The 'Unified airway': The RCPCH care pathway for asthma/or rhinitis

Abstract Aims The Royal College of Paediatrics and Child Health (RCPCH) Science and Research Department was commissioned by the Department of Health to develop national care pathways for children with allergies: the asthma/rhinitis care pathway is the third such pathway. Asthma and rhinitis have been considered together. These conditions co-exist commonly, have remarkably similar immuno-pathology and an integrated management approach benefits symptom control. Method The asthma/rhinitis pathway was developed by a multidisciplinary working group and was based on a comprehensive review of evidence. The pathway was reviewed by a broad group of stakeholders including the public and was approved by the Allergy Care Pathways Project Board and the RCPCH Clinical Standards Committee. Results The pathway entry points are defined by symptom type and severity at presentation. Acute severe rhinitis and life-threatening asthma are presented as distinct entry routes to the pathway, recognising that initial care of these conditions requires presentation-specific treatments. However, the pathway emphasises that ideal long term care should take account of both conditions in order to achieve maximal improvements in disease control and quality of life. Conclusions The pathway recommends that acute presentations of asthma and/or rhinitis should be treated separately. Where both conditions exist, ongoing management should address the upper and lower airways. The authors recommend that this pathway is implemented locally by a multidisciplinary team (MDT) with a focus on creating networks. The MDT within these networks should work with patients to develop and agree on care plans that are age and culturally appropriate.

Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway

We describe in this report the characterization of the recently discovered N-linked glycosylation locus of the human bacterial pathogen Campylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five individual glycosyltransferases in the biosynthesis of the N-linked heptasaccharide. Furthermore, we show that C. jejuni- and E. coli-derived pathways can interact in the biosynthesis of N-linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP-GlcNAc: undecaprenylphosphate GlcNAc-1-phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N-linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni-derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N-linked glycan occurs on a lipid-linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N-linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.

Identification of a general secretory pathway in a human isolate of Burkholderia vietnamiensis (formerly B. cepacia complex genomovar V) that is required for the secretion of hemolysin and phospholipase C activities

Members of the Burkholderia cepacia complex can secrete proteases, lipases, and hemolysins. We report in this study the identification of a general secretory pathway present in a B. vietnamiensis (formerly genomovar V) clinical isolate, which is required for the efficient secretion of phospholipase C and hemolysin activities. Southern blot hybridization experiments revealed that this general secretion pathway is highly conserved among the different genomovars of the B. cepacia complex and is homologous to a similar system described in B. pseudomallei. We also show that this pathway appears not to be necessary for intracellular survival of B. vietnamiensis within Acanthamoeba polyphaga.

Biosynthesis pathway of ADP-L-glycero-beta-D-manno-heptose in Escherichia coli

The steps involved in the biosynthesis of the ADP-L-glycero-beta-D-manno-heptose (ADP-L-beta-D-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-D-beta-D-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-D-beta-D-heptose. Our results demonstrate that the synthesis of ADP-D-beta-D-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional D-beta-D-heptose 7-phosphate kinase/D-beta-D-heptose 1-phosphate adenylyltransferase), and GmhB (D,D-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-L-beta-D-heptose precursor utilized in the assembly of inner core LPS.

Pharmacogenomic Profiling and Pathway Analyses Identify MAPK-Dependent Migration as an Acute Response to SN38 in p53 Null and p53-Mutant Colorectal Cancer Cells.

The topoisomerase I inhibitor irinotecan is used to treat advanced colorectal cancer and has been shown to have p53-independent anticancer activity. The aim of this study was to identify the p53-independent signaling mechanisms activated by irinotecan. Transcriptional profiling of isogenic HCT116 p53 wild-type and p53 null cells was carried out following treatment with the active metabolite of irinotecan, SN38. Unsupervised analysis methods showed that p53 status had a highly significant impact on gene expression changes in response to SN38. Pathway analysis indicated that pathways involved in cell motility [adherens junction, focal adhesion, mitogen-activated protein kinase (MAPK), and regulation of the actin cytoskeleton] were significantly activated in p53 null cells, but not p53 wild-type cells, following SN38 treatment. In functional assays, SN38 treatment increased the migratory potential of p53 null and p53-mutant colorectal cancer cell lines, but not p53 wild-type lines. Moreover, p53 null SN38-resistant cells were found to migrate at a faster rate than parental drug-sensitive p53 null cells, whereas p53 wild-type SN38-resistant cells failed to migrate. Notably, cotreatment with inhibitors of the MAPK pathway inhibited the increased migration observed following SN38 treatment in p53 null and p53-mutant cells. Thus, in the absence of wild-type p53, SN38 promotes migration of colorectal cancer cells, and inhibiting MAPK blocks this potentially prometastatic adaptive response to this anticancer drug.

Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle

This study assessed the contribution of L-type Ca2+ channels and other Ca2+ entry pathways to Ca2+ store refilling in choroidal arteriolar smooth muscle. Voltage-clamp recordings were made from enzymatically isolated choroidal microvascular smooth muscle cells and from cells within vessel fragments (containing <10 cells) using the whole-cell perforated patch-clamp technique. Cell Ca2+ was estimated by fura-2 microfluorimetry. After Ca2+ store depletion with caffeine (10 mM), refilling was slower in cells held at -20 mV compared to -80 mV (refilling half-time was 38 +/- 10 and 20 +/- 6 s, respectively). To attempt faster refilling via L-type Ca2+ channels, depolarising steps from -60 to -20 mV were applied during a 30 s refilling period following caffeine depletion. Each step activated L-type Ca2+ currents and [Ca2+]i transients, but failed to accelerate refilling. At -80 mV and in 20 mM TEA, prolonged caffeine exposure produced a transient Ca2+-activated Cl- current (I(Cl)(Ca)) followed by a smaller sustained current. The sustained current was resistant to anthracene-9-carboxylic acid (1 mM; an I(Cl)(Ca) blocker) and to BAPTA AM, but was abolished by 1 microM nifedipine. This nifedipine-sensitive current reversed at +29 +/- 2 mV, which shifted to +7 +/- 5 mV in Ca2+-free solution. Cyclopiazonic acid (20 microM; an inhibitor of sarcoplasmic reticulum Ca2+-ATPase) also activated the nifedipine-sensitive sustained current. At -80 mV, a 5 s caffeine exposure emptied Ca2+ stores and elicited a transient I(Cl)(Ca). After 80 s refilling, another caffeine challenge produced a similar inward current. Nifedipine (1 microM) during refilling reduced the caffeine-activated I(Cl)(Ca) by 38 +/- 5 %. The effect was concentration dependent (1-3000 nM, EC50 64 nM). In Ca2+-free solution, store refilling was similarly depressed (by 46 +/- 6 %). Endothelin-1 (10 nM) applied at -80 mV increased [Ca2+]i, which subsided to a sustained 198 +/- 28 nM above basal. Cell Ca2+ was then lowered by 1 microM nifedipine (to 135 +/- 22 nM), which reversed on washout. These results show that L-type Ca2+ channels fail to contribute to Ca2+ store refilling in choroidal arteriolar smooth muscle. Instead, they refill via a novel non-selective store-operated cation conductance that is blocked by nifedipine.

Towards a better understanding of the nature and contribution of the interflow to catchment dynamics.

A detailed understanding of flow and contaminant transfer along each of the key hydrological pathways within a catchment is critical for designing and implementing cost effective Programmes of Measures under the Water
Framework Directive.
The Contaminant Movement along Pathways Project (’The Pathways Project’) is an Irish, EPA STRIVE funded, large multi-disciplinary project which is focussed on understanding and modelling flow and attenuation along each of these pathways for the purposes of developing a catchment management tool. The tool will be used by EPA and RBD catchment managers to assess and manage the impacts of diffuse contamination on stream aquatic ecology. Four main contaminants of interest — nitrogen, phosphorus, sediment and pathogens — are being
investigated in four instrumented test catchments. In addition to the usual hydrological and water chemistry/quality parameters typically captured in catchment studies, field measurements at the test catchments include ecological
sampling, sediment dynamics, soil moisture dynamics, and groundwater levels and chemistry/quality, both during and between significant rainfall events. Spatial and temporal sampling of waters directly from the pathways of
interest is also being carried out.
Sixty-five percent of Ireland is underlain by poorly productive aquifers. In these hydrogeological settings, the main pathways delivering flow to streams are overland flow, interflow and shallow bedrock flow. Little is
known about the interflow pathway and its relative importance in delivery of flow and contaminants to the streams. Interflow can occur in both the topsoil and subsoil, and may include unsaturated matrix flow, bypass or macropore
flow, saturated flow in locally perched water tables and artificial field drainage.
Results to date from the test catchment experiments show that artificial field drains play an important role in the delivery of interflow to these streams, during and between rainfall events when antecedent conditions are
favourable. Hydrochemical mixing models, using silica and SAC254 (the absorbance of UV light at a wavelength of 254 nm which is a proxy for dissolved organic matter) as tracers, show that drain flow is an important end
member contributing to the stream and that proportionally, its contribution is relatively high.
Results from the study also demonstrate that waters originating from one pathway often mix with the waters from another, and are subsequently delivered to the stream at rates, and with chemical/quality characteristics,
that are not typical of either pathway. For example, pre-event shallow groundwater not far from the catchment divide comes up to the surface as rejected recharge during rainfall events and is rapidly delivered to the stream
via overland flow and/or artificial land drainage, bringing with it higher nitrate than would often be expected from a quickflow pathway contribution. This is contrary to the assumption often made in catchment studies that the
deeper hydrological pathways have slower response times in stream hydrographs during a rainfall event, and it emphasizes that it is critical to have a strong three-dimensional conceptual model as the basis for the interpretation
of catchment data.

Pathways for delivery of surface water nutrients to receptors

Diffuse contaminants can make their way into rivers via a number of different pathways, including overland flow, interflow, and shallow and deep groundwater. Identification of the key pathway(s) delivering contaminants to a receptor is important for implementing effective water management strategies. The ‘Pathways Project’, funded by the Irish Environmental Protection Agency, is developing a catchment management tool that will enable practitioners to identify the critical source areas for diffuse contaminants, and the key pathways of interest in assessing contaminant problems on a catchment and sub-catchment scale.
One of the aims of the project is to quantify the flow and contaminant loadings being delivered to the stream via each of the main pathways. Chemical separation of stream event hydrographs is being used to supplement more traditional physical hydrograph separation methods. Distinct, stable chemical signatures are derived for each of the pathway end members, and the proportion of flow from each during a rainfall event can be determined using a simple mass balance approach.
Event sampling was carried out in a test catchment underlain by poorly permeable soils and bedrock, which is predominantly used for grazing with a number of one-off rural residential houses. Results show that artificial field drainage, which includes subterranean land drains and collector drains around the perimeters of the 1 to 10 ha fields, plays an important role in the delivery of flow and nutrients to the streams in these types of hydrogeological settings.
Nitrate infiltrates with recharge and is delivered to the stream primarily via the artificial drains and the shallow groundwater pathway. Longitudinal stream profiles show that the nitrate load input is relatively uniform over the 8 km length of the stream at high flows, suggesting widespread diffuse contaminant input. In contrast, phosphorus is adsorbed in the clay-rich soil and is transported mainly via the overland flow pathway and the artificial drains. Longitudinal stream profiles for phosphorus suggest a pattern of more discrete points of phosphorus inputs, which may be related to point sources of contamination.
These techniques have application elsewhere within a toolkit of methods for determining the key pathways delivering contaminants to surface water receptors.