Adaptive immunity rather than viral cytopathology mediates polyomavirus-associated nephropathy in mice.

Nephropathy associated with BK polyomavirus causes kidney allograft dysfunction and failure. Understanding the pathogenesis of polyomavirus-associated allograft nephropathy (PVAN) is hampered by the species specificity of Polyomaviridae family members. Using a mouse polyomavirus (MPyV) kidney transplant model, we investigated clinically relevant variables that may contribute to PVAN. We found that the timing and source (i.e. donor vs. recipient) of MPyV infection and the titer of the viral inoculum have significant effects on the extent of allograft injury, with acute infection of the recipient by high-titer MPyV inoculums producing the most profound PVAN. In contrast, altering the degree of MHC matching or increasing ischemia/reperfusion injury by prolonging the cold ischemic time of the allograft did not affect the severity of PVAN. Survival correlated positively with serum creatinine levels, but not with viral loads in the kidney allograft. Using splenectomized alymphoplasia mice, which are unable to mount primary adaptive immune responses, we further demonstrate that persistent high viral loads in the kidney are not sufficient to cause advanced PVAN. These findings suggest that the mechanism of PVAN in mice is not a direct consequence of viral cytopathology, but rather involves interplay between viral infection and the recipient antidonor immune response.

Comparing the cost of delayed and immediate autologous breast reconstruction in Belgium.

This study documents the cost of immediate and delayed DIEP flap breast reconstruction. Immediate reconstruction is more attractive from an economic perspective since it only requires one operation, one anaesthetic procedure and one recovery period in hospital. From the perspective of healthcare budget management, assessing the possible cost savings from immediate reconstruction yields interesting results. Since charges do not reflect the real costs of providing care, we calculated resource costs using the micro-costing method. About 95% of the initial mastectomy costs could be saved when performing an immediate breast reconstruction. This was about 35% of total standard direct and indirect costs due to mastectomy and delayed breast reconstruction. In a growing cost conscious environment of managed care, the economic evaluation should, therefore, encourage the trend towards more immediate reconstructions.

Oxygen regulation of the Escherichia coli cytochrome d oxidase (cydAB) operon: roles of multiple promoters and the Fnr-1 and Fnr-2 binding sites.

The Escherichia coli cydAB operon encodes the high-affinity terminal oxidase of the oxygen respiratory chain, cytochrome d oxidase. The sensor-regulator pair, ArcB-ArcA, is responsible for the microaerobic activation of the cydAB operon, whereas the anaerobic regulator Fnr represses its expression in the absence of oxygen. Fnr binds in vitro at two sites within the cydAB promoter element. To discern whether these two regions have an in vivo function in the anaerobic regulation of cydAB, the Fnr-binding motifs were mutagenized individually and in combination. The effects of these mutations on in vivo gene expression were determined by lac fusion and primer extension analysis. Our results show that the Fnr-2 site is critical for Fnr-mediated anaerobic repression of the two main cydAB promoters, P1 and P2. In contrast, the Fnr-1 site has an auxiliary role in the anaerobic repression of P1, but not of P2. Transcription from P1 did not affect ArcA-mediated activation or Fnr-mediated repression of P2, indicating that oxygen regulation is exerted on both promoters in an independent fashion. In addition, three new promoters were identified in the cydAB control region, and the 5' ends of the corresponding transcripts were mapped. Two of these promoters, designated P3 and P4, are co-ordinately regulated with P1 and P2 in response to oxygen, ArcA and Fnr. The P5 promoter is not Fnr regulated and is only weakly activated by ArcA. The contribution of these three additional promoters to the overall cydAB expression is most relevant under aerobic conditions. Our results suggest a unique repression model, in which one Fnr dimer bound to one single site (Fnr-2) is sufficient to downregulate transcription from four cydAB promoters. In conclusion, transcription of the cydAB operon is driven by a complex regulatory element containing at least five promoters that act in unison to provide adequate oxygen control of gene expression.

Liquid chromatographic method for determining added folic acid in fortified cereal products.

Reversed-phase ion-pair high-performance liquid chromatography (HPLC) was coupled with detection by UV absorption (280 nm) for separation and quantitation of added folic acid (FA) in fortified cereal based foods. A simple and rapid liquid-solid extraction method, combined with enzymatic digestion, to recover FA from the sample matrices is also presented. The quantitation of added FA was achieved in products including corn (maize), wheat-, rice- and oat-based cereal breakfast foods fortified at 25% and 100% of the reference daily intake (RDI). The retention time for FA was ca. 15 min, and the detection limit was 2 ng/20 microliters injection for standard FA. When FA was added to unfortified samples of wheat flour at concentrations of 3.08 or 20.0 micrograms/g, recoveries were 93% and 96%, respectively. Comparison of HPLC results with those of a standard microbiological assay has shown quite good agreement (r = 0.998). A solid-phase extraction clean-up procedure has also been developed for use with samples fortified with low levels of FA, where interferences may otherwise hinder quantitation.

Aerobic regulation of cytochrome d oxidase (cydAB) operon expression in Escherichia coli: roles of Fnr and ArcA in repression and activation.

The cydAB operon of Escherichia coli encodes the cytochrome d oxidase complex, one of two aerobic terminal oxidases that catalyses the oxidation of ubiquinol-8 and the reduction of oxygen to water. This enzyme has a higher affinity for oxygen than the cytochrome o oxidase complex and accumulates as oxygen becomes limiting. Expression of the cydAB operon is microaerobically controlled by the ArcA/ArcB two-component regulatory system and by Fnr. To understand how ArcA and Fnr contribute to this control, a set of cyd-lacZ reporter fusions were constructed and analysed in vivo. Two cydAB promoters, designated P1 and P2, were identified by primer extension analysis and are located 288 and 173 bp upstream of the start of cydA translation respectively. Transcription from promoter P1 was shown to be regulated by both Fnr and ArcA in response to anaerobiosis. DNasel footprint experiments revealed the locations of two Fnr binding sites at the P1 promoter: one is centred at the start of cyd transcription, while the other is positioned 53.5 bp upstream. A single ArcA-phosphate binding site of 49 bp, centred 93 bp upstream of promoter P1, was identified to be sufficient for the activation of cydAB expression. Based on the results of the in vitro and in vivo studies, a working model for ArcA activation and Fnr repression of cydAB transcription is proposed.