance for renal epithelial cells, which form a highly regulated barrier along the nephron. Here, we investigated the effect of the calcium chelator EGTA on morphological alterations and changes in pro-fibrotic gene expression in proximal renal tubular epithelial cells. METHODS: Epithelial cells were exposed to EGTA alone or in combination with TGFβ-1, followed by the analysis ofN-cadherin and paxillin localization and cytoskeletal rearrangement. The expression of connective tissue growth factor (CTGF), a pro-fibrotic protein …regulated by alterations of cell morphology, was investigated. RESULTS: Even low concentrations of EGTA, which did not lead to the loss of cell-cell interactions or cell detachment, were sufficient to reduce N-cadherin binding, to rearrange focal adhesion, to induce CTGF expression, and to strongly enhance TGFβ-1-mediated signaling. These data indicate a substantial crosstalk between the changes in cell cytoskeletal structures at the cell-cell adhesions and the signaling of soluble mediators. CONCLUSIONS: By linking cell-cell and cell-matrix interactions with the regulation of pro-fibrotic signaling, our studies provide deeper insights into the physiological mechanisms underlying the natural regeneration and repair processes in the kidney. Show more

Keywords: Cadherins, calcium chelation, connective tissue growth factor, cytoskeletal reorganization, TGFβ-1, tubular epithelial cells

DOI: 10.3233/JCB-15024

Citation: Journal of Cellular Biotechnology, vol. 2, no. 1, pp. 23-33, 2016

Authors: George, S. | Rödiger, S. | Schröder, C. | Knaut, M. | Küpper, J.-H.

Article Type: Research Article

Abstract: Severe heart diseases such as myocarditis and cardiomyopathy are often characterized by progressive damages of contractile heart tissue which ultimately can lead to terminal heart failure. There is a need for relevant in vitro cultures of human cardiomyocytes to study pathogenic processes and to perform pharmacological testing of new heart drugs. By using the upcyte/EPCC (enhanced primary cell culture) approach for direct multiplication of organ-specific cells, we established proliferating human cardiomyocyte cultures derived from atrial appendages. For qualitative cardiac expression profiling we established a comprehensive set of multiplex PCR assays, selected from a panel of 32 genes, to rapidly …screen changes at the transcriptional level in human ventricular and atrial cardiomyocytes. Our multiplex PCR approach revealed some donor variability of native atrial heart tissue that need to be confirmed by further studies with more samples. Our initial studies further indicated that characteristic heart muscle cell markers such as MLC-2 a , MLC-2v , CHRM2 , ADRB1 , DES , EDRNB , C x40 and KCNA5 were down-regulated when isolated cardiomyocytes were taken into primary cell culture. Compared to native heart tissue, proliferating atrial cardiomyocytes lacked expression of those cardiac markers but still expressed MYCD , GATA-4 , Cx43 , SERCA2 , BNP , Tbx5 , EDNRA and ACTB . Surprisingly, atrium-derived cardiomyocytes started to express NFAc4 in passage three, and cardiomyocyte marker expressions of Cx43 and BNP were even increased over cultivation time. In conclusion, our novel multiplex PCR assays should be useful for expression profiling of native heart tissues from patients with different disease conditions and for characterization of in vitro cardiomyocyte cultures. Show more

Keywords: Atrial appendages, ACTA1, ACTB ACTC1, ADRB1, ADRB2, ATP2A2, atrial fibrillation, BMP2, CHRM2, cluster analysis, DES, EDNRA, EDNRB, enhanced primary cell cultures (EPCC), GAPDH, GATA4, gene expression, GJA1, GJA5, HPRT1, KCNA5, KCNH2, multiplex PCR, MYL2, MYL7, MYOCD, NFATC4, NKX2-5, NPPA, NPPA, PLN, qPCR, reference genes, RPLP0, SCN5A, TBX5, TNNI3, TNNT2, upcyte genes, VIM

DOI: 10.3233/JCB-15025

Citation: Journal of Cellular Biotechnology, vol. 2, no. 1, pp. 35-55, 2016

Authors: Meyer, R.G. | Meyer-Ficca, M.L. | Küpper, J.-H.

Article Type: Research Article

Abstract: Loss of the DNA homologous recombination repair pathway (DHR) sensitizes cells to the effects of clastogenic chemotherapy agents when the alternative base excision DNA repair pathway (BER) is blocked by inhibitors of poly(ADP-ribose) polymerases (PARP). The mutation of BRCA is an example of defective DHR, where several PARP inhibitors are now in advanced stages of clinical trials due to their ability to form synthetic lethality with chemotherapy drugs in breast and ovarian cancer. Because the inability of cells to perform DHR is the determining factor for successful PARP inhibitor administration in a chemotherapy protocol, testing of patient cells for sensitization …by PARP inhibitors would be advantageous for the prediction of therapy outcome. This proof-of-principle study set out to design recombinant adenoviruses as a simple tool for the functional block of PARP1-mediated DNA strand break repair important for the execution of BER that could potentially be used to test cultured cells from patients for the sensitizing effects of PARP inhibitors in a chemotherapy setting. Recombinant adenoviruses for the expression of the PARP1 DNA binding domain (DBD) as a dominant-negative PARP1 mutant, for full-length PARP1 expression and other control viruses were generated and used to infect normal human fibroblasts. Transgene expression and the modulation of poly(ADP-ribose) (PAR) were studied using immunofluorescence and immunoblotting techniques. Overexpression of PARP1 increased, but expression of the PARP1 DBD blocked PAR formation in response to methylnitronitrosoguanidine (MNNG) treatment of virally transduced cells. Ectopic DBD expression caused the formation of caspase 3 cleavage products of PARP1 protein, which are a marker of apoptotic cell death. Cells infected with control virus without PARP DBD did not induce caspase 3-mediated cleavage of PARP1. In conclusion, the set of adenoviral vectors presented in this study can be used to modulate PARP activity in normal human cells and may therefore be used as a tool for testing cellular sensitization by blocking DNA BER by administration of PARP inhibitors and to gain better understanding of potential PARP inhibitor resistance. Show more

Keywords: PARP, poly(ADP-ribose), synthetic lethality, inhibitor, base excision repair, homologous recombination, BRCA, chemotherapy

DOI: 10.3233/JCB-15026

Citation: Journal of Cellular Biotechnology, vol. 2, no. 1, pp. 57-68, 2016

Authors: Pfeiffer, S. | Baum, A. | Kehlen, A. | Hopperdietzel, C. | Gemeinhardt, O. | Jung, F. | Unger, J. | Kekulé, A.S. | Hiebl, B.

Article Type: Research Article

Abstract: Health care associated infections are the fourth leading cause of disease in industrialized countries, and the most common complication affecting hospitalized patients. Recently a polymerized siloxane coating substrate was suggested as a promising candidate for the coating of materials which are aimed to be used in cardiovascular tissue engineering. To reduce the risk of tissue remodeling failure after implantation of such engineered tissue implants, coatings substrates for tissue engineering scaffolds might be helpful to lower the risk of bacterial attachment and growth during and after implantation. In the presented study a coating based on polymerized siloxanes was tested with …respect to these properties. Tests were performed with aerobic nosocomial bacteria (Escherichia coli and Staphylococcus epidermidis ) and an exemplary selection of materials (stainless steel, polycarbonate, soda-lime glass). The siloxane coating was able to significantly reduce bacterial adherence, and it is supposed that this effect is mainly a result of the high hydrophobicity of the coating substrate. Show more

Keywords: Biomaterial, siloxanes, coating, bacterial growth, biofilm

DOI: 10.3233/JCB-15027

Citation: Journal of Cellular Biotechnology, vol. 2, no. 1, pp. 69-75, 2016

Authors: Tsompos, C. | Panoulis, C. | Toutouzas, K. | Triantafyllou, A. | Zografos, G. | Papalois, A.

Article Type: Research Article

Abstract: OBJECTIVE: This experimental study examined the effect of erythropoietin (epo) in a rat model and particularly in a hypoxia-reoxygenation (HR) protocol. The effect of epo was studied hematologically using the blood mean corpuscular volume (MCV) levels. MATERIALS AND METHODS: 40 rats of mean weight 247.7 ± 34.991 g were used in the study. MCV levels were measured at 60 min (groups A and C) and at 120 min (groups B and D) of reoxygenation. Epo was administered only in groups C and D. RESULTS: Epo administration increased non-significantly the predicted MCV levels by 0.56% ± 0.66% (p  = 0.3549). The …reperfusion time decreased non-significantly the predicted MCV levels by 0.55% ± 0.65% (p  = 0.3721). Epo administration and reperfusion time together did also not induce an increase of the MCV levels (0.30% ± 0.39%; p  = 0.4430). CONCLUSIONS: Epo administration, reperfusion time and their interaction had no short – term effect on MCV within the time of 2 hours. Show more

Keywords: Hypoxia, mean corpuscular volume, erythropoietin, reoxygenation

DOI: 10.3233/JCB-15028

Citation: Journal of Cellular Biotechnology, vol. 2, no. 1, pp. 77-83, 2016