Co-culture of human fibroblasts,smooth muscle and endothelial cells promotes osteopontin induction in hypoxia

Arteriovenous fistulas (AVFs) are the preferred vascular access for haemodialysis of patients suffering from end-stage renal disease, a worldwide public health problem. However, they are prone to a high rate of failure due to neointimal hyperplasia and stenosis. This study aimed to determine if osteopontin (OPN) was induced in hypoxia and if OPN could be responsible for driving AVF failure. Identification of new factors that participate in remodelling of AVFs is a challenge. Three cell lines representing the cells of the three layers of the walls of arteries and veins, fibroblasts, smooth muscle cells and endothelial cells, were tested in mono- and co-culture in vitro for OPN expression and secretion in normoxia compared to hypoxia after silencing the hypoxia-inducible factors (HIF-1α, HIF-2α and HIF-1/2α) with siRNA or after treatment with an inhibitor of NF-kB. None of the cells in mono-culture showed OPN induction in hypoxia, whereas cells in co-culture secreted OPN in hypoxia. The changes in oxygenation that occur during AVF maturation up-regulate secretion of OPN through cell-cell interactions between the different cell layers that form AVF, and in turn, these promote endothelial cell proliferation and could participate in neointimal hyperplasia.     

Discovery of P2X3 selective antagonists for the treatment of chronic pain

Purinergic receptor P2X3 has been linked to analgesia in a number of pre-clinical models of pain, and is expressed in the human pain perception pathway. Only few P2X3-selective antagonists have been reported to date. This Letter describes the SAR and in vivo analgesic profile of a novel scaffold of selective P2X3 antagonists.     

Effect of N-acetyl- -cysteine on peroxynitrite and superoxide anion production of lung alveolar macrophages in systemic sclerosis

Lung macrophages may play a relevant role in oxidative processes producing both superoxide anion (O(2)(-)) and NO. In this view, an antioxidant therapy can be useful in the treatment of systemic sclerosis (SSc) patients. N-Acetylcysteine (NAC) is able to expand natural antioxidant defenses by increasing intracellular gluthatione concentration and it has been proposed as an antioxidant therapy in respiratory distress syndromes. The aim of our study was to determine whether lung macrophages obtained from SSc patient bronchoalveolar lavage (BAL) express the inducible form of nitric oxide synthase (iNOS) and whether NAC can reduce the peroxynitrite (ONOO(-)) and O(2)(-) production of these cells. Alveolar macrophages were isolated from BAL of 32 patients and used for the immunocytochemical determination of iNOS, and the production of ONOO(-) and O(2)(-) was measured by fluorimetric or spectrophotometric methods, respectively. Lung macrophages obtained from SSc patients expressed a higher level of iNOS compared to healthy subject cells. NAC preincubation (5 x 10(-5)M, 24h) significantly reduced (-21%) the ONOO(-) production in formyl Met-Leu-Phe (fMLP)-activated cells and slightly reduced it under resting conditions, whereas NAC preincubation was unable to modify the release of O(2)(-) both in basal condition and in fMLP-stimulated cells. We conclude that since SSc lung macrophages express high levels of iNOS and produce a significant quantity of ONOO(-), NAC administration reduces ONOO(-) production and can be an useful treatment to alleviate SSc symptoms.     

Reduced Susceptibility to Praziquantel among Naturally Occurring Kenyan Isolates of Schistosoma mansoni

Background

The near exclusive use of praziquantel (PZQ) for treatment of human schistosomiasis has raised concerns about the possible emergence of drug-resistant schistosomes.

Methodology/Principal Findings

We measured susceptibility to PZQ of isolates of Schistosoma mansoni obtained from patients from Kisumu, Kenya continuously exposed to infection as a consequence of their occupations as car washers or sand harvesters. We used a) an in vitro assay with miracidia, b) an in vivo assay targeting adult worms in mice and c) an in vitro assay targeting adult schistosomes perfused from mice. In the miracidia assay, in which miracidia from human patients were exposed to PZQ in vitro, reduced susceptibility was associated with previous treatment of the patient with PZQ. One isolate (“KCW”) that was less susceptible to PZQ and had been derived from a patient who had never fully cured despite multiple treatments was studied further. In an in vivo assay of adult worms, the KCW isolate was significantly less susceptible to PZQ than two other isolates from natural infections in Kenya and two lab-reared strains of S. mansoni. The in vitro adult assay, based on measuring length changes of adults following exposure to and recovery from PZQ, confirmed that the KCW isolate was less susceptible to PZQ than the other isolates tested. A sub-isolate of KCW maintained separately and tested after three years was susceptible to PZQ, indicative that the trait of reduced sensitivity could be lost if selection was not maintained.

Conclusions/Significance

Isolates of S. mansoni from some patients in Kisumu have lower susceptibility to PZQ, including one from a patient who was never fully cured after repeated rounds of treatment administered over several years. As use of PZQ continues, continued selection for worms with diminished susceptibility is possible, and the probability of emergence of resistance will increase as large reservoirs of untreated worms diminish. The potential for rapid emergence of resistance should be an important consideration of treatment programs.     

Genetic stability and xanthan gum production in Xanthomonas campestris pv. campestris NRRL B1459

A transposon (Tn5-SC) was constructed that can be used to quantify genetic deletions or amplifications. This transposon was used to evaluate the genomic stability of Xanthomonas campestris pv. campestris NRRL B1459 and we found that the genome of this bacterium is as stable as other Gram-negative bacteria or even more stable. Homologous recombination between plasmid sequences was determined in strain NRRL B1459 and was found to occur at a similar level to that reported for other Gram-negative bacteria. We report here that in X.c.c. NRRL B1459 there is no straightforward correlation between the occurrence of genetic rearrangements and frequency of homologous recombination. These data are discussed with respect to the reported instability of strain NRRL B1459 for xanthan gum production.     

The mechanism of chemical delivery to the vomeronasal organs in squamate reptiles: a comparative morphological approach

Vomeronasal chemoreception, an important chemical sense in squamate reptiles (lizards and snakes), is mediated by paired vomeronasal organs (VNOs), which are only accessible via ducts opening through the palate anteriorly. We comparatively examined the morphology of the oral cavity in lizards with unforked tongues to elucidate the mechanism of stage I delivery (transport of chemical-laden fluid from the tongue tips to the VNO fenestrae) and to test the generality of the Gillingham and Clark (1981. Can J Zool 59:1651-1657) hypothesis (based on derived snakes), which suggests that the sublingual plicae act as the direct conveyors of chemicals to the VNOs. At rest, the foretongue lies within a chamber formed by the sublingual plicae ventrally and the palate dorsally, with little or no space around the anterior foretongue when the mouth is closed. There is a remarkable conformity between the shape of this chamber and the shape of the foretongue. We propose a hydraulic mechanism for stage I chemical transport in squamates: during mouth closure, the compliant tongue is compressed within this cavity and the floor of the mouth is elevated, expressing fluid from the sublingual glands within the plicae. Chemical-laden fluid covering the tongue tips is forced dorsally and posteriorly toward the VNO fenestrae. In effect, the tongue acts as a piston, pressurizing the fluid surrounding the foretongue so that chemical transport to the VNO ducts is effected almost instantaneously. Our findings falsify the Gillingham and Clark (1981. Can J Zool 59:1651-1657) hypothesis for lizards lacking forked, retractile tongues.