RIPK1- and RIPK3-induced cell death mode is determined by target availability.

Both receptor-interacting protein kinase 1 (RIPK1) and RIPK3 can signal cell death following death receptor ligation. To study the requirements for RIPK-triggered cell death in the absence of death receptor signaling, we engineered inducible versions of RIPK1 and RIPK3 that can be activated by dimerization with the antibiotic coumermycin. In the absence of TNF or other death ligands, expression and dimerization of RIPK1 was sufficient to cause cell death by caspase- or RIPK3-dependent mechanisms. Dimerized RIPK3 induced cell death by an MLKL-dependent mechanism but, surprisingly, also induced death mediated by FADD, caspase 8 and RIPK1. Catalytically active RIPK3 kinase domains were essential for MLKL-dependent but not for caspase 8-dependent death. When RIPK1 or RIPK3 proteins were dimerized, the mode of cell death was determined by the availability of downstream molecules such as FADD, caspase 8 and MLKL. These observations imply that rather than a 'switch' operating between the two modes of cell death, the final mechanism depends on levels of the respective signaling and effector proteins.

Infection and sudden unexpected death in infancy: a systematic retrospective case review.

BACKGROUND: The cause and mechanism of most cases of sudden unexpected death in infancy (SUDI) remain unknown, despite specialist autopsy examination. We reviewed autopsy results to determine whether infection was a cause of SUDI. METHODS: We did a systematic retrospective case review of autopsies, done at one specialist centre between 1996 and 2005, of 546 infants (aged 7-365 days) who died suddenly and unexpectedly. Cases of SUDI were categorised as unexplained, explained with histological evidence of bacterial infection, or explained by non-infective causes. Microbial isolates gathered at autopsy were classified as non-pathogens, group 1 pathogens (organisms usually associated with an identifiable focus of infection), or group 2 pathogens (organisms known to cause septicaemia without an obvious focus of infection). FINDINGS: Of 546 SUDI cases, 39 autopsies were excluded because of viral or pneumocystis infection or secondary bacterial infection after initial collapse and resuscitation. Bacteriological sampling was done in 470 (93%) of the remaining 507 autopsies. 2079 bacteriological samples were taken, of which 571 (27%) were sterile. Positive cultures yielded 2871 separate isolates, 484 (32%) of which showed pure growth and 1024 (68%) mixed growth. Significantly more isolates from infants whose deaths were explained by bacterial infection (78/322, 24%) and from those whose death was unexplained (440/2306, 19%) contained group 2 pathogens than did those from infants whose death was explained by a non-infective cause (27/243, 11%; difference 13.1%, 95% CI 6.9-19.2, p<0.0001 vs bacterial infection; and 8.0%, 3.2-11.8, p=0.001 vs unexplained). Significantly more cultures from infants whose deaths were unexplained contained Staphylococcus aureus (262/1628, 16%) or Escherichia coli (93/1628; 6%) than did those from infants whose deaths were of non-infective cause (S aureus: 19/211, 9%; difference 7.1%, 95% CI 2.2-10.8, p=0.005; E coli: 3/211, 1%, difference 4.3%, 1.5-5.9, p=0.003). INTERPRETATION: Although many post-mortem bacteriological cultures in SUDI yield organisms, most seem to be unrelated to the cause of death. The high rate of detection of group 2 pathogens, particularly S aureus and E coli, in otherwise unexplained cases of SUDI suggests that these bacteria could be associated with this condition.

Alkynylcyclohexanol chairs and twist-boats: Co(2)(CO)(6) as a conformational switch.

Treatment of 1-[axial]-(trimethylsilylethynyl)cyclohexan-1-ol with dicobalt octacarbonyl results in a conformational ring flip such that the bulky dicobalt-alkyne cluster moiety now occupies the favored equatorial site. However, when a 4-tert-butyl substituent is present, the coordinated alkynyl group retains its original axial or equatorial position. Complexation of trans-[diaxial]-1,4-bis(triphenylsilylethynyl)cyclohexane-1,4-diol brings about a chair-to-chair conformational inversion such that both cluster fragments now occupy equatorial sites. In contrast, cis-1,4-bis(triphenylsilylethynyl)cyclohexane-1,4-diol reacts with Co(2)(CO)(8) to yield the twist-boat conformer in which the two axial hydroxy substituents exhibit intra-molecular hydrogen bonding. Likewise, the corresponding reaction of cis-1,4-bis(trimethylsilylethynyl)cyclohexane-1,4-diol with Co(2)(CO)(8) leads to a twist-boat, but in this case, the molecules are linked through inter-molecular hydrogen bonds. Eight of these cobalt clusters have been characterized by X-ray crystallography, and the potential use of twist-boats in synthesis is discussed.

Regional differences in health care delivery: implications for a national resource allocation formula.

In several countries formulae for allocating resources to regions are derived using national average relationships between population characteristics and health service use. However, there may be significant regional heterogeneity in health care delivery which, has two main implications for a national resource allocation formula. First, it offers alternative ways of measuring the relative needs of different population groups. Since the primary focus of research and policy is on the difficulty of targeting resources at high-need populations, it is proposed that progressivity in the delivery of health care could be seen as a frontier problem analogous to efficiency. The effects of using the slope parameters from the most progressive region are simulated. Second, regional heterogeneity may thwart the objective of the formula of securing equitable use of resources by different population groups. An adjustment mechanism is developed to illustrate the trade-off between the levels of geographical and vertical equity achieved. A locus of equity possibilities for acute care in Scotland is derived. Traditional formulae represent a corner solution indicating extreme relative aversion to geographical inequity. Because regional variation in need dominates regional variation in progressivity in Scotland, high-need rather than progressive regions gain from the pursuit of vertical equity.

Does waiting matter? A randomized controlled trial of new non-urgent rheumatology out-patient referrals.

OBJECTIVE: To examine the effect of waiting times on the health status of patients referred for a non-urgent rheumatology opinion. METHODS: The study was a randomized controlled clinical study evaluating a 'fast track' appointment with a 6-week target waiting time against an 'ordinary' appointment in the main city out-patient clinic of the rheumatology service for the Lothian and Borders region (population approximately 1 million). Health status was measured using the SF12 physical and mental summary component T-scores and pain was measured with a 100 mm visual analogue pain scale. Secondary outcomes were health utility and perceived health both measured with the EuroQol instrument, mental health measured with the Hospital Anxiety and Depression scale, disability with the modified Health Assessment Questionnaire and economic costs measured from a societal perspective. RESULTS: Mean waiting times were 43 days (sigma = +/-16) and 105 days (sigma = +/-51) for 'fast track' and 'ordinary' appointments, respectively. Both groups showed significant improvements in mean [95% confidence interval (CI)] scores for pain: 11 (7, 16)(P < 0.001); physical health status: 4 (2, 5) (P < 0.001); mental health status: 2 (0.1, 4) (P < 0.02); and health utility: 0.11 (0.07, 0.16) (P < 0.001) by the end of the 15-month period of the study, but there was no significant difference between either arm of the study. CONCLUSIONS: Rationing by delay was not detrimental to either mental or physical health and patients in both arms of the study showed significant and similar improvement in health by 15 months. Expenditure of resources on waiting times without regard to clinical outcomes is likely to be wasteful and additional resources should be directed at achieving the greatest clinical benefit. More research into effective methods of controlling demand and better identification of those who would benefit from access to specialist care is needed.

The hidden cost of clinical audit: a questionnaire study of NHS staff.

OBJECTIVES: To determine the full cost of clinical audit in one health board area and extrapolate the result of Scotland. METHODS: A questionnaire was sent ot a representative sample of NHS staff to determine time spent on clinical audit. This was combined with cost data from clinical audit budgets and unit cost data for staff time. RESULTS: Seventy-two percent of staff participated in clinical audit at some point in time. Medical staff were significantly more likely to participate in audit than non medical staff (P <0.0001). Those who participated in clinical audit devoted only a small proportion of time to it. However, due to the high participation rates in clinical audit, this aggregated to a significant amount. In Forth Valley the total cost was estimated to be pound 1.72m (pound 1.37m-pound 2.10m) and in Scotland pound 36.3m (pound 29.6m-pound 44.0m). Staff time accounted for over 80% of the total cost of clinical audit. CONCLUSIONS: Clinical audit is widespread within the Scotish NHS and the total cost of staff time devoted to audit is substantial. Research is needed into the value of clinical audit and the potential cost implications of clinical governance need to be explicitly recognised.

Independent SH2-binding sites mediate interaction of Dok-related protein with RasGTPase-activating protein and Nck.

A murine embryonic cDNA library was screened for potential substrates of the Src family kinase, Lyn, using a phosphorylation-screening strategy. One cDNA that we identified encodes Dok-related protein (DokR), a protein with homology to p62(dok) (Dok), and members of the insulin receptor substrate-1 family of proteins. Analysis of murine tissue extracts with DokR-specific antisera revealed that DokR protein is expressed at highest levels in lymphoid tissues. Co-expression of a FLAG epitope-tagged form of DokR (FLAG-DokR) with Lyn in embryonic kidney 293T cells resulted in constitutive phosphorylation of FLAG-DokR on tyrosine residues and consequential physical association with RasGTPase-activating protein (GAP) and the Nck adaptor protein. Stimulation of BaF/3 hematopoietic cells co-expressing the epidermal growth factor (EGF) receptor tyrosine kinase and FLAG-DokR with EGF also induced phosphorylation of FLAG-DokR and promoted its association with GAP. Immunoprecipitation experiments using DokR-specific antibodies revealed an interaction between endogenous DokR and a 150-kDa protein that is tyrosine-phosphorylated in EGF-stimulated BaF/3 cells. The molecular basis of the interactions involving DokR with GAP and Nck was investigated using a novel glutathione S-transferase fusion protein binding assay and/or site-directed mutagenesis. Tandem SH2-binding sites containing Tyr-276 and Tyr-304 were shown to mediate binding of DokR to GAP, whereas Tyr-351 mediated the binding of DokR to Nck. These results suggest that DokR participates in numerous signaling pathways.

A new method for isolating tyrosine kinase substrates used to identify fish, an SH3 and PX domain-containing protein, and Src substrate.

We describe a method for identifying tyrosine kinase substrates using anti-phosphotyrosine antibodies to screen tyrosine-phosphorylated cDNA expression libraries. Several potential Src substrates were identified including Fish, which has five SH3 domains and a recently discovered phox homology (PX) domain. Fish is tyrosine-phosphorylated in Src-transformed fibroblasts (suggesting that it is a target of Src in vivo) and in normal cells following treatment with several growth factors. Treatment of cells with cytochalasin D also resulted in rapid tyrosine phosphorylation of Fish, concomitant with activation of Src. These data suggest that Fish is involved in signalling by tyrosine kinases, and imply a specialized role in the actin cytoskeleton.

HDL and apolipoprotein A-I protect erythrocytes against the generation of procoagulant activity.

The appearance of anionic lipids on the extracellular surface of cells is required for the formation of the procoagulant complex that leads to the activation of prothrombin. Procoagulant activity would be expected to be inhibited by substances that stabilize the membrane structure and hence inhibit the transbilayer diffusion of phosphatidylserine from the cytoplasmic to the extracellular surface of the plasma membrane. The generation of procoagulant activity in human erythrocytes by A23187 and Ca2+ is inhibited by apolipoprotein A-I, its amphipathic peptide analogues, and high-density lipoprotein (HDL). These agents do not inhibit the Ca2+ loading of erythrocytes by A23187, nor do they inhibit the activation of prothrombin once the cells have been incubated at 37 degrees C with A23187 and Ca2+. Transbilayer diffusion of fluorescently labeled phosphatidylserine is inhibited by apolipoprotein A-I. These findings indicate that class A amphipathic helixes as well as lipoprotein particles and liposomes inhibit the transbilayer diffusion of phospholipids and procoagulant activity. This activity may contribute to the protective role of HDL against arteriosclerosis and thrombosis.

Complex binding of leukemia inhibitory factor to its membrane-expressed and soluble receptors.

The complex interaction of leukemia inhibitory factor (LIF) with its specific receptor present on the cell surface, in isolated membranes and in solution, has been examined in detail. Several aspects of this complexity have been highlighted, including the presence of high- and low-affinity murine LIF receptors, biphasic dissociation of human LIF from apparently homogeneous high- or low-affinity human LIF receptors, and unusual species cross-reactivity. The unusual species cross-reactivity observed between murine and human LIF has also been exploited to map an important receptor binding epitope on human LIF.

Cross-species receptor binding characteristics of human and mouse leukemia inhibitory factor suggest a complex binding interaction.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine whose activities appear to be mediated through a single heterodimeric receptor complex. Human LIF (hLIF) can bind to and activate mouse LIF (mLIF) receptors but mLIF is unable to bind to hLIF receptors. Cross-species competition of mLIF and hLIF for binding to the mLIF receptor was found to be dependent on which ligand was used as the radioactive tracer (Layton, M. J., Cross, B. A., Metcalf, D., Ward, L. D., Simpson, R. J., and Nicola, N. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8616-8620), and this phenomenon was investigated in the present study. We found that hLIF bound to the low affinity mLIF receptor with a 100-500-fold higher primary affinity and lower kinetic dissociation rate than mLIF, but both ligands displayed a single rate of ligand dissociation. In contrast, the binding of hLIF to low and high affinity hLIF receptors revealed two classes of binding site. The observed tracer-dependent phenomena suggested that both mLIF and hLIF interfere with the binding of each other to the mLIF receptor. A model is presented in which hLIF binds to two sites on mLIF and hLIF receptors, one of which interferes with the common site for mLIF. This model may reconcile some of the observed complexities of LIF/LIF receptor interactions.

CD4-mediated enhancement or inhibition of T cell activation does not require the CD4:p56lck association.

CD4 is the coreceptor molecule expressed on the surface of T cells specific for or restricted by class II molecules of the major histocompatibility complex (MHC). Its expression on T cells is required for an optimal response to antigen (Ag). Three mechanisms have been invoked for the involvement of CD4 in T cell activation. First, it was shown that CD4 binds to MHC class II molecules on antigen presenting cells (APCs) thereby favoring an adhesion between effector cells and APCs. Association of CD4 to the T cell receptor and to the tyrosine kinase p56lck have also been shown to be critically involved in the positive function of CD4. Here, we demonstrate that the interaction of CD4 with p56lck is not required to enhance the response of two CD4-dependent, Ag-specific T cell hybridomas. Mutant forms of CD4 (TCD4), which lose association to p56lck, were expressed in these T cells and were shown to enhance the Ag-specific response as efficiently as the wild-type CD4. Moreover both CD4-dependent and independent T cell responses were inhibited by CD4-specific mAbs even when CD4 was not associated with p56lck. These results indicate that mechanisms distinct from sequestration of p56lck and/or negative signaling operate in these inhibitions. Results demonstrating enhancement of TCR-mediated signaling by the coaggregation of TCD4 mutant to the TCR further confirm that the association of p56lck to CD4 is not absolutely required for the regulatory functions of CD4. Our results suggest that the mechanisms implicated in the enhancement of T cell stimulation via CD4 depend solely on the extracellular and transmembrane domains of CD4.

Receptor insertion into factor-dependent murine cell lines to develop specific bioassays for murine G-CSF and M-CSF and human GM-CSF.

cDNAs encoding the receptors for murine G-CSF, M-CSF or human GM-CSF were inserted into the murine hemopoietic continuous cell lines Ba/F3 or FDC-P1 and sublines selected that were then responsive to proliferative stimulation by these growth factors. When used in microwell assays the Ba/F3 G-CSF receptor-expressing cell line was able to detect 100 pg G-CSF per ml, the Ba/F3 M-CSF receptor-expressing cell line 100-400 pg M-CSF per ml and the FDC-P1 line expressing the alpha- and beta-chains of the human GM-CSF receptor detected 5-10 pg/ml of GM-CSF in test material. These cell lines appear satisfactory for use as selective bioassays for these colony stimulating factors in material potentially containing a mixture of growth factors.

Reliability of the Spanish version of the Nottingham Health Profile in patients with stable end-stage renal disease.

OBJECTIVE: Since reproducibility of results is a basic prerequisite of health status measures for its use in prospective and evaluative studies, the reliability of the Spanish version of the Nottingham Health Profile (NHP), a multi-dimensional perceived health status measure, was assessed in a sample of stable end-stage renal disease (ESRD) patients. METHODS: The NHP was administered on two occasions four weeks apart to a group of hospital hemodialysis program patients who were clinically stable according to their physicians. Correlations of scores and agreement of first and second administrations were assessed together with internal consistency. Afterwards, analyses were repeated taking into account the time (before, during or after the dialysis) and the method of administration (self vs interviewer), and the interviewer. RESULTS: Spearman correlation coefficients (rs) between responses to the first and to the second administration were > 0.6 for all of the six dimensions of the NHP (range = 0.69-0.85) and in every sub-group analyzed (P < 0.01). Agreement percent (AP) between items was > 0.4 (0.48-0.65). Internal consistency was 0.91 for the whole profile and > 0.5 (0.58-0.86) when analyzed by individual dimensions. Reliability did not vary significantly with the time nor the method of administration (self or interviewer). CONCLUSIONS: Overall, results suggest that the Spanish version of NHP is sufficiently reliable to be used in ESRD patients. While a higher reliability would have been achieved by a shorter retest period, the study provides a realistic approximation to the reliability of the questionnaire in actual research and clinical applications.

Histidine-367 of the human common beta chain of the receptor is critical for high-affinity binding of human granulocyte-macrophage colony-stimulating factor.

High-affinity receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3, and interleukin 5 consist of ligand-specific alpha chains (low-affinity subunits) and a common beta chain (beta c) that converts each complex to a high-affinity form. Although beta c alone has no detectable cytokine-binding activity, amino acid substitutions for Glu-21 of human GM-CSF significantly reduce high-affinity but not low-affinity binding, implying that beta c interacts directly with GM-CSF during formation of the high-affinity receptor but only in the presence of the alpha chain. A potential GM-CSF-binding determinant was identified in the second hemopoietin domain of beta c, and the role of individual residues within this region was investigated by determining the ability of mutated beta c chains to confer high-affinity binding when coexpressed with the alpha subunit of the GM-CSF receptor in COS cells. Substitutions involving Met-363, Arg-364, Tyr-365, and Glu-366 did not affect high-affinity binding. However, substitution of His-367 by lysine or glutamine abolished high-affinity binding, suggesting that this residue may form an important part of the high-affinity GM-CSF-binding determinant. Consistent with the loss of high-affinity binding, higher concentrations of human GM-CSF were required to stimulate proliferation of CTLL-2 cell lines transfected with cDNAs for GM-CSF receptor alpha chain and His-367 beta c mutant than those expressing GM-CSF receptor alpha subunit and beta c wild type.

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF-R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins.

Lipopolysaccharide- and interferon-gamma-induced expression of hck and lyn tyrosine kinases in murine bone marrow-derived macrophages.

We have examined the role of tyrosine phosphorylation during the course of macrophage activation. Initial experiments indicated that vanadate, a known phosphotyrosine phosphatase inhibitor, enhanced the phorbol 12-myristate 13-acetate (PMA)-triggered respiratory burst and potentiated the priming effects of bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), suggesting that tyrosine phosphorylation may be important in these end cell functions. As src-related kinases have been implicated in the activation of cells of other haemopoietic lineages, we examined the relationship between the activity of two such kinases, hck and lyn, and priming of the respiratory burst. We found that the level of hck and lyn is increased following exposure of bone marrow-derived macrophages (BMM) to LPS or IFN-gamma. The induction of both of these kinases follows similar kinetics with maximal activity occurring at 24-48 h. Interestingly, the kinetics of induction of hck and lyn kinase activity in BMM demonstrated a close temporal relationship with the priming effects of LPS and IFN-gamma on the macrophage respiratory burst. Collectively, these observations raise the possibility that modulation of expression of hck and lyn is involved in the regulation of the respiratory burst.

Two isoforms of murine hck, generated by utilization of alternative translational initiation codons, exhibit different patterns of subcellular localization.

Mammalian hck, a member of the src family of tyrosine kinases, is expressed predominantly in cells of the myeloid and B-lymphoid lineages. Using mutational analysis, we have investigated the molecular basis of two immunoreactive forms of murine hck of 56 and 59 kDa found in numerous hemopoietic cell types. Our results indicate that translation of murine p59hck initiates from a CTG codon located 21 codons 5' of an ATG that is utilized to generate p56hck. We provide evidence that two human hck isoforms are generated by the same mechanism. Subcellular fractionation studies reveal that while p59hck and p56hck are associated with membranes of various murine B-lymphoid and myeloid cell lines, p59hck alone is also located in the cytosol. In contrast to membrane-associated p59hck, which is metabolically labeled with [3H]myristic acid and exhibits amphiphilic properties in Triton X-114 detergent, cytosolic p59hck is hydrophilic, suggesting that it is not acylated. Possible mechanisms are proposed to account for these observations.