Development of New PCR Assay with SYBR Green I for Detection of Mycoplasma, Acholeplasma, and Ureaplasma sp. in Cell Cultures.

Mycoplasma, Acholeplasma, and Ureaplasma sp. are atypical bacteria responsible for in vitro cell culture contaminations that can warp the results. These bacteria also cause human and animal infections and may lead to chronic diseases. In developed polymerase chain reaction (PCR) in this study a quantitative PCR with SYBR Green I fluorochrome was applied to facilitate the Mycoplasma, Acholeplasma, and Ureaplasma sp. DNA detection and identification. Screening Test-1 v.1 (triplex qPCR) allowed for the detection of 11 species. Test-1 v.2 (three single qPCRs) pre-identified three subgroups, allowing for the reduction of using single qPCRs in Test-2 for species identification. The range of both tests was consistent with pharmacopeial requirements for microbial quality control of mammal cells and included detection of M. arginini, M. orale, M. hyorhinis, M. fermentans, M. genitalium, M. hominis, M. pneumoniae, M. salivarium, M. pirum, A. laidlawii, and U. urealyticum. Limit of detection values varied between 125-300 and 50-100 number of copies per milliliter in Test-1 and Test-2, respectively. Test-1 and Test-2 showed fully concordant results, allowed for time-saving detection and/or identification of selected species from Mycoplasma, Acholeplasma, and Ureaplasma in tested cell cultures.

ERCC1-deficient cells and mice are hypersensitive to lipid peroxidation.

Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1-/- mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1-/- cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1-/- cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats.

[Incidence of methicillin-resistant Staphylococcus aureus strains (MRSA) and difficulties in treatment. Own experience]. / Wystepowanie szczepów metycylinoopornych gronkowców zlocistych (MRSA) oraz trudnosci w leczeniu wywolanych przez nie infekcji u dzieci hospitalizowanych w klinice.

THE AIM: To estimate the incidence of MRSA strains in hospitalized children, the analysis of antibiotic treatment necessity and its effectiveness. RESULTS: In 54 cases biological material obtained from hospitalized children revealed the culture of S. aureus, 22% of them were MRSA strains. Micro-organisms were cultured from throat swabs, faeces, purulent skin lesions, blood, cerebrospinal fluid. Antibiotic treatment was applied in 3 cases. Vancomycin (Edicin) was used as the first-choice option. Symptoms of drug intolerance was observed in all three cases after the first or the second dose, must have been immediately withdrawn. CONCLUSIONS: MRSA strains are common in children population, they are not only nosocomial strains. Infections caused by S. aureus MRSA may result in life-threatening diseases. The generic formula of Vancomycin, that is used in Poland may cause serious side effects, which restricts narrow range of therapeutic options to eradicate MRSA strains.

Immunogenicity of inactivated hepatitis A vaccine in children with chronic liver disease.

One and 6 months after vaccination with hepatitis A virus vaccine (HAVRIX 720 Junior), immunologic responses (anti-hepatitis A virus >/=20 mIU/ml) in children with chronic hepatitis C infection seroprotection were 92% (23/25) and 75% (9/12) in children with chronic hepatitis B infection 87% (21/24) and 88% (15/17) and in healthy children 91% (20/22) at both times. A booster dose induced seroprotection in all children.