Impact of antibiotics on the proliferation and differentiation of human adipose-derived mesenchymal stem cells

Adipose tissue is a promising source of mesenchymal stem cells. Their potential to differentiate and regenerate other types of tissues may be affected by several factors. This may be due to in vitro cell-culture conditions, especially the supplementation with antibiotics. The aim of our study was to evaluate the effects of a penicillin-streptomycin mixture (PS), amphotericin B (AmB), a complex of AmB with copper (II) ions (AmB-Cu2+) and various combinations of these antibiotics on the proliferation and differentiation of adipose-derived stem cells in vitro. Normal human adipose-derived stem cells (ADSC, Lonza) were routinely maintained in a Dulbecco’s Modified Eagle Medium (DMEM) that was either supplemented with selected antibiotics or without antibiotics. The ADSC that were used for the experiment were at the second passage. The effect of antibiotics on proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine-B (SRB) tests. Differentiation was evaluated based on Alizarin Red staining, Oil Red O staining and determination of the expression of ADSC, osteoblast and adipocyte markers by real-time RT-qPCR. The obtained results indicate that the influence of antibiotics on adipose-derived stem cells depends on the duration of exposure and on the combination of applied compounds. We show that antibiotics alter the proliferation of cells and also promote natural osteogenesis, and adipogenesis, and that this effect is also noticeable in stimulated osteogenesis

Structure and properties of slow-resorbing nanofibers obtained by (co-axial) electrospinning as tissue scaffolds in regenerative medicine

With the rapid advancement of regenerative medicine technologies, there is an urgent need for the development of new, cell-friendly techniques for obtaining nanofibers—the raw material for an artificial extracellular matrix production. We investigated the structure and properties of PCL10 nanofibers, PCL5/PCL10 core-shell type nanofibers, as well as PCL5/PCLAg nanofibres prepared by electrospinning. For the production of the fiber variants, a 5–10% solution of polycaprolactone (PCL) (Mw = 70,000–90,000), dissolved in a mixture of formic acid and acetic acid at a ratio of 70:30 m/m was used. In order to obtain fibers containing PCLAg 1% of silver nanoparticles was added. The electrospin was conducted using the above-described solutions at the electrostatic field. The subsequent bio-analysis shows that synthesis of core-shell nanofibers PCL5/PCL10, and the silver-doped variant nanofiber core shell PCL5/PCLAg, by using organic acids as solvents, is a robust technique. Furthermore, the incorporation of silver nanoparticles into PCL5/PCLAg makes such nanofibers toxic to model microbes without compromising its biocompatibility. Nanofibers obtained such way may then be used in regenerative medicine, for the preparation of extracellular scaffolds: (i) for controlled bone regeneration due to the long decay time of the PCL, (ii) as bioscaffolds for generation of other types of artificial tissues, (iii) and as carriers of nanocapsules for local drug delivery. Furthermore, the used solvents are significantly less toxic than the solvents for polycaprolactone currently commonly used in electrospin, like for example chloroform (CHCl3), methanol (CH3OH), dimethylformamide (C3H7NO) or tetrahydrofuran (C4H8O), hence the presented here electrospin technique may allow for the production of multilayer nanofibres more suitable for the use in medical field

Comprehensive molecular and clinical analysis of adalimumab and etanercept therapeutic potentialin patients with psoriatic arthritis

Introduction: Adalimumab and etanercept are drugs used in anti-TNF therapy in patients with psoriasis and psoriatic arthritis. Despite the molecular targeting of these drugs, the loss of pharmacological response to treatment is observed in patients. The development of personalized medicine makes it possible to use not only clinical parameters of disease severity, but also molecular marker systems. Aim: The aim of the study was to evaluate the changes in TNF-α, TNFR1, and TNFR2 expression in relation to parameters of disease severity (PASI, BSA, DAS28) in patients treated with adalimumab and etanercept. We have attempted to determine whether changes in the TNF-α, TNFR1, and TNFR2 expression profile may be a useful molecular marker of the therapeutic potential of anti-TNF drugs. Material and methods: The study group consisted of 3 patients initially treated with adalimumab, followed by etanercept. The control group included 20 healthy volunteers. The expression profile of TNFR1 and TNFR2 was determined at the mRNA level, while TNF-α expression was evaluated at the transcriptome and proteome levels using the RT-qPCR method (transcriptional activity assay) and MALDI-TOF MS (protein level assessment). Results: Depending on the drug, different expression profiles of the studied cytokines are observed. Conclusions: The obtained data indicate that TNF-α, TNFR1, and TNFR2 may be useful markers of the efficacy of anti-TNF therapy, thus complementing clinical parameters

Treatment seeking for problematic pornography use among women

Background and aims Previous studies examined psychological factors related to treatment seeking for problematic pornography use (PU) among males. In this study, we focused on females who seek treatment for problematic PU and compared them with non-problematic pornography users with regard to variables related to problematic PU. Second, we investigated the relationships between critical constructs related to problematic PU with the path analysis method, emphasizing the predictors for treatment seeking among women. We also compared our results with previous studies on males. Methods A survey study was conducted on 719 Polish-speaking Caucasian females, 14–63 years old, including 39 treatment seekers for problematic PU. Results The positive relationship between the mere amount of PU and treatment seeking loses its significance after introducing two other predictors of treatment-seeking: religiosity and negative symptoms associated with PU. This pattern is different from the results obtained in previous studies on males. Discussion Different from previous studies on male samples, our analysis showed that in the case of women, mere amount of PU may be related to treatment-seeking behavior even after accounting for negative symptoms associated with PU. Moreover, religiousness is a significant predictor of treatment seeking among women, which may indicate that in the case of women, treatment seeking for problematic PU is motivated not only by experienced negative symptoms of PU but also by personal beliefs about PU and social norms. Conclusion For females, negative symptoms associated with PU, the amount of PU and religiosity is associated with treatment seeking. Those factors should be considered in treatment

Adipose-Derived Stem Cells undergo differentiation after co-culture with porcine Limbal Epithelial Stem Cells

Mesenchymal stem cells (MSCs) are objects of interest in regenerative medicine. They are used for various therapies such as for the regeneration of bone, chondrocytes and other tissues. Adipose derived stem cells (ADSCs) inter alia are particularly easy to access, they are relatively abundant in fat tissue. ADSCs could be differentiated into many types of cells. To date, it has been proven that ADSCs only differentiate into mesodermal cell lineages. In this study, we present the differentiation of ADSCs into the corneal epithelium. Human ADSCs were placed in a co-culture with porcine limbal epithelial stem cells (LESCs). After 14 days of cultivation, total RNA was extracted for the analysis of the molecular markers (expression of genes of interest). The gene expression was assessed by real-time RT-qPCR. The expression of the surface molecular markers of ADSCs is modulated after co-culturing. We have observed the decrease in CD73, CD90 and CD105 mRNA expression, while the expression of mRNA coding for CK3 and CK12 mRNA was increased in ADSCs co-cultured with porcine limbal epithelial stem cells as compared to the control. We conclude that the coculture of LESCs and ADSCs changed ADSCs’ molecular markers gene expression indicating initiation of differentiation towards limbal cells

Survivin expression at the mRNA level in tumors and the protein concentration in the serum and peritoneal fluid in patients with serous ovarian tumors

Objectives: Ovarian cancer is one of the gynecological cancers that have the worst prognosis. The expression of the proteins from the IAP family (inhibitor of apoptosis protein), including survivin, is observed in many types of cancer. The aim of the study was to evaluate survivin at the mRNA level in tumors and the protein concentration in the serum and peritoneal fluid of patients with serous ovarian cancer in order to assess the relationship between the concentration of survivin and the histological subtypes of cancer. Material and methods: The study group consisted of 55 women, including patients with serous ovarian cancer (n = 30, nine low-grade serous carcinoma LGSC, 21 high-grade serous carcinoma HGSC), serous cysts (n = 10) and the control group (n = 15). The concentration of protein in the peritoneal fluid and serum was assessed using ELISA tests. The expression of survivin gene BIRC5 in the tumors was assessed using the RT-qPCR method. Results: The data that was obtained indicated that the concentration of survivin was higher in the serum of the women with serous ovarian cancer compared those that had benign tumors (p < 0.05) and the control group (p < 0.001). The survivin concentration was also higher in both the serum and peritoneal fluid in the HGSC group compared to the LGSC group (p < 0.001). The mRNA level was highest in the HGSC group, and there was a statistically significant difference compared to those in the benign tumor group and HGSC group ( p < 0.05). Conclusions: The observed changes prove that the expression level increases significantly in HGSC in both the protein and mRNA levels. Based on these findings, it can be assumed that assessing this parameter could be a useful additional indicator of the progression and differentiation of this type of cancer. However, this requires further research in a larger group of patients and possibly in other types of ovarian cancer

Transformujący czynnik wzrostu beta w raku podstawnokomórkowym, kolczystokomórkowym i rogowiaku kolczystokomórkowym

Introduction. Transforming Growth Factor β (TGFβ) activates signaling cascades which regulate cell proliferation, differentiation, apoptosis, inflammatory response and angiogenesis. In the early stages of malignant transformation this cytokine acts as an inhibitor of tumour growth. In the advanced stages of malignant transformation TGFβ acts as a promoter of metastasis. Changes in the expression of genes associated with TGFβactivity could provide a new strategy of molecularly targeted therapy.Aim. The aim of this study was to compare the mRNA profile of genes associated with TGFβ signaling pathways in non-melanoma skin pathologies biopsy specimens of bas-al cell carcinoma (BCC), squamous cell carcinoma (SCC) and keratoacanthoma (KA) in comparison to normal skin.Material and methods. Tissue samples of KA, SCC and BCC were obtained from the central part of tumours. Healthy skin margins comprised the control group. mRNA profile of genes coding TGFβ and proteins involved in TGFβ-induced signaling pathways was determined using oligonucleotide microarrays (Affymetrix).Results. Microarray analysis showed changes in profile of genes coding proteins in-volved in TGFβ-induced signaling pathways. In SCC TGFβ-1 (TGFB1) was upregulated, comparing to controls. Both in KA and SCC, the most statistically significant change re-ferred to TGFBR3 (Transforming Growth Factor beta Receptor III) mRNA.Conclusions. mRNA profile of genes coding proteins involved in TGFβ-induced signal-ization reveals strong molecular similarity of SCC and KA

Psoriasis Treatment Changes the Expression Profile of Selected Caspases and their Regulatory MicroRNAs

Background/Aims: Psoriasis, an autoimmune diseases of the skin, characterized by patches of abnormal/inflammed skin, although not usually life-threatening, it causes severe discomfort, esthetic impairments, and may lead to impaired social functions and social withdrawal. Besides UV-phototherapy, various anti-inflammatory treatments are applied, depending on the severity of symptoms. In 2008, adalimumab (fully humanized human anti-TNF antibody) was launched for the treatment of psoriasis. In the quest to better understand the pathomechanism of adalimumab’s therapeutic effects, and the acquired resistance to the drug, we have investigated how its administration affect the regulation of the expression of selected caspases, including those activated by inflammosome. Methods: The research was initially carried out on normal human dermal fibroblasts (NHDF) treated with adalimumab for 2, 8 and 24 hours in vitro. Then, expression profile of genes encoding caspases and their regulatory micro-RNAs was determined with the use of oligonucleotide microarray. The validation of the microarray results was carried out by qRT-PCR. The in vitro study was followed by ex-vivo investigation of adalimumab’s effects on the expression of caspase-6 in blood of the psoriatic patients. The samples were collected before, and 2 hours after adalimumab’s administration and the analysis was determined by qRT-PCR. Results: The result of the analysis indicated that introduction of adalimumab to the NHDF culture resulted in the change of the transcription activity of genes encoding caspases and genes encoding miRNAs. The analysis revealed 5 different miRNA molecules regulating the expression of: CASP2, CASP3 and CASP6. There were no statistically significant differences in the expression of gene encoding caspase-6 in the patients’ blood before and 2 hours after the anti-TNF drug administration. Conclusion: We have found that adalimumab administration affects caspases expression, thus they may be used as molecular markers for monitoring the therapy with the use of an anti-TNF drugs, including adalimumab. It is likely that the mechanisms responsible for changed expression profiles of genes encoding caspase-2,-3, and -6, may be caused by the upregulation of the respective microRNA molecules. Increased expression of genes encoding specific caspases may induce inflammatory processes, as well as trigger apoptosis. Furthermore, the proapoptotic activity of caspases may be enhanced by miRNA molecules, which exhibit proapoptotic function. The overexpression of such miRNAs was observed in our study