Birmingham and Lambeth Liver Evaluation Testing Strategies (BALLETS): a prospective cohort study.

OBJECTIVE: To evaluate mildly abnormal liver function test (LFT) results in general practice among patients who do not have known liver disease. DESIGN: Prospective cohort study of people with abnormal LFT results identified in primary care. Participants were intensively investigated using a common protocol and followed up for 2 years. Substudies investigated the psychological sequelae of abnormal test results, clinicians' reasons for testing, decision options when LFT results were abnormal and early detection of liver fibrosis. SETTING: Eleven primary-care practices: eight in Birmingham and three in Lambeth. PARTICIPANTS: Adults with abnormal LFT results who did not have pre-existing or obvious liver disease. Eight analytes were included in the panel of LFTs. MAIN OUTCOME MEASURES: Statistical tests were used to identify the interactions between clinical features, the initial pattern of abnormal LFT results and (1) specific viral, genetic and autoimmune diseases, such as viral hepatitis, haemochromatosis and primary biliary cirrhosis; (2) a range of other serious diseases, such as metastatic cancer and hypothyroidism; (3) 'fatty liver' not associated with the above; and (4) the absence of detectable disease. RESULTS: Fewer than 5% of people with abnormal LFT results had a specific disease of the liver, and many of these were unlikely to need treatment. The diagnostic potential of the LFT panel is largely subsumed into just two analytes: alanine aminotransferase (ALT) and alkaline phosphatase (ALP). Gamma-glutamyltransferase (GGT) offers a small increase in sensitivity at the margin at the cost of a large loss of specificity. Eighty-four per cent of abnormal LFT results remain abnormal on retesting 1 month later. In many cases, carrying out a definitive or specific test will be more efficient than repeating LFTs, with a view to specific testing only if the test remains abnormal. An ultrasound diagnosis of 'fatty liver' was present in nearly 40% of patients with abnormal LFTs and a small amount of weight loss over 2 years was associated with a reduced incidence of liver fat. There was a J-shaped relationship between alcohol intake and fatty liver in men. An abnormal LFT result causes temporary anxiety, which does not appear to promote sustained behaviour change. CONCLUSIONS: Liver disease is rare among people with abnormal LFT results in primary care. Only two analytes (ALT and ALP) are helpful in identifying the majority of liver disease. GGT adds little information in return for a high false-positive rate but it is sensitive to alcohol intake. LFT results seldom revert from abnormal to normal over a 1-month period, and modelling shows that repeating an abnormal LFT panel, as recommended in the current guidelines, is inefficient. LFTs are often undertaken to meet perceived patient need for a blood test, but as they are neither specific nor indicative of any particular disease they are among the least suitable tests for this purpose. Obesity and raised ALT provide strong evidence for a presumptive diagnosis of 'fatty' liver. Abnormal LFTs and 'fatty' liver provoke only short-term anxiety and neither is associated with sustained weight loss. Even a small amount of weight loss reduces liver fat. FUTURE WORK RECOMMENDATIONS: (1) the cases of 'fatty liver' and controls should be followed up in the long term to identify features that predict development of hepatosteatosis and then cirrhosis; (2) the acceptability of replacing the traditional six- to eight-analyte LFT panel with a drop down menu including the ALT/ALP combination should be evaluated. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

Tissue predilection sites and effect of dose on Mycobacterium avium subs. paratuberculosis organism recovery in a short-term bovine experimental oral infection model.

The objective of this study was to develop a short-term experimental infection model for Mycobacterium avium subsp. paratuberculosis (MAP) in cattle, using small oral doses of organisms. Specifically, the effect of dose size was evaluated, as well as specific tissue predilection sites for recovery of MAP. Oral doses as low as 1.5 x 10(6) CFU reliably produced infection that could be detected 3 weeks following infection. Detection of infection required culture of multiple intestinal samples (jejunum and ileum) for MAP. Histological examination did not permit detection at this early stage. Results from this study suggest intestinal mucosa, rather than tonsil, as the primary portal of entry for MAP. The experimental infection model described here is useful for studying the early effects of preventive and therapeutic interventions for paratuberculosis in cattle.

Groundwater: the processes and global significance of aquifer degradation.

The exploitation of groundwater resources for human use dates from the earliest civilizations, but massive resource development has been largely restricted to the past 50 years. Although global in scope, the emphasis of this paper is on groundwater-based economies in a developing nation context, where accelerated resource development has brought major social and economic benefits over the past 20 years. This results from groundwater's significant role in urban water supply and in rural livelihoods, including irrigated agriculture. However, little of the economic benefit of resource development has been reinvested in groundwater management, and concerns about aquifer degradation and resource sustainability began to arise. A general review, for a broad-based audience, is given of the mechanisms and significance of three semi-independent facets of aquifer degradation. These are (i) depletion of aquifer storage and its effects on groundwater availability, terrestrial and aquatic ecosystems; (ii) groundwater salinization arising from various different processes of induced hydraulic disturbance and soil fractionation; and (iii) vulnerability of aquifers to pollution from land-use and effluent discharge practices related to both urban development and agricultural intensification. Globally, data with which to assess the status of aquifer degradation are of questionable reliability, inadequate coverage and poor compilation. Recourse has to be made to 'type examples' and assumptions about the extension of similar hydrogeological settings likely to be experiencing similar conditions of groundwater demand and subsurface contaminant load. It is concluded that (i) aquifer degradation is much more than a localized problem because the sustainability of the resource base for much of the rapid socio-economic development of the second half of the twentieth century is threatened on quite a widespread geographical basis; and (ii) major (and long overdue) investments in groundwater resource and quality protection are urgently needed. These investments include appropriate institutional provisions, demand-side management, supply-side enhancement and pollution control.

A field study to assess the degradation and transport of diuron and its metabolites in a calcareous soil.

An experimental plot has been established on a calcareous soil in southern England to investigate the fate and transport of diuron (N'-[3,4-dichlorophenyl]-N,N-dimethylurea), a commonly used phenylurea herbicide. An agricultural grade of diuron was applied to the soil surface at a rate of 6.7 kg/ha along with a potassium bromide conservative tracer applied at 200 kg/ha, in early January, 2001. Hand augured samples were taken at regular intervals over the next 50 days, with samples collected down to 54 cm. Porewaters were extracted from the soil cores by using high speed centrifugation and the supernatant fluids were retained for analysis by HPLC, for diuron and three of its metabolites, N'-[3,4-dichlorophenyl]-N,N-methylurea (DCPMU), N'-3,4-dichlorophenylurea (DCPU) and 3,4-dichloroaniline (DCA). The centrifuged soil was retained and then extracted with methanol prior to HPLC analysis for the same suite of phenylureas. A mass balance approach showed large variations in diuron distribution, but on average accounted for 104% of the diuron applied. Concentrations of diuron and its metabolites were roughly five times higher in the soil than in the soil porewaters. After 50 days, metabolites comprised 10% of the total diuron present in the porewater and 20% of the total diuron sorbed to the soil matrix. After 36 days, a large pulse of diuron and DCPMU appeared in the porewaters and soil matrix at a depth of 54 cm, travelling an average of 0.15 cm/day faster than Br. A preferential route for diuron transport is suggested. There is evidence to suggest that degradation occurs at depth as well as at the soil surface. Metabolites generally appear to move more slowly than the parent compound. All metabolites were encountered, but interpreting transport and degradation processes simultaneously proved beyond the scope of the study. Diuron was detected once in a shallow (5 m) observation well, situated on the experimental plot. High concentrations of diuron and metabolites were still present in the soil and soil solutions after 50 days and remain as a source of potential groundwater contamination.

Bone marrow cell graft engineering: from bench to bedside.

Bone marrow transplantation (BMT) has the potential to treat hemoglobinopathies (sickle cell and thalassemia) autoimmunity (diabetes, lupus, multiple sclerosis, rheumatoid arthritis, Crohn's colitis) and enzyme deficiency states. Graft versus host disease (GVHD) is a major complication and limitation to the therapeutic application of BMT. There have been many clinical trials and experimental animal models that have attempted to control GVHD through the engineering of the donor bone marrow cells (BMC). Historically, several methods have demonstrated effectiveness in controlling GVHD; however they were also associated with a marked increase in the rate of graft failure. Highly purified hematopoietic stem cells (HSC) engraft quite readily in genetically-matched recipients while they do not engraft as easily in MHC-disparate recipients. The numbers of HSC must be increased 100-200 fold in order to overcome the allogeneic barrier. We were the first to phenotypically and to functionally characterize a novel cell in the bone marrow that enables engraftment of highly purified HSC in allogeneic recipients. The discovery of graft facilitating cell populations has resulted in the restoration of the engraftment-potential of purified HSC between genetically-disparate individuals. The addition of facilitating cells (FC) to T cell-depleted BMC grafts results in allogeneic engraftment without GVHD or graft failure. New strategies of BMC engineering that retain FC and HSC but avoid GVHD have allowed successful engraftment in mismatched and older recipients. These techniques have expanded the therapeutic potential of BMT to virtually every candidate as well as to non-malignant diseases in which the morbidity associated with conventional BMT could not be accepted. This article reviews the transition of the FC technology from bench to bedside and discuss the potentially broad-reaching applications of BMT and mixed chimerism.

Assessing herbicide concentrations in the saturated and unsaturated zone of a Chalk aquifer in southern England.

The behavior of the herbicides isoproturon (IPU) and chlortoluron (CTU) in ground water and shallow unsaturated zone sediments were evaluated at a site situated on the Chalk in southern England. Concentrations of IPU in ground water samples varied from < 0.05 to 0.23 microgram/L over a five-year period of monitoring, and were found to correlate with application of the pesticide. Concentrations of pesticides in ground water samples collected during periods of rising water table were significantly higher than pumped samples and suggest that rapidly infiltrating recharge water contains higher herbicide concentrations than the native ground water. Significant variations in herbicide concentrations were observed over a three-month period in ground water samples collected by an automated system, with concentrations of IPU ranging from 0.1 to 0.5 microgram/L, and concentrations of a recent application of CTU ranging from 0.2 to 0.8 microgram/L. Different extraction methods were used to assess pore water concentrations of herbicides in the unsaturated zone, and samples were analyzed by standard HPLC analysis and immunoassay (ELISA) methods. These data indicated highly variable concentrations of herbicide ranging from 4 to 200 g/ha for HPLC and 0.01 to 0.04 g/ha for ELISA, but indicate a general pattern of decreasing concentrations with depth. The results of this study indicate that transport of IPU and CTU through the unsaturated zone to shallow ground water occurs and that this transport increases immediately following herbicide application. Measured concentrations of herbicides are generally lower than specified by the European Union Drinking Water Directive, but are observed to spike above this limit. These results imply that, while delivery of pesticides to ground water can occur as a result of normal agricultural practices, the impact on potable supplies is likely to be negligible due to the potential for degradation during the relatively long travel time through the unsaturated zone and high degree of dilution that occurs within the aquifer. As a result of the wide variation in concentrations detected by different techniques, it is suggested that for future site investigations more than one sampling strategy be employed to characterize the occurrence of pesticide residues and elucidate the transport mechanisms.

An audit of oral care practice and staff knowledge in hospital palliative care.

Mouth care is considered one of the most basic of nursing activities, and palliative care patients are especially vulnerable to oral problems (Macmillan Practice Development Unit, 1995). This article describes a project on developing oral care practice and staff knowledge, by nursing staff and Macmillan nurses at a hospital in central England. A baseline audit (audit I) was carried out to examine all aspects of current oral care practice and nursing knowledge, including assessment, implementation, prescribing and evaluation of care. Oral care guidelines and a programme of ward-based teaching were then introduced. Several months later a follow-up audit (audit II) was conducted. Results showed an improvement in all aspects of oral care and staff knowledge. Additional benefits of this process included improved professional relationships and the promotion of further audits in hospital palliative care. Recommendations include the need for further nursing research into oral care to build the evidence base further. Additionally, it is suggested that nurses must recognize their important and central role in improving this aspect of palliative care. Education and training is pivotal to this process.

Biosynthetic requirements for the repair of membrane damage in pressure-treated Escherichia coli.

Cells of Escherichia coli that survived pressure treatment at 400 MPa showed increased sensitivity to sodium deoxycholate or sodium chloride in the plating medium, implying that homeostatic or barrier functions associated with outer and cytoplasmic membranes, respectively, were impaired. Repair of such sublethal membrane damage occurred when cells were incubated at 37 degrees C in tryptone soya broth. Inhibitor studies indicated that repair of cytoplasmic membrane damage was energy-dependent and required RNA and protein synthesis, whereas repair of outer membrane damage occurred with no requirement for energy or RNA or protein synthesis.

The role of alphabeta- and gammadelta-T cells in allogenic donor marrow on engraftment, chimerism, and graft-versus-host disease.

BACKGROUND: We previously characterized a facilitating cell (FC) in mouse marrow that enables engraftment of allogeneic hematopoietic stem cells (HSCs) without causing graft-versus-host disease (GVHD). The FC shares some cell surface molecules with T cells (Thy1+, CD3epsilon+, CD8+, CD5+, and CD2+) but is T-cell receptor (TCR) negative. Historically, depletion of CD3+ or CD8+ cells from rat marrow was associated with an increased rate of failure of engraftment. In this study, we evaluated whether depletion of alphabeta- and gammadelta-TCR(+) T cells from donor marrow would retain engraftment potential yet avoid GVHD. METHODS: Wistar-Furth rats were conditioned with 950 cGy of total body irradiation and transplanted with ACI bone marrow processed to remove either alphabeta-TCR(+), gammadelta-TCR(+), or alphabeta- plus gammadelta-TCR(+) T cells. Recipients were typed for chimerism at 28 days and monthly thereafter. RESULTS: Recipients of marrow depleted of alphabeta- (group A), gammadelta- (group B), or alphabeta- and gammadelta-TCR(+) T cells (group C) engrafted and had an average chimerism level of 73.0+/-8.3%, 92.3+/-9.2%, and 46.3+/-32.8%, respectively. Aggressive T-cell depletion did not remove the FC population (CD8+/CD3+/TCR(-)). Group A and group B both developed GVHD, with a higher incidence of GVHD in group B compared to group A. None of the recipients in group C developed GVHD. CONCLUSIONS: These data demonstrate that depletion of T cells from rat marrow does not impair engraftment of HSCs, indirectly supporting the existence of FCs in rat marrow. Moreover, donor alphabeta- and gammadelta-TCR(+) T cells contribute to GVHD in a nonredundant fashion, although alphabeta-TCR(+) T cells are more potent as the effector cells. Finally, the level of donor chimerism is influenced by the composition of the graft, because recipients of marrow that contain alphabeta-TCR(+) T cells exhibited significantly higher donor chimerism compared to recipients of marrow depleted of both alphabeta- and gammadelta-TCR(+) T cells.

Regulation of the production of soluble IL-4 receptors in murine cutaneous leishmaniasis. The roles of IL-12 and IL-4.

These studies were undertaken with the purpose of elucidating the key signals involved in the regulation of the production of soluble interleukin-4 receptors (sIL-4R) in mice during Th1 and Th2 responses to infection with the parasite Leishmania major. Our results showed that the production of sIL-4R was consistently higher in lymph node cell cultures from animals mounting a predominant Th2 response (BALB/c mice), and that sIL-4R production paralleled that of IL-4 in both mouse strains, even in the presence of a dominant Th1 response (C3H/FeJ mice). Consistently, administration of anti-IL-12 antibodies to infected C3H/ FeJ mice induced a switch from a Th1- to a Th2-type response and resulted in enhanced production of sIL-4R. Addition of rIL-12 to splenic cell cultures, however, was found not to have a direct effect on sIL-4R production induced by IL-4 or T cell mitogens. Moreover, the production of sIL-4R appears to be little influenced by Th1-produced cytokines, inasmuch as recombinant interferon-gamma or supernatants derived from antigen-stimulated Th1 clones did not affect the production of sIL-4R by activated splenic cultures. Despite its correlation with Th2 responses, the presence of IL-4 was not an absolute requirement for the up-regulation of the expression of sIL-4R because increased levels could be induced on cells obtained from IL-4-/- mice. These results indicate that, although enhanced sIL-4R production is a feature related to the activation and/or generation of Th2 responses, it is not absolutely dependent on IL-4 or directly inhibited by IL-12 or Th1 cytokines.

Regulation of the expression of the soluble and membrane forms of the murine IL-4 receptor.

The actions of interleukin-4 (IL-4) in vivo are likely to be positively influenced by the expression of membrane IL-4 receptors (mIL-4R) on target cells and negatively by the concentration of soluble IL-4 receptors (sIL-4R) in the extracellular environment. Inasmuch as the two forms of the mouse IL-4R are differentially encoded by alternatively spliced mRNA transcripts, the purpose of this work was to determine how their expression is regulated by IL-4 and T cell activation and whether there is preferential expression of one type of transcript over the other. In this study, the expression of sIL-4R and mIL-4R transcripts was analyzed by a semiquantitative RT-PCR method in resting and mitogen-activated splenic cells. Irrespectively of the state of cell activation, IL-4 up-regulated the levels of both types of mRNA with similar kinetics and dose-response curves. In contrast, ConA failed to enhance the steady-state levels of sIL-4R or mIL-4R transcripts despite increased expression at the protein level, suggesting that sIL-4R expression is also regulated at levels other than transcription. Western blot analysis of supernatants of IL-4- and ConA-stimulated spleen cells substantiated the presence of sIL-4R molecules derived by translation of sIL-4R-specific transcripts, thus confirming the importance of this mechanism for the generation of sIL-4R molecules in normal cells. These results indicate that the sIL-4R- and mIL-4R-specific transcripts are normally regulated in a parallel manner and further suggest that expression of both forms of the IL-4R is controlled at multiple levels (i.e., transcriptional and posttranscriptional).

The production of soluble interleukin 4 receptors is preferentially regulated by the murine Th2 cell subset.

In order to understand how the endogenous production of soluble IL-4 receptors (sIL-4r) is regulated, the authors tested prototypic clones of Th1 and Th2 murine CD4+ T cell subsets for their ability to regulate their expression of sIL-4r. Results showed that although both types of clones produced low levels of sIL-4r under resting conditions, only the Th2 clones upregulated sIL-4r expression following antigenic stimulation. Inhibition of endogenous IL-4 with a neutralizing anti-IL-4 mAb had only a minor (approximately 20%) inhibitory effect on sIL-4r production by the Th2 cells, and addition of rIL-4 to Th1 cells resulted only in a modest two-fold increase in sIL-4r levels, suggesting that IL-4 is not the only factor that regulates sIL-4r production and that the ability of Th2 clones to upregulate sIL-4r expression can be relatively independent of IL-4. Indeed, the production of sIL-4r by Th2 cells was found to be regulated by cell contact and/or IL-1 mediated signals. Transcripts for both sIL-4r and mIL-4r were detected by RT-PCR on both resting and activated Th1 and Th2 cells, with the relative levels of expression being moderately higher in the Th2 clones. Moreover, the expression of sIL-4r-specific transcripts appeared to increase to a greater extent than those of mIL-4r after activation of Th2 cells with APCs, both in the presence and absence of antigen. Taken together, these results predict that increased sIL-4r production in vivo might be preferentially associated with Th2-type responses and indicate that even though the production of IL-4 and sIL-4r is mediated by the same cells (i.e. Th2 cells), the synthesis of sIL-4r can be regulated independently from that of IL-4 through alternative signals such as cell contact and/or IL-1. These properties may allow for changing ratios of sIL-4r to IL-4 and sIL-4r to mIL-4r during different phases of an immune response and are consistent with a regulatory role for sIL-4r on IL-4 activity in vivo.

Control of the production of soluble interleukin-4 receptors: implications in immunoregulation.

Soluble cytokine receptors (sCR) are generated in vivo through proteolytic cleavage of the membrane-bound receptors or by direct translation of mRNAs specifically encoding the soluble forms. Despite their widespread presence in biological fluids, the physiological role of endogenous sCR as immunoregulatory molecules is not yet well understood. In vivo, exogenous soluble interleukin-4 receptors (sIL-4R) have been shown to have both agonistic and antagonistic effects on IL-4 responses, depending on the relative concentration ratios of sIL-4R to IL-4. In an effort to elucidate the potential role of endogenous sIL-4R in the regulation of IL-4 responses, the mechanisms controlling the production of sIL-4R have been investigated. Although many cell types are able to constitutively produce low levels, production of sIL-4R is significantly up-regulated in vitro by T cell activation and IL-4. The ability of splenic cells to produce sIL-4R and the serum levels of sIL-4R have consistently been found to be increased during immune responses characterized by T cell activation and IL-4 secretion (Th2 responses). In agreement, clones of Th2, but not Th1, cells were found to significantly up-regulate sIL-4R production following antigenic stimulation. However, the production of sIL-4R by Th2 cells appears to be independent from that of IL-4 and can also be induced by cell contact and/or IL-1-dependent pathways. Taken together, these observations suggest that the production of sIL-4R in vivo is closely associated with the secretion of IL-4, and are consistent with the notion that endogenous sIL-4R are involved in the regulation of IL-4 activity during immune responses.

Linked in vivo expression of soluble interleukin-4 receptor and interleukin-4 in murine schistosomiasis.

Soluble interleukin-4 receptors (sIL-4R) are truncated IL-4R molecules that are secreted into biological fluids. To gain an insight into the mechanisms that control sIL-4R synthesis in vivo and their role in the regulation of immune responses, the expression and secretion of sIL-4R in mice infected with Schistosoma mansoni was studied. Splenocytes from infected animals responded to schistosomal antigen preparations with increased production of both IL-4 and sIL-4R. The synthesis of sIL-4R by spleen cells peaked at 8 weeks following infection and coincided with maximum levels of sIL-4R in serum and sIL-4R-specific mRNA in the liver of infected mice. The expression of IL-4-specific mRNA in the liver was different from that of IL-4R, reaching its peak approximately 2 weeks earlier. A relationship between sIL-4R production and the development and activation of Th2 cells was suggested by the findings that: (a) in vivo administration of anti-IL-4 antibodies (11B11) impaired the ability of splenic cells to secrete either IL-4 or sIL-4R; and (b) splenic cells from mice vaccinated with irradiated cercariae, which tend to develop much weaker Th2 responses than mice injected with live cercariae, expressed reduced levels of sIL-4R when challenged with schistosomal antigens. Moreover, a direct role for IL-4 in regulating the expression of sIL-4R was suggested by the ability of anti-IL-4 antibodies to inhibit sIL-4R synthesis in vitro. These data provide the first evidence demonstrating that the production of sIL-4R in vivo is up-regulated during immune responses, especially during those characterized by the development and activation of Th2 cells and IL-4 secretion. The association between sIL-4R and IL-4 synthesis is consistent with a potential role for sIL-4R in the regulation of IL-4 activity in vivo.

Production of soluble IL-4 receptors by murine spleen cells is regulated by T cell activation and IL-4.

Many cytokine receptors exist naturally as both membrane-bound and soluble forms. Whereas the membrane receptors have an obvious role in signal transduction, the putative immunoregulatory role played by the soluble receptors remains unclear. Although natural forms of soluble IL-4R (sIL-4R) are known to be present in the biologic fluids of normal mice, the mechanisms regulating the production of sIL-4R have not been characterized. In this study, we have developed an ELISA that allows the measurement of sIL-4R without interference from endogenous IL-4, and have analyzed the effect of cellular activation and several cytokines on the secretion of sIL-4R by murine splenic cells. Although normal spleen cells in culture produced low, but detectable levels of sIL-4R under basal conditions, stimulation with the T cell-mitogens, Con A or soluble anti-CD3 antibodies, caused a 10- to 40-fold increase in the production of sIL-4R. Stimulation of B lymphocytes with LPS, however, did not result in significant up-regulation of sIL-4R secretion. Moreover, IL-4, but not other cytokines, was also a potent inducer of sIL-4R production by spleen cells, even in the absence of other stimuli. Blocking experiments with an anti-IL-4 antibody, 11B11, demonstrated that the effect of T cell-mitogens is partially mediated by endogenously produced IL-4. Cell depletion experiments suggested that although the effect of T cell-mitogens was dependent on the presence of viable T cells, all major cell types including T cells, B cells, and macrophages, either resting or activated, were able to up-regulate their secretion of sIL-4R in response to IL-4. Unlike many activities of IL-4, the secretion of sIL-4R by IL-4-stimulated splenic cells was not antagonized by IFN-gamma. These results suggest that the production of sIL-4R is regulated by stimuli leading to T cell activation and IL-4 secretion and are consistent with sIL-4R having an important role in the regulation of IL-4 activity in vivo.