Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.
BackgroundThe cell membrane water channel protein, aquaporins (AQPs), regulate cellular water transport and cell volume and play a key role in water homeostasis. Recently, AQPs are considered as important players in the field of reproduction. In previous studies, we have established the presence of AQP1 and 5 in porcine uterus. Their expression at protein level altered in distinct tissues of the female reproductive system depending on the phase of the estrous cycle. However, the regulation of aquaporin genes and proteins expression has not been examined in porcine uterine tissue. Therefore, we have designed an in vitro experiment to explain whether steroid hormones, progesterone (P4) and estradiol (E2), and other factors: oxytocine (OT), arachidonic acid (AA; substrate for prostaglandins synthesis) as well as forskolin (FSK; adenylate cyclase activator) and cAMP (second messenger, cyclic adenosine monophosphate) may impact AQPs expression.MethodsUterine tissues were collected on Days 10–12 and 14–16 of the estrous cycle representing the mid-luteal phase and luteolysis. Real-time PCR and Western blot analysis were performed to examine the expression of porcine AQP1 and AQP5. Their expression in the uterine explants was also evaluated by immunohistochemistry.ResultsThe results indicated that uterine expression of AQP1 and AQP5 potentially remains under control of steroid hormones and AA-derived compounds (e.g. prostaglandins). P4, E2, AA, FSK and cAMP cause translocation of AQP5 from apical to the basolateral plasma membrane of the epithelial cells, which might affect the transcellular water movement (through epithelial cells) between uterine lumen and blood vessels. The AC/cAMP pathway is involved in the intracellular signals transduction connected with the regulation of AQPs expression in the pig uterus.ConclusionsThis study documented specific patterns of AQP1 and AQP5 expression in response to P4, E2, AA, FSK and cAMP, thereby providing new indirect evidence of their role in maintaining the local fluid balance within the uterus during the mid-luteal phase of the estrous cycle and luteolysis in pigs.
Female undernutrition during early pregnancy may affect the physiological pattern of genomic DNA methylation. We hypothesised that in utero DNA methylation may be impaired in females fed a restrictive diet in early pregnancy. In this study we evaluated whether poor maternal nutritional status, induced by applying a restricted diet during the peri-conceptional period, may influence: (1) the potential for in utero DNA methylation, expressed as changes in the mRNA expression and protein abundance of methyltransferases: DNA methyltransferase 1 (DNMT1) and DNMT3a in the endometrium and the myometrium, (2) the intrauterine microenvironment, measured as oestradiol 17β (E) and progesterone (P) concentrations in uterine flushings and (3) plasma concentration of E and P during the peri-implantation period. Our results indicate that maternal peri-conceptional undernutrition affects maintenance and de novo DNA methylation in the endometrium, de novo methylation in the myometrium and a results in a decrease in intrauterine E concentration during the peri-implantation period. The intrauterine concentration of P and plasma concentrations of E and P did not change. These findings suggest that undernutrition during the earliest period of pregnancy, and perhaps the pre-pregnancy period, may create changes in epigenetic mechanisms in the uterus and intrauterine milieu of E during the peri-implantation period.
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