CD39 abrogates platelet-derived factors induced IL-1β expression in the human placenta

Tissue insults in response to inflammation, hypoxia and ischemia are accompanied by the release of ATP into the extracellular space. There, ATP modulates several pathological processes, including chemotaxis, inflammasome induction and platelet activation. ATP hydrolysis is significantly enhanced in human pregnancy, suggesting that increased conversion of extracellular ATP is an important anti-inflammatory process in preventing exaggerated inflammation, platelet activation and hemostasis in gestation. Extracellular ATP is converted into AMP, and subsequently into adenosine by the two major nucleotide-metabolizing enzymes CD39 and CD73. Here, we aimed to elucidate developmental changes of placental CD39 and CD73 over gestation, compared their expression in placental tissue from patients with preeclampsia and healthy controls, and analyzed their regulation in response to platelet-derived factors and different oxygen conditions in placental explants as well as the trophoblast cell line BeWo. Linear regression analysis showed a significant increase in placental CD39 expression, while at the same time CD73 levels declined at term of pregnancy. Neither maternal smoking during first trimester, fetal sex, maternal age, nor maternal BMI revealed any effects on placental CD39 and CD73 expression. Immunohistochemistry detected both, CD39 and CD73, predominantly in the syncytiotrophoblast layer. Placental CD39 and CD73 expression were significantly increased in pregnancies complicated with preeclampsia, when compared to controls. Cultivation of placental explants under different oxygen conditions had no effect on the ectonucleotidases, whereas presence of platelet releasate from pregnant women led to deregulated CD39 expression. Overexpression of recombinant human CD39 in BeWo cells decreased extracellular ATP levels after culture in presence of platelet-derived factors. Moreover, platelet-derived factors-induced upregulation of the pro-inflammatory cytokine, interleukin-1β, was abolished by CD39 overexpression. Our study shows that placental CD39 is upregulated in preeclampsia, suggesting an increasing demand for extracellular ATP hydrolysis at the utero-placental interface. Increased placental CD39 in response to platelet-derived factors may lead to enhanced conversion of extracellular ATP levels, which in turn could represent an important anti-coagulant defense mechanism of the placenta

The Impact of Different Antibiotic Regimens on the Emergence of Antimicrobial-Resistant Bacteria

Backgroud: The emergence and ongoing spread of antimicrobial-resistant bacteria is a major public health threat. Infections caused by antimicrobial-resistant bacteria are associated with substantially higher rates of morbidity and mortality compared to infections caused by antimicrobial-susceptible bacteria. The emergence and spread of these bacteria is complex and requires incorporating numerous interrelated factors which clinical studies cannot adequately address. Methods/Principal Findings: A model is created which incorporates several key factors contributing to the emergence and spread of resistant bacteria including the effects of the immune system, acquisition of resistance genes and antimicrobial exposure. The model identifies key strategies which would limit the emergence of antimicrobial-resistant bacterial strains. Specifically, the simulations show that early initiation of antimicrobial therapy and combination therapy with two antibiotics prevents the emergence of resistant bacteria, whereas shorter courses of therapy and sequential administration of antibiotics promote the emergence of resistant strains. Conclusions/Significance: The principal findings suggest that (i) shorter lengths of antibiotic therapy and early interruption of antibiotic therapy provide an advantage for the resistant strains, (ii) combination therapy with two antibiotics prevents the emergence of resistance strains in contrast to sequential antibiotic therapy, and (iii) early initiation of antibiotics is among the most important factors preventing the emergence of resistant strains. These findings provide new insights into strategies aimed at optimizing the administration of antimicrobials for the treatment of infections and the prevention of the emergence of antimicrobial resistance

Gene Expression Profiling via Multigene Concatemers

We established a novel method, Gene Expression Profiling via Multigene Concatemers (MgC-GEP), to study multigene expression patterns simultaneously. This method consists of the following steps: (1) cDNA was obtained using specific reverse primers containing an adaptor. (2) During the initial 1–3 cycles of polymerase chain reaction (PCR), the products containing universal adaptors with digestion sites at both termini were amplified using specific forward and reverse primers containing the adaptors. (3) In the subsequent 4–28 cycles, the universal adaptors were used as primers to yield products. (4) The products were digested and ligated to produce concatemers. (5) The concatemers were cloned into the vector and sequenced. Then, the occurrence of each gene tag was determined. To validate MgC-GEP, we analyzed 20 genes in Saccharomyces cerevisiae induced by weak acid using MgC-GEP combined with real-time reverse transcription (RT)-PCR. Compared with the results of real-time RT-PCR and the previous reports of microarray analysis, MgC-GEP can precisely determine the transcript levels of multigenes simultaneously. Importantly, MgC-GEP is a cost effective strategy that can be widely used in most laboratories without specific equipment. MgC-GEP is a potentially powerful tool for multigene expression profiling, particularly for moderate-throughput analysis

Bacterial Community Profiling of Milk Samples as a Means to Understand Culture-Negative Bovine Clinical Mastitis

Inflammation and infection of bovine mammary glands, commonly known as mastitis, imposes significant losses each year in the dairy industry worldwide. While several different bacterial species have been identified as causative agents of mastitis, many clinical mastitis cases remain culture negative, even after enrichment for bacterial growth. To understand the basis for this increasingly common phenomenon, the composition of bacterial communities from milk samples was analyzed using culture independent pyrosequencing of amplicons of 16S ribosomal RNA genes (16S rDNA). Comparisons were made of the microbial community composition of culture negative milk samples from mastitic quarters with that of non-mastitic quarters from the same animals. Genomic DNA from culture-negative clinical and healthy quarter sample pairs was isolated, and amplicon libraries were prepared using indexed primers specific to the V1–V2 region of bacterial 16S rRNA genes and sequenced using the Roche 454 GS FLX with titanium chemistry. Evaluation of the taxonomic composition of these samples revealed significant differences in the microbiota in milk from mastitic and healthy quarters. Statistical analysis identified seven bacterial genera that may be mainly responsible for the observed microbial community differences between mastitic and healthy quarters. Collectively, these results provide evidence that cases of culture negative mastitis can be associated with bacterial species that may be present below culture detection thresholds used here. The application of culture-independent bacterial community profiling represents a powerful approach to understand long-standing questions in animal health and disease