ABSTRACT
BACKGROUND: A new form of amphotericin B (AmB)- complex with copper (II) ions (AmB-Cu2+) - is less toxic to human renal cells. Cytokines, including Tumor Necrosis Factor (TNF), are responsible for nephrotoxicity observed in patients treated with AmB. Another problem during therapy is the occurrence of oxidized forms of AmB (AmB-ox) in patients' circulation. To elucidate the molecular mechanism responsible for the reduction of the toxicity of AmB-Cu2+, we evaluated the expression of genes encoding TNF and its receptors alongside encoding proteins involved in TNF-induced signalization. METHODS: Renal cells (RPTECs) were treated with AmB, AmB-Cu2+ or AmB-ox. The expression of TNF and its receptors was evaluated by ELISA tests and real-time RT-qPCR. The expression of TNF-related genes was appointed using oligonucleotide microarrays. RESULTS: Only sTNFR1 was detected, and its level was lower in AmB-Cu2+- and AmB-ox-treated cells. TNFR1 mRNA was downregulated in AmB-ox, while TNFR2 mRNA was upregulated in AmB and AmB-Cu2+. Several changes in the expression of TNF-related genes coincided with changes in the expression of TNF receptors. CONCLUSIONS: The lower toxicity of AmB-Cu2+ could result from the changes in the expression of TNF receptors, which coincided with the changes in the expression of genes encoding proteins involved in TNF-induced pathways. This situation might subsequently result in a changes in intracellular signalization and influence the toxicity of tested forms of AmB on renal cells.
Subject(s)
Amphotericin B/pharmacology , Copper/pharmacology , Down-Regulation/drug effects , Receptors, Tumor Necrosis Factor, Type I/genetics , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/genetics , Amphotericin B/chemistry , Antifungal Agents/pharmacology , Cell Line , Copper/chemistry , Down-Regulation/physiology , Humans , Receptors, Tumor Necrosis Factor, Type I/antagonists & inhibitors , Receptors, Tumor Necrosis Factor, Type I/metabolism , Signal Transduction/physiology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Pigs seem to be the answer to worldwide organ donor shortage. Porcine skin may also be applied as a dressing for severe burns. Genetic modifications of donor animals enable reduction of immune response, which prolongs xenograft survival as temporary biological dressing and allows achieving resistance against xenograft rejection. The risk posed by porcine endogenous retroviruses (PERVs) cannot be eliminated by breeding animals under specific-pathogen-free conditions and so all recipients of porcine graft will be exposed to PERVs. Therefore our study has been focused on the assessment of PERV DNA and mRNA level in skin samples of transgenic pigs generated for xenotransplantation. Porcine skin fragments were obtained from 3- to 6-month-old non-transgenic and transgenic Polish Landrace pigs. Transgenic pigs were produced by pronuclear DNA microinjection and were developed to express the human α-galactosidase and the human α-1,2-fucosyltransferase gene. The copy numbers of PERV DNA and RNA were evaluated using real-time Q-PCR and QRT-PCR. Comparative analysis of all PERV subtypes revealed that PERV-A is the main subtype of PERVs in analyzed skin samples. There was no significantly different copy number of PERV-A, PERV-B and PERV-C between non-transgenic pigs, pigs with the human α-galactosidase and pigs expressing the human α-1,2-fucosyltransferase gene, except of PERV-C DNA. It brings the conclusion, that transgenesis process exerts no influence on PERVs transinfection. That is another step forward in the development of pig skin xenografts as burn wounds dressing.