Novel C5a receptor antagonists regulate neutrophil functions in vitro and in vivo.

Novel recombinant human C5a receptor antagonists were discovered through modification of the C terminus of C5a. The C5a1-71T1M,C27S,Q71C monomer, (C5aRAM; CGS 27913), was a pure and potent functional antagonist. The importance of a C-terminal cysteine at position 71 to antagonist properties of C5aRAM was confirmed by studying C5a1-71 derivatives with replacements of Q71, C5a derivatives of various lengths (70-74) with C-terminal cysteines, and C5a derivatives of various lengths (71-74) with Q71C replacements. The majority of C5a1-71Q71 derivatives were agonists (C5a-like) in the human neutrophil C5a-induced intracellular calcium mobilization assay. The C5a1-71Q71C derivative was an antagonist. C5a derivatives of lengths 73 and 74 with C-terminal cysteines were agonists, while lengths 70 to 72 were antagonists. C5a derivatives of lengths 72, 73, and 74 with Q71C replacements were agonists, while, again, C5a1-71Q71C was an antagonist. C5aRAM and its adducts, including its dimer, C5aRAD (CGS 32359), were pure antagonists. Additionally, CSaRAM and CSaRAD inhibited binding of 125I-labeled recombinant human C5a to neutrophil membranes (Ki = 79 and 2 pM, respectively), C5a-stimulated neutrophil intracellular calcium mobilization (8 and 13 nM), CD11b integrin up-regulation (10 and 1 nM), superoxide generation (182 and 282 nM), lysozyme release (1 and 2 microM), and chemotaxis (11 and 7 microM). In vivo, intradermal injection of C5aRAM inhibited C5a-induced dermal edema in rabbits. Furthermore, a 5-mg/kg i.v. bolus of C5aRAD significantly inhibited C5a-induced neutropenia in micropigs when challenged with C5a 30 min after C5aRAD administration. C5aRAM and C5aRAD are novel, potent C5a receptor antagonists devoid of agonist or proinflammatory activity with demonstrated efficacy in vitro and in vivo.

Exposure of human vascular smooth muscle cells to Raf-1 antisense oligodeoxynucleotides: cellular responses and pharmacodynamic implications.

To characterize the pharmacodynamic properties of CGP 69846A/ISIS 5132, an antisense oligodeoxynucleotide directed against the mitogenic signal transducer Raf-1 kinase, we investigated the elicited biological responses in human coronary artery smooth muscle cells. Cell exposure to CGP 69846A resulted in a reversible time- and concentration-dependent down-regulation of cellular Raf-1 gene expression and, ultimately, inhibition of cell cycle progression. The highest potencies of this compound to reduce Raf-1 mRNA and protein levels were observed after 24 and 48 hr of cell exposure, respectively, with corresponding IC50 values of approximately 100 and approximately 300 nM. Proliferation was inhibited with an IC50 value of approximately 300 nM after 72 hr. We interpreted the recovery rate of Raf-1 mRNA after cell exposure to antisense ODNs as the half-life (t1/2 approximately 50 hr) of active intracellular CGP 69846A in our cell culture system. The endogenous Raf-1 turnover half-life of approximately 30 hr, as assessed by monitoring metabolically labeled Raf-1 protein, correlated kinetically with the antisense-induced protein decay rate (50% decay in approximately 33 hr), indicating that the efficiency of CGP 69846A in decreasing Raf-1 protein levels was rate-limited by the endogenous protein turnover rate. The pharmacodynamic effects of CGP 69846A antisense ODNs are therefore limited by the duration of its intracellular activity rather than by its ability to transiently decrease mRNA levels. Local steady state exposure to CGP 69846A may represent a new approach to prevent the transition of quiescent vascular smooth muscle cells into the pathologically hyperproliferating cells seen after angioplasty.

Medical decision support for remote general practitioners using telemedicine.

A telemedicine link was set up between the casualty department of a remote community hospital and the accident and emergency department of a large urban hospital. The telemedicine link comprised teleradiology, videoconferencing and telepresence. The system was connected by ISDN (128 kbit/s) and also by a satellite link (64 kbit/s). During a one-year clinical trial, 120 teleconsultations took place between the community hospital and the specialist trauma centre, 110 using ISDN and 10 using the satellite link. Teleradiology was used in 116 teleconsultations, videoconferencing in 76, and telepresence in four. Survey results indicated that both the general practitioners running the community hospital and accident and emergency consultants felt that teleconsultation had improved patient care. Communication between clinicians using the telemedicine link avoided the transfer of 70 patients, representing an estimated cost saving of Pounds 65,000.

Establishing a postgraduate qualification in remote health care.

The British Antarctic Survey (BAS) provides medical care for the scientists and support staff working on British scientific bases and research vessels in the Antarctic. The BAS directs significant resources towards medical research, so a doctor who does not complete the research component of the programme of training and medical duties represents a partially wasted investment. Additionally, the professional experience gained by the doctor is appropriate for a postgraduate qualification. For these reasons, the training, clinical placement and research undertaken by doctors were formalized as a masters degree in 1992. The objectives of the MSc degree were to optimize the benefits of the training and research for Antarctic doctors and their patients, and to improve the quality of the research output. In the three years before the degree was introduced, only 25% of doctors produced a useful research output. Following the introduction of the MSc, this figure rose to 88%.

Night-shift work in Antarctica: sleep characteristics and bright light treatment.

Changes in sleep parameters during and after night-shift and the effects of bright white (2500-3000 1x) and dim red (> 500 1x) light treatment on re adaptation after night-shift during winter were studied in 14 men on the British Antarctic Survey Base of Halley (75 degrees south). Subjects kept daily sleep diaries and mood ratings from one week before to three weeks after night-shift and received either full-spectrum white or dim red light treatment from 1100 to 1300 h daily during the first week after night-shift. Plasma melatonin (for 24 h at the end of weeks 1, 2 and 4), and urinary 6-sulfatoxymelatonin (aMT6s, for 48 h weekly) were measured. A significant (MANOVA; p < 0.05) improvement in sleep was seen during night shift (latency and duration) and with bright light treatment (latency). Melatonin and aMT6s rhythms delayed by 7-8 h during night-shift. The white light group readapted slowly, apparently by phase delay, as assessed by aMT6s measurement. The red light group readapted slightly, but significantly (ANOVA, p < 0.01) faster than the white light group.

Telemedicine in the British Antarctic Survey Medical Unit.

The system of telemedical care practised by the British Antarctic Survey has developed over a period of 50 years. It is a system that deals with everyday routine medical problems as well as occasional emergencies. It is tried and tested, but undergoes continual modification. Although the Antarctic stations represent a unique setting, the system has the potential for being adapted to many different situations, wherever there are small groups in remote areas needing medical backup. Initial telemedicine work conducted in the Antarctic has led to projects to improve primary care in Scottish communities some distance from specialist centres. As telecommunication links to the Antarctic stations improve, in future the lessons learnt in UK-based projects can be applied in the Antarctic. The evolutionary process will thus continue.

The pharmacophore of the human C5a anaphylatoxin.

We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.

LTB4-induced transient neutropenia in the rat: a model for evaluating efficacy and bioavailability of LTB4 receptor antagonists.

An animal model of leukotriene B4- (LTB4) induced neutropenia has been developed to evaluate LTB4 receptor antagonists in vivo. LTB4, a potent chemotactic inflammatory mediator, when administered intravenously, induces a profound, rapid, and transient redistribution of blood neutrophils from the circulating pool to the marginated pool. This phenomenon is applied in the neutropenia model whereby circulating blood neutrophil counts prior to and after intravenous infusion of LTB4 are compared. Kinetics of LTB4-induced neutrophil responses are determined through the use of a Technicon H*1 automated blood cell analyzer. LTB4 receptor antagonists are identified by inhibition of LTB4-induced neutropenia. Standard antiinflammatory compounds including BW-755C, Abbott A-64077 (zileuton), dexamethasone-21-acetate, indomethacin, and naproxen did not affect LTB4-induced neutropenia. A potent LTB4 receptor antagonist, designated "RPR," inhibited LTB4-induced neutropenia following oral administration in a dose-dependent fashion.

Characterization of the binding of Bolton-Hunter labeled [125I]C5a to human neutrophil, monocyte and U-937 cell membranes.

The fifth component of the complement cascade, C5a, was iodinated using the Bolton-Hunter reagent. Results from the present study, using the high affinity ligand, [125I]Bolton-Hunter-labeled C5a ([125I]BH-C5a), revealed a single binding site on membranes prepared from human neutrophils, U-937 cells and human monocytes. Saturation studies demonstrated Bmax values in these cells of 11.5, 47.3 and 16.6 fmol/10(6) cells, respectively. The C5a receptor demonstrated a very high affinity for [125I]BH-C5a of approximately 4 pM in each cell type. Competition studies using analogs of C5a generated a similar order of potency in each of the cell types of C5a > or = C5a(1-74), Ser66Ala > C5a(1-73) > C5a(1-69). These studies indicate that [125I]BH-C5a labels a similar receptor in neutrophil, U-937 cell and monocyte membranes. Furthermore, C5a(1-73) produced shallow inhibition curves in competition experiments in each cell type. Computer analysis of the binding data revealed two components of binding. When 10 nM unlabeled C5a was used to initiate dissociation of [125I]BH-C5a binding in neutrophil membranes, two binding components were observed. In addition, dissociation of [125I]BH-C5a binding by 10 nM unlabeled C5a in the presence of 1 mM GppNHp decreased the percentage of binding to the slowly dissociating, high affinity binding component from 84 to 58%. These results suggest that multiple states of the C5a receptor exist.

Platelet activating factor stimulates intracellular calcium transients in human neutrophils: involvement of endogenous 5-lipoxygenase products.

Stimulation of human neutrophils with platelet activating factor (PAF) resulted in a transient elevation of free cytosolic calcium. Neutrophils exhibited a two-component calcium response observed as a double peak when stimulated with greater than 5 nM PAF. In contrast, leukotriene B4 (LTB4), C5a, or formylmethionyl-leucyl-phenylalanine stimulated only a single-peak calcium response. The double-peak calcium response was not elicited in human monocytes or differentiated U937 cells, which demonstrated a single peak. Pretreatment of neutrophils with a 5-lipoxygenase inhibitor or a specific LTB4-receptor antagonist selectively blocked the second calcium peak. These results suggest that PAF-mediated activation of human neutrophils results in the activation of the 5-lipoxygenase and the subsequent generation of LTB4. This LTB4 in turn elicits a secondary rise in calcium, which contributes to the overall response of neutrophils of PAF. These results demonstrate how LTB4 participates in the cellular responses elicited by PAF in human neutrophils.

Relationship of blood markers to disease severity and drug efficacy in rat adjuvant arthritis.

Rat adjuvant arthritis (AA) was used as a model to evaluate several blood markers as possible predictive indicators of drug efficacy. AA was induced in Sprague-Dawley rats by the injection of complete Freund's adjuvant into the right hind foot pad. The rats were dosed p.o. from day 18 to day 31 with levamisole (10 mg/kg), indomethacin (1 mg/kg), diclofenac sodium (0.5 & 1 mg/kg), and prinomide (10 & 20 mg/kg). Disease severity was assessed by paw circumference on day 31. The following blood markers were analyzed: hyaluronate by ELISA, prostaglandin E2 by RIA, ESR by micro-dispette, total PMN by Technicon H-1, and albumin by BCG dye. Blood marker correlation (r) to disease severity was: hyaluronate (0.71), prostaglandin E2 (0.58), ESR (0.52), PMN (0.58), and albumin (-0.71). The relative rank order of drug efficacy (indomethacin, diclofenac sodium, and prinomide) did not differ using the change in paw circumference (day 31-day 17) or blood markers. Levamisole exacerbated the disease as measured by all the above parameters. Thus, these blood markers provide additional information for the statistical evaluation of drugs in rat adjuvant arthritis.

Evidence for membrane differentiation in polarised leucocytes: the distribution of surface antigens analysed with Ig-gold labelling.

The distribution of a number of leucocyte surface antigens was studied on both round and polarised neutrophil or mononuclear leucocytes using Ig-gold conjugates with transmission electron microscopy. Thin sections of cells, which had been lightly fixed before antibody labelling, were analysed using a statistical method to determine: (1) whether the antigens had a non-random distribution or 'clustering' over the cell surface; and (2) whether there was any overall bias in labelling to give a polarised distribution. Comparison between the results of this analysis and cell morphology were made. The results indicated that with the antigens investigated here, CD45, CD15, HLA-DR and CR3, the majority of polarised cells had a calculated direction of overall asymmetry of gold particles that was aligned with the long axis of morphological polarity. Maximal asymmetry was seen in polarised cells labelled for CD45 and HLA-DR, with labelling ratios of up to 6:1 between the front and back of the cell. A number of round mononuclear cells demonstrated significant polarisation of gold particles but this had no apparent morphological correlation and, in general, round cells showed a low degree of asymmetry. However, there was evidence that both round and polarised cells had a non-random distribution or 'clustering' of gold particles, which was more marked in morphologically polarised cells and particularly significant in polarised neutrophil leucocytes labelled for CR3. The significance of these results for models of cell locomotion involving membrane flow is discussed.

Effects of phorbol esters on shape and locomotion of human blood lymphocytes.

The effects of phorbol esters on shape change and locomotion of human blood lymphocytes were studied both immediately after separating the cells from blood and after overnight culture. Phorbol myristate acetate (PMA), phorbol dibutyrate (PDB) and related esters produced complex shape changes in lymphocytes at both times. These shapes were analysed quantitatively using objective measurements derived from the moments of cell shapes. Immediately after removal from blood, many lymphocytes (20-60% of the total) protruded and retracted veils or spikes at more than one point on the cell surface. The morphology of these cells was not typical of locomotor cells. Usually, formation of a veil was not followed by a contraction wave moving down the cell, though some cells did show contraction waves, and some moved into collagen gels or filters. After overnight culture, a high proportion (70-80%) of cells had changed shape in PMA and PDB. Although the shapes were still atypical, they resembled classical locomotor morphology more closely; veils formed at one point on the cell surface tended to persist, and contraction waves and constriction rings were seen in many cells. These cells moved in large numbers into collagen gels or filters. Comparison of the paths traversed by PMA-treated lymphocytes in collagen gels suggested that cells cultured in PMA for 24 h pursued more persistent paths that those in short-term culture, but the difference was not marked. We suggest that phorbol esters induce immediate shape change without inducing the complete sequence of motor events necessary for efficient locomotion, whereas after prolonged culture in phorbol esters, locomotion is more efficient, possibly because phorbol esters, like other growth activators, stimulate events during the G1 phase of growth that are necessary for full expression of locomotor capacity.