Dynamics of the bacterial gut microbiota in preterm and term infants after intravenous amoxicillin/ceftazidime treatment

BACKGROUND: It is important to understand the consequences of pre-emptive antibiotic treatment in neonates, as disturbances in microbiota development during this key developmental time window might affect early and later life health outcomes. Despite increasing knowledge regarding the detrimental effect of antibiotics on the gut microbiota, limited research focussed on antibiotic treatment duration. We determined the effect of short and long amoxicillin/ceftazidime administration on gut microbiota development during the immediate postnatal life of preterm and term infants. METHODS: Faeces was collected from 63 (pre) term infants at postnatal weeks one, two, three, four and six. Infants received either no (control), short-term (ST) or long-term (LT) postpartum amoxicillin/ceftazidime treatment. RESULTS: Compared to control infants, ST and LT infants' microbiota contained significantly higher abundance of Enterococcus during the first two postnatal weeks at the expense of Bifidobacterium and Streptococcus. Short and long antibiotic treatment both allowed for microbiota restoration within the first six postnatal weeks. However, Enterococcus and Bifidobacterium abundances were affected in fewer ST than LT infants. CONCLUSIONS: Intravenous amoxicillin/ceftazidime administration affects intestinal microbiota composition by decreasing the relative abundance of Escherichia-Shigella and Streptococcus, while increasing the relative abundance of Enterococcus and Lactobacillus species during the first two postnatal weeks. Thriving of enterococci at the expense of bifidobacteria and streptococci should be considered as aspect of the cost-benefit determination for antibiotic prescription.</p

Serial selection for invasiveness increases expression of miR-143/miR-145 in glioblastoma cell lines

<p>Abstract</p> <p>Background</p> <p>Glioblastoma multiforme (GBM) is the most common primary central nervous system malignancy and its unique invasiveness renders it difficult to treat. This invasive phenotype, like other cellular processes, may be controlled in part by microRNAs - a class of small non-coding RNAs that act by altering the expression of targeted messenger RNAs. In this report, we demonstrate a straightforward method for creating invasive subpopulations of glioblastoma cells (IM3 cells). To understand the correlation between the expression of miRNAs and the invasion, we fully profiled 1263 miRNAs on six different cell lines and two miRNAs, miR-143 and miR-145, were selected for validation of their biological properties contributing to invasion. Further, we investigated an ensemble effect of both miR-143 and miR-145 in promoting invasion.</p> <p>Methods</p> <p>By repeated serial invasion through Matrigel^®-coated membranes, we isolated highly invasive subpopulations of glioma cell lines. Phenotypic characterization of these cells included <it>in vitro </it>assays for proliferation, attachment, and invasion. Micro-RNA expression was compared using miRCURY arrays (Exiqon). In situ hybridization allowed visualization of the regional expression of miR-143 and miR-145 in tumor samples, and antisense probes were used investigate <it>in vitro </it>phenotypic changes seen with knockdown in their expression.</p> <p>Results</p> <p>The phenotype we created in these selected cells proved stable over multiple passages, and their microRNA expression profiles were measurably different. We found that two specific microRNAs expressed from the same genetic locus, miR-143 and miR-145, were over-expressed in our invasive subpopulations. Further, we also found that combinatorial treatment of these cells with both antisense-miRNAs (antimiR-143 and -145) will abrogated their invasion without decreasing cell attachment or proliferation.</p> <p>Conclusions</p> <p>To best of our knowledge, these data demonstrate for the first time that miR-143 and miR-145 regulate the invasion of glioblastoma and that miR-143 and -145 could be potential therapeutic target for anti-invasion therapies of glioblastoma patients.</p

P2Y1 and P2Y12 receptor cross-talk in calcium signalling: Evidence from nonstarved and long-term serum-deprived glioma C6 cells

The current work presents results of experiments on the calcium response evoked by the stimulation by extracellular nucleotides occurring in control, nonstarved glioma C6 cells and in cells after long-term (96 h) serum starvation. Three nucleotide receptors were studied: P2Y1, P2Y2 and P2Y12. Two of them, P2Y1 and P2Y2, directly stimulate calcium response. The protein level of the P2Y2 receptor did not change during the serum starvation, while P2Y1 protein level fell dramatically. Observed changes in the calcium response generated by P2Y1 are directly correlated with the receptor protein level as well as with the amount of calcium present in the intracellular calcium stores, partially depleted during starvation process. The third receptor, P2Y12, did not directly evoke calcium response, however it is activated by the same ligand as P2Y1. The experiments with AR-C69941MX, the P2Y12-specific antagonist, indicated that in control and serum-starved cells, calcium response evoked by P2Y1 receptor is potentiated by the activity of P2Y12-dependent signaling pathways. This potentiation may be mediated by P2Y12 inhibitory effect on the plasma membrane calcium pump. The calcium influx enhanced by the cooperation of P2Y1 and P2Y12 receptor activity directly depends on the capacitative calcium entrance mechanism

Modulation of purinergic signaling by NPP-type ectophosphodiesterases

Extracellular nucleotides can elicit a wide array of cellular responses by binding to specific purinergic receptors. The level of ectonucleotides is dynamically controlled by their release from cells, synthesis by ectonucleoside diphosphokinases and ectoadenylate kinases, and hydrolysis by ectonucleotidases. One of the four structurally unrelated families of ectonucleotidases is represented by the NPP-type ectophosphodiesterases. Three of the seven members of the NPP family, namely NPP1–3, are known to hydrolyze nucleotides. The enzymatic action of NPP1–3 (in)directly results in the termination of nucleotide signaling, the salvage of nucleotides and/or the generation of new messengers like ADP, adenosine or pyrophosphate. NPP2 is unique in that it hydrolyzes both nucleotides and lysophospholipids and, thereby, generates products that could synergistically promote cell motility. We review here the enzymatic properties of NPPs and analyze current evidence that links their nucleotide-hydrolyzing capability to epithelial and neural functions, the immune response and cell motility

Multiple P2Y receptors couple to calcium-dependent, chloride channels in smooth muscle cells of the rat pulmonary artery

BACKGROUND: Uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP) act via P2Y receptors to evoke contraction of rat pulmonary arteries, whilst adenosine 5'-triphosphate (ATP) acts via P2X and P2Y receptors. Pharmacological characterisation of these receptors in intact arteries is complicated by release and extracellular metabolism of nucleotides, so the aim of this study was to characterise the P2Y receptors under conditions that minimise these problems. METHODS: The perforated-patch clamp technique was used to record the Ca(2+)-dependent, Cl(- )current (I(Cl,Ca)) activated by P2Y receptor agonists in acutely dissociated smooth muscle cells of rat small (SPA) and large (LPA) intrapulmonary arteries, held at -50 mV. Contractions to ATP were measured in isolated muscle rings. Data were compared by Student's t test or one way ANOVA. RESULTS: ATP, UTP and UDP (10(-4)M) evoked oscillating, inward currents (peak = 13–727 pA) in 71–93% of cells. The first current was usually the largest and in the SPA the response to ATP was significantly greater than those to UTP or UDP (P < 0.05). Subsequent currents tended to decrease in amplitude, with a variable time-course, to a level that was significantly smaller for ATP (P < 0.05), UTP (P < 0.001) and UDP (P < 0.05) in the SPA. The frequency of oscillations was similar for each agonist (mean≈6–11.min(-1)) and changed little during agonist application. The non-selective P2 receptor antagonist suramin (10(-4)M) abolished currents evoked by ATP in SPA (n = 4) and LPA (n = 4), but pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (10(-4)M), also a non-selective P2 antagonist, had no effect (n = 4, 5 respectively). Currents elicited by UTP (n = 37) or UDP (n = 14) were unaffected by either antagonist. Contractions of SPA evoked by ATP were partially inhibited by PPADS (n = 4) and abolished by suramin (n = 5). Both antagonists abolished the contractions in LPA. CONCLUSION: At least two P2Y subtypes couple to I(Cl,Ca )in smooth muscle cells of rat SPA and LPA, with no apparent regional variation in their distribution. The suramin-sensitive, PPADS-resistant site activated by ATP most resembles the P2Y(11 )receptor. However, the suramin- and PPADS-insensitive receptor activated by UTP and UDP does not correspond to any of the known P2Y subtypes. These receptors likely play a significant role in nucleotide-induced vasoconstriction

Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks

The role of nucleotides in intracellular energy provision and nucleic acid synthesis has been known for a long time. In the past decade, evidence has been presented that, in addition to these functions, nucleotides are also autocrine and paracrine messenger molecules that initiate and regulate a large number of biological processes. The actions of extracellular nucleotides are mediated by ionotropic P2X and metabotropic P2Y receptors, while hydrolysis by ecto-enzymes modulates the initial signal. An increasing number of studies have been performed to obtain information on the signal transduction pathways activated by nucleotide receptors. The development of specific and stable purinergic receptor agonists and antagonists with therapeutical potential largely contributed to the identification of receptors responsible for nucleotide-activated pathways. This article reviews the signal transduction pathways activated by P2Y receptors, the involved second messenger systems, GTPases and protein kinases, as well as recent findings concerning P2Y receptor signalling in C6 glioma cells. Besides vertical signal transduction, lateral cross-talks with pathways activated by other G protein-coupled receptors and growth factor receptors are discussed

In vivo glioblastoma growth is reduced by apyrase activity in a rat glioma model

BACKGROUND: ATP is an important signalling molecule in the peripheral and central nervous system. Both glioma growth and tumor resection induces cell death, thus liberating nucleotides to the extracellular medium. Nucleotides are hydrolyzed very slowly by gliomas when compared with astrocytes and induce neuronal cell death and glioma proliferation. The objective of the present study was to test the involvement of extracellular ATP in glioblastoma growth in a rat glioma model. METHODS: To deplete the extracellular ATP, the enzyme apyrase was tested on the treatment of gliomas implanted in the rats CNS. One million glioma C6 cells in 3 microliters of DMEM/FCS were injected in the right striata of male Wistar rats, 250–270 g. After 20 days, the rats were decapitated and the brain sectioning and stained with hematoxylin and eosine. We performed immunohistochemical experiments with Ki67, CD31 and VEGF. Total RNA was isolated from cultured glioma C6 cells and the cDNA was analyzed by Real Time-PCR with primers for the NTPDase family. RESULTS: C6 glioma cells effectively have a low expression of all NTPDases investigated, in comparison with normal astrocytes. The implanted glioma co-injected with apyrase had a significant reduction in the tumor size (p < 0.05) when compared with the rats injected only with gliomas or with gliomas plus inactivated apyrase. According to the pathological analysis, the malignant gliomas induced by C6 injection and co-injected with apyrase presented a significant reduction in the mitotic index and other histological characteristics that indicate a less invasive/proliferative tumor. Reduction of proliferation induced by apyrase co-injection was confirmed by counting the percentage of Ki67 positive glioma cell nuclei. According to counts with CD31, vessel density and neoformation was higher in the C6 group 20 days after implantation. Confirming this observation, rats treated with apyrase presented less VEGF staining in comparison to the control group. CONCLUSION: These results indicate that the participation of extracellular ATP and the ecto-nucleotidases may be associated with the development of this type of brain tumor in an in vivo glioma model