Lysozyme as a cotreatment during antibiotics use against vaginal infections: An in vitro study on Gardnerella vaginalis biofilm models

Bacterial vaginoses are frequent in women, most of them involving Gardnerella vaginalis. In more than 50% of the cases, usual antibiotic treatments are not capable of eliminating completely the infection, leading to recurrent vaginosis. In addition to the appearance of antibiotic resistance, recurrence can be due to the development of a biofilm by G. vaginalis. In vitro experiments on G. vaginalis biofilms showed that the biofilm protected bacteria from the antibiotic clindamycin. Also, recombinant human lysozyme (rhLys) was able to both degrade biofilms and prevent their formation. This degradation effect persisted whenever other vaginal commensal or pathogenic microorganisms were added to the culture and on each tested clinical biofilm-producing strain of G. vaginalis. The co-administration of rhLys and clindamycin or metronidazole improved both antibiotics’ efficiency and lysozyme-driven biofilm degradation. The comparison of both clindamycin and metronidazole antibacterial spectra showed that metronidazole was preferable to treat vaginosis. This suggests that human lysozyme could be added as an anti-biofilm cotreatment to vaginal antibiotherapy, preferably metronidazole, against Gardnerella vaginalis infection in vivo. [Int Microbiol 19(2): 101-107 (2016)]Keywords: Gardnerella vaginalis · recombinant human lysozyme · clindamycin · metronidazole · biofilms in pathogen

Expression of Growth Hormone Receptors by Lymphocyte subpopulations in the Human tonsil

The ability of human tonsillar lymphoid cells to express growth hormone receptor (hGH-N-R) was analyzed by flow cytometry. FITC-coupled recombinant human growth hormone (hGH-N) was used to reveal the receptors, in combination with phenotype markers

Tolerance to the Foeto-Placental 'Graft': Ten Ways to Support a Child for Nine Months

Tolerance to the foetal 'allograft' has been extensively studied in the past few years, providing interesting new insights. In addition to a potential role for HLA-G, which has been widely discussed, there are hypotheses suggesting roles for several other molecules or cells: leukemia inhibitory factor and its receptor; indoleamine 2. 3-dioxygenase; the Th1/Th2 balance; suppressor macrophages; hormones such as progesterone or the placental growth hormone; CD95 and its ligand; and, as recently proposed, annexin II. Tolerance of the foetal allograft is probably the consequence of a wide panel of mechanisms that may or may not be pregnancy-specific, that are of major or secondary importance and that may be interconnected

Determination of qPCR Reference Genes Suitable for Normalizing Gene Expression in a Canine Model of Duchenne Muscular Dystrophy

Background:Dogs with dystrophin-deficient muscular dystrophy are valuable models of the equivalent human disease, Duchenne Muscular Dystrophy (DMD): unlike the mdx mouse, these animals present a disease severity and progression that closely matches that found in human patients. Canine models are however less thoroughly characterised than the established mdx mouse in many aspects, including gene expression. Analysis of expression in muscle plays a key role in the study of DMD, allowing monitoring and assessment of disease progression, evaluation of novel biomarkers and gauging of therapeutic intervention efficacy. Appropriate normalization of expression data via carefully selected reference genes is consequently essential for accurate quantitative assessment. Unlike the expression profile of healthy skeletal muscle, the dystrophic muscle environment is highly dynamic: transcriptional profiles of dystrophic muscle might alter with age, disease progression, disease severity, genetic background and between muscle groups. Objectives:The aim of this work was to identify reference genes suitable for normalizing gene expression in healthy and dystrophic dogs under various comparative scenarios. Methods:Using the delta-E50 MD canine model of DMD, we assessed a panel of candidate reference genes for stability of expression across healthy and dystrophic animals, at different ages and in different muscle groups. Results:We show that the genes HPRT1, SDHA and RPL13a appear universally suitable for normalizing gene expression in healthy and dystrophic canine muscle, while other putative reference genes are exceptionally poor, and in the case of B2M, actively disease-correlated. Conclusions:Our findings suggest consistent cross-sample normalization is possible even throughout the dynamic progression of dystrophic pathology, and furthermore highlight the importance of empirical determination of suitable reference genes for neuromuscular diseases

Four-color multiplex real-Time PCR assay prototype targeting azithromycin resistance mutations in Mycoplasma genitalium

peer reviewedBackground: The worldwide expansion of macrolide-resistant Mycoplasma genitalium (MG) in cases of genital infections has led to an increased recurrence rate of these infections after first-line azithromycin treatment. By detecting the presence of azithromycin-resistant MG, the patient's antibiotic treatment can be targeted and the spread of resistance prevented. With this aim in mind, macrolide-resistance detection kits are helpful tools for the physician. Methods: Azithromycin resistance mutations in MG are targeted using a four-color multiplex real-Time RT-PCR assay. Tested targets include plasmid DNA (as positive controls) as well as macrolide-sensitive and macrolide-resistant genomic DNA from characterized cell lines and clinical samples. Results: The analytical data presented here were generated from plasmid DNA and genomic RNA/DNA and include adaptation to an internal control, specificity between targets, specificity vs non-MG species, limit of detection (LoD) and interference studies (co-infection and endogenous substances). The clinical data were based on the application of the assay to clinical samples characterized by sequencing. Conclusions: A new NAAT (nucleic acid amplification test) prototype has been developed in collaboration with the Diagenode s.a. company, this prototype targets MG and azithromycin-resistance mutations in that pathogen. © 2019 The Author(s)

Cyclosporin A Treatment Modulates Cytokine mRNA Expression by Inflammatory Cells Extracted from the Spinal Cord of Rats with Experimental Autoimmune Encephalomyelitis Induced by Inoculation with Myelin Basic Protein

In Lewis rats, treatment with high doses of cyclosporin A (CsA) suppresses clinical signs of experimental autoimmune encephalomyelitis (EAE), although disease occurs when treatment is ceased. Treatment with low doses of CsA causes EAE to take a chronic relapsing course. We have previously shown that CsA treatment causes a decline in the number of T cells and increased inflammatory cell apoptosis in the spinal cord. The present study was undertaken to assess whether CsA therapy also modulates cytokine mRNA expression by inflammatory cells in the spinal cord of rats with EAE, looking for changes that might contribute to the observed effects of CsA on the course of EAE. EAE was induced in Lewis rats by inoculation with myelin basic protein and adjuvants. At the peak of neurological signs, on day 14 after inoculation, rats were given a single intraperitoneal injection of saline, or CsA at a dose of 8, 16, 32 or 64 mg/kg. The next day, rats were sacrificed, the spinal cords removed, inflammatory cells were extracted from the cords, and mRNA isolated from these cells. Expression of cytokine mRNA was assessed by semi-quantitative reverse transcription polymerase chain reaction (PCR) and by quantitative real-time PCR. With both techniques, we found that CsA suppressed the expression of interferon-gamma mRNA and interleukin-2 (IL-2) mRNA. With real-time PCR, we found that CsA caused significantly increased expression of transforming growth factor-beta mRNA. With the different techniques, we observed no consistent pattern of alteration of expression of interleukin-10 or interleukin-4 mRNA. It is possible that these changes in cytokine mRNA expression contribute to the modulation of the clinical course of EAE that is produced by CsA treatment

Impaired expression of NER gene network in sporadic solid tumors

Nucleotide repair genes are not generally altered in sporadic solid tumors. However, point mutations are found scattered throughout the genome of cancer cells indicating that the repair pathways are dysfunctional. To address this point, in this work we focus on the expression pathways rather than in the DNA structure of repair genes related to either genome stability or essential metabolic functions. We present here a novel statistical analysis comparing ten gene expression pathways in human normal and cancer cells using serial analysis of gene expression (SAGE) data. We find that in cancer cells nucleotide-excision repair (NER) and apoptosis are the most impaired pathways and have a highly altered diversity of gene expression profile when compared to normal cells. We propose that genome point mutations in sporadic tumors can be explained by a structurally conserved NER with a functional disorder generated from its entanglement with the apoptosis gene network

Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood

The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells

Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans

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