Accurate test of chiral dynamics in the γp→π0p reaction.

A precision measurement of the differential cross sections dσ/dΩ and the linearly polarized photon asymmetry Σ≡(dσ⊥-dσ∥)/(dσ⊥+dσ∥) for the γp→π0p reaction in the near-threshold region has been performed with a tagged photon beam and almost 4π detector at the Mainz Microtron. The Glasgow-Mainz photon tagging facility along with the Crystal Ball/TAPS multiphoton detector system and a cryogenic liquid hydrogen target were used. These data allowed for a precise determination of the energy dependence of the real parts of the S- and all three P-wave amplitudes for the first time and provide the most stringent test to date of the predictions of chiral perturbation theory and its energy region of agreement with experiment.

Development of an instrument to measure face validity, feasibility and utility of patient questionnaire use during health care: the QQ-10.

OBJECTIVE: To develop and establish the psychometric properties of an instrument to measure face validity, feasibility and utility of patient questionnaires used during health care. DESIGN: Our instrument, QQ-10, is a 10-item self-completed questionnaire, which was developed during the evaluation of another questionnaire (ePAQ-PF), to assess patients' views on questionnaire use during health care. SETTING: Urogynaecology Department, Royal Hallamshire Hospital, Sheffield, UK. PARTICIPANTS: The Sheffield maternity patient user group identified 10 key themes relating to patients' views on using questionnaires; these themes translated into 10 statements, each using the same 5-point Likert response scale. INTERVENTION: Not applicable. Outcome Measures Principal component analysis established the factor structure of our instrument. Internal reliability was assessed using Cronbach's alpha. Construct validity was assessed using Spearman's rho. RESULTS: Factor analysis yielded two meaningful factors: Value and Burden, both achieving Cronbach's alpha scores >0.7. Significant correlations were found between scores for Value and communication experience and between scores for Burden and barriers to health care. CONCLUSIONS: Our instrument offers a valid, reliable measure of patients' views relating to value and burden of using health-related quality of life questionnaires. Its two domains show good internal reliability and with its free text items, it may offer a valuable, standardized assessment of face validity and utility of other questionnaires used in health care.

Human neutrophils have a novel purinergic P2-type receptor linked to calcium mobilization.

Stimulation of suspensions of fura-2-loaded human neutrophils with ATP resulted in an elevation in cytosolic free calcium concentration ([Ca2+]i) from a basal value of 0.1 microM to a transient peak of 1 microM. The response is due to Ca2+ release from intracellular stores and influx of extracellular Ca2+. Release from intracellular stores is shown by the response in the absence of extracellular Ca2+. The greater and more maintained response in the presence of extracellular Ca2+ is indicative of stimulated Ca2+ entry and a stimulated influx pathway was confirmed by using Mn2+ as a surrogate for Ca2+. A variety of purinergic agonists were used to characterize the pharmacology of this [Ca2+]i response. Their rank order of potency was ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) greater than ADP much greater than 2-methylthioadenosine 5'-triphosphate (2Me-SATP), where ATP had an EC50 value of 3 microM and 2MeSATP had an EC50 value of 1000 microM. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), adenylyl (alpha,beta-methylene)- diphosphonate (AMPCPP) and adenosine were inactive at 1 mM. These results suggest that neutrophils have a novel type of purinergic P2 receptor that is neither P2x nor P2y.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.

Diffuse interstitial pneumonitis. Clinicopathologic correlations in 20 patients treated with prednisone/azathioprine.

Twenty patients with diffuse interstitial pulmonary disease diagnosed by open lung biopsy received combined prednisone/azathioprine therapy. Twelve patients demonstrated improvement with therapy. Each patient's clinical presentation, roentgenologic features and pathologic findings were correlated with their therapeutic response. Patients with an illness of one year's duration or less had a more favorable response to therapy than patients with a greater than two year duration of illness. Patients with associated extrathoracic abnormalities (anemia, glomerulitis, hepatopathy) exhibited a better therapeutic response that those with only pulmonary disease. The biopsy material from each patient was quantitatively graded on 20 morphologic variables. Statistical analysis using multiple linear regression revealed that a single variable, degree of interstitial fibrosis, was more that 90 per cent accurate in separating those responsive to therapy from those who failed to respond. Patients who respond to treatment had less interstitial fibrosis. Neither the amount of alveolar septal inflammation nor intra-alveolar cellular reaction was discriminatory in predicting response to therapy. A beneficial response to therapy was reflected in both improved lung volumes and gas exchange. Eight patients appeared to have a selective beneficial effect from azathioprine.

The effects of siguazodan, a selective phosphodiesterase inhibitor, on human platelet function.

1. The effects of siguazodan (SK&F 94836) a selective phosphodiesterase (PDE) inhibitor with inotropic and vasodilator activity, were studied on human platelets. 2. Siguazodan selectively inhibited the major cyclic AMP-hydrolysing PDE in human platelet supernatants. The inhibited enzyme has been variously termed cyclic GMP-inhibited PDE or PDE-III. 3. In platelet-rich plasma (PRP), siguazodan inhibited U46619-induced aggregation more potently than that induced by ADP and collagen. Treatment of the PRP with aspirin had no effect on the potency of siguazodan. 4. In washed platelets, siguazodan increased cyclic AMP levels and reduced cytoplasmic free calcium [( Ca2+]i). ADP decreased the ability of siguazodan to raise cyclic AMP and this may explain its lower potency in inhibiting responses to ADP. 5. Siguazodan has anti-platelet actions over the same concentration range that it is an inotrope and vasodilator.

Selective binding of the truncated form of the chemokine CKbeta8 (25-99) to CC chemokine receptor 1(CCR1).

Human CC chemokine receptor 1 (CCR1) has been proposed as a receptor for CKbeta8. To obtain conclusive evidence, binding-displacement studies of 125I-CKbeta8 (25-99) were performed on membranes of Chinese hamster ovary cells expressing human CCR1. The Ic50 for displacement of 125I-CKbeta8 (25-99) with CKbeta8 (25-99) was 0.22 nM. The longer forms of CKbeta8 (24-99 and 1-99) also displaced 125I-CKbeta8, with Ic50 values of 6.5 and 16 nM, respectively. Displacement profiles of 125I-CKbeta8 (25-99) on freshly prepared human monocytes indicated that CCR1 was the major receptor for CKbeta8. We conclude that CCR1 is a receptor for different-length CKbeta8 and that CKbeta8 (25-99) has a similar affinity for CCR1 as macrophage inflammatory protein-1alpha (MIP-1alpha). The longer variants of CKbeta8 are significantly less potent than CKbeta8 (25-99) and MIP-1a on CCR1 and monocytes (P < 0.05).

The role of fractalkine in the recruitment of monocytes to the endothelium.

Recombinant fractalkine possesses both chemoattractive and adhesive properties in vitro. Previous studies have demonstrated an upregulation of this molecule on the membranes of activated human endothelial cells and hypothesised that fractalkine plays a role in the recruitment and adherence of monocytes to the activated endothelium. Here we present data analysing both the adhesive and chemoattractive properties of this chemokine expressed by activated human umbilical vein endothelial cells. We demonstrate that both recombinant fractalkine and endogenously produced fractalkine function as adhesion molecules, tethering monocytes to the endothelium. However, our data demonstrate that although recombinant fractalkine has the potential to function as a potent monocyte chemoattractant, the endogenous fractalkine cleaved from activated human umbilical vein endothelial cells is not responsible for the observed chemotaxis in this model. Instead, we show that monocyte chemoattractant protein-1 (MCP-1), secreted from the activated human umbilical vein endothelial cells, is responsible for the chemotaxis of these monocytes.

Cortical network dynamics during verbal working memory function.

This study is an exploratory investigation of the regional timing of cortical activity associated with verbal working memory function. ERP activity was obtained from a single subject using a 124-channel sensor array during a task requiring the monitoring of imageable words for occasional targets. Distributed cortical activity was estimated every 2.5 ms with high spatial resolution using real head, boundary element modelling of non-target activity. High-resolution structural MRI was used for segmentation of tissue boundaries and co-registration to the scalp electrode array. The inverse solution was constrained to the cortical surface. Cortical activity was observed in regions commonly associated with verbal working memory function. This included: the occipital pole (early visual processing); the superior temporal and inferior parietal gyrus bilaterally and the left angular gyrus (visual and phonological word processing); the dorsal lateral occipital gyrus (spatial processing); and aspects of the bilateral superior parietal lobe (imagery and episodic verbal memory). Activity was also observed in lateral and superior prefrontal regions associated with working memory control of sensorimotor processes. The pattern of cortical activity was relatively stable over time, with variations in the extent and amplitude of contributing local source activations. By contrast, the pattern of concomitant scalp topography varied considerably over time, reflecting the linear summation effects of volume conduction that often confound dipolar source modelling.

Lipoxin A4 elevates cytosolic calcium in human neutrophils.

Lipoxin A4 (LXA4), a lipoxygenase-derived metabolite of arachidonic acid, stimulated a dose-dependent elevation in cytosolic free Ca2+ concentration, [Ca2+]i, in fura-2-loaded human neutrophils, with an EC50 of 0.4-0.5 microM. The time for [Ca2+]i to peak was also dose-dependent. In the presence of extracellular Ca2+ (CaDT-PA added), the rise in [Ca2+]i was due to a combination of Ca2+ release from internal stores and influx of extracellular Ca2+. In the absence of extracellular Ca2+, the rise in [Ca2+]i was due to release from internal stores, which then became depleted. No response to LXA4 was seen in the absence of divalent cation chelators (EGTA or DTPA); this is presumably because LXA4 forms an inactive complex with heavy metal cations. In the presence of extracellular Ca2+, LXA4 had no effect on the subsequent response of neutrophils to the chemotactic peptide fmetleu-phe (fmlp). In the absence of extracellular Ca2+, LXA4 dose-dependently reduced the subsequent response of neutrophils to fmlp; this is presumably because LXA4 discharges the store, and so reduces the amount of Ca2+ available for subsequent release by fmlp.

Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+.

A description is given of the methodology, and problems encountered, for the use of a new fluorescent Ca2(+)-indicator dye, fluo-3, in neutrophils and platelets. The higher Kd and longer excitation wavelength of fluo-3 can have significant advantages over fura-2. Although neutrophils and platelets are used as examples, these observations will be applicable to other cell types. The Kd of fluo-3 for binding Ca2+ at 37 degrees C was measured and found to be 864 nM; the previously published value was 400 nM at 22 degrees C. The Kd of fluo-3, like that of fura-2, is therefore very temperature-dependent. Protocols for loading cells, and preventing leakage of fluo-3, are described; probenecid, known to inhibit fura-2 leakage from cells, was found to be essential to get good fluo-3 signals from platelets. Calibration of fluo-3 fluorescence signals to [Ca2+] and methods for obtaining maximum and minimum fluorescence signals are described; these methods differ from those used with fura-2. Agonist-stimulated responses of fluo-3-loaded neutrophils and platelets are shown, and the calculated cytosolic [Ca2+] is comparable with that previously obtained with fura-2. Responses of cells in the presence of plasma are also shown; such measurements, unobtainable with quin2, fura-2 or indo-1, are possible with fluo-3, owing to its longer excitation wavelengths. Co-loading of cells with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid and fluo-3 is included as an example of how cytosolic [Ca2+] can be buffered and manipulated. Many of these observations will be of value when using fluo-3 (or other Ca2(+)-indicator dyes) in most cell types.

Fractalkine cleavage from neuronal membranes represents an acute event in the inflammatory response to excitotoxic brain damage.

Fractalkine is a recently identified chemokine that exhibits cell adhesion and chemoattractive properties. It represents a unique member of the chemokine superfamily because it is located predominantly in the brain in which it is expressed constitutively on specific subsets of neurons. To elucidate the possible role of neuronally expressed fractalkine in the inflammatory response to neuronal injury, we have analyzed the regulation of fractalkine mRNA expression and protein cleavage under conditions of neurotoxicity. We observed that mRNA encoding fractalkine is unaffected by experimental ischemic stroke (permanent middle cerebral artery occlusion) in the rat. Similarly, in vitro, levels of fractalkine mRNA were unaffected by ensuing excitotoxicity. However, when analyzed at the protein level, we found that fractalkine is rapidly cleaved from cultured neurons in response to an excitotoxic stimulus. More specifically, fractalkine cleavage preceded actual neuronal death by 2-3 hr, and, when evaluated functionally, fractalkine represented the principal chemokine released from the neurons into the culture medium upon an excitotoxic stimulus to promote chemotaxis of primary microglial and monocytic cells. We further demonstrate that cleavage of neuron-derived, chemoattractive fractalkine can be prevented by inhibition of matrix metalloproteases. These data strongly suggest that dynamic proteolytic cleavage of fractalkine from neuronal membranes in response to a neurotoxic insult, and subsequent chemoattraction of reactive immune cells, may represent an early event in the inflammatory response to neuronal injury.

Involvement of calcium in modulation of neutrophil function by phorbol esters that activate protein kinase C isotypes and related enzymes.

In this study, the effects of a series of phorbol esters with different spectra of biological activities and different patterns of activation of the isoenzymes of protein kinase C (PKC) have been studied in human neutrophils. The aim was to gain more information on which isoenzymes of PKC are involved in neutrophil activation, specifically inhibition of fMet-Leu-Phe (fMLP)-stimulated bivalent cation influx and stimulation of O2-. release (either alone or potentiation of the response to fMLP). Prior addition of both phorbol 12-myristate 13-acetate (PMA) and sapintoxin A (SAPA) inhibited fMLP-stimulated Mn2+ influx. Higher concentrations of resiniferatoxin (RX) were also inhibitory, inhibition being more apparent at longer preincubation times. However, 12-deoxyphorbol 13-O-phenylacetate (DOPPA) showed only a slight inhibitory effect and required a prolonged preincubation. PMA, SAPA and RX, but not DOPPA, stimulated O2-. release by themselves. Lower concentrations of PMA, SAPA and RX, which were ineffective alone, considerably potentiated O2-. release stimulated by fMLP, whereas DOPPA had little or no effect. These results rule out a major role for PKC-delta (not activated by SAPA) and PKC-beta 1 (activated by DOPPA), but suggest the involvement of RX kinase in addition to PKC in the inhibition of fMLP-stimulated Mn2+ influx and potentiation of fMLP-stimulated O2-. release. However, when the cytosolic free Ca2+ concentration ([Ca2+]i) was elevated with the Ca2+ ionophore ionomycin, DOPPA was able to stimulate O2-. release, which probably reflects the known Ca2+ requirement for activation of PKC-beta 1 by DOPPA in vitro. The effects of the other phorbols were also enhanced when [Ca2+]i was elevated; all of the phorbols synergize, to variable extents, with Ca2+ to activate PKC in vitro. Enhancement of RX-stimulated O2- release by elevation of [Ca2+]i was unexpected, since RX kinase has been reported to be inhibited by high concentrations of Ca2+ in vitro. Finally, use of fura-2 and SK&F 96365 to manipulate the fMLP-stimulated rise in [Ca2+]i showed that when fMLP was able to evoke its normal rise in [Ca2+]i (to a peak of 700-900 nM), O2-. release was potentiated by PMA, SAPA and RX. However, when fMLP was only able to evoke a small increase in [Ca2+]i (to a peak of 400 nM), potentiation by PMA was unaffected but potentiation by SAPA and RX was considerably reduced. This observation agrees with published data demonstrating that activation of PKC in vitro by SAPA is more Ca(2+)-dependent than activation by PMA.(ABSTRACT TRUNCATED AT 400 WORDS)