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Biomedical subjects

A D Watts

Publications and source records attributed to A D Watts.

At least 19 recordsLinked to original sources
PubMed1999

Use of fixed cells in cell contact-dependent cytotoxicity assays for TNF: a cautionary report.

The use of fixed effector cells or cell membrane preparations in assays to study cell surface TNF-mediated immunological effects has been widespread for more than a decade. The assumption has always been made that observed effects of fixed cells are due to the cell surface TNF molecule. Here we report that paraformaldehyde-fixed, LPS-stimulated RAW264.7 cells release a factor that causes cytotoxicity against TNF sensitive WEHI-164 cells. The factor was neutralized by soluble TNF receptors as well as antibody and could not be removed by ultracentrifugation at 100,000 x g, and is therefore likely to be a form of soluble TNF. This has important implications for the interpretation of these assays, given that cell surface TNF is thought to exert biological effects through a different signaling mechanism than soluble TNF.

Animals↗
PubMed1999

A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in 'reverse signalling'.

We have identified a putative signalling feature of the cytoplasmic domains of the tumour necrosis factor (TNF) family members based on available amino acid sequence data. A casein kinase I (CKI) consensus sequence is conserved in the cytoplasmic domain of six of 15 members of the type II integral membrane TNF ligand family. We examined the phosphorylation state of transmembrane tumour necrosis factor-alpha (mTNF) with [32P]orthophosphate labelling and in vitro kinase assays, in lipopolysaccharide-stimulated RAW264.7 cells. A dimeric form of the type I soluble TNF receptor (sTNFR) was found to dephosphorylate mTNF. This effect could be prevented by treatment with phosphatase inhibitors. Recombinant CKI was able to phosphorylate mTNF that had been dephosphorylated by sTNFR ligation in vivo, and this was less effective if phosphatase inhibitors had been used to prevent mTNF dephosphorylation. A mutated form of mTNF, lacking the CKI recognition site, cannot be phosphorylated by the enzyme. Binding of sTNFR to mTNF induced an increase in intracellular calcium levels in RAW264.7 cells, implying the presence of an associated signalling pathway. We predict that this CKI motif is phosphorylated in other TNF ligand members, and that it represents a new insight into the mechanism of 'reverse signalling' in this cytokine family.

Amino Acid Sequence↗
PubMed1999

Soluble TNF-alpha receptors bind and neutralize over-expressed transmembrane TNF-alpha on macrophages, but do not inhibit its processing.

Tumor necrosis factor alpha (TNF-alpha) is initially synthesized as a type II integral membrane protein (transmembrane TNF-alpha) after macrophage activation. In this study we have investigated some aspects of the regulation of expression and biological activity of transmembrane TNF-alpha by both soluble TNF-alpha receptors (sTNF-alphaR) and inhibitors of TNF-alpha processing. We show, using the technique of receptor-mediated ligand precipitation (RMLP), that a dimeric construct of the type I sTNF-alphaR binds to transmembrane TNF-alpha, expressed on the mouse macrophage cell line RAW264.7, under cell culture conditions. This interaction between sTNF-alphaR and transmembrane TNF-alpha does not prevent processing and release of soluble TNF-alpha. A specific hydroxamic acid-based inhibitor of processing, BB1101 (British Biotech), was found to increase the total cellular levels of whole-cell, 26-kDa, precursor TNF-alpha by 2.2-fold. However, the inhibitor increased the levels of precursor TNF-alpha present solely on the cell surface (i.e., transmembrane TNF-alpha) by 5.1- to 7.5-fold. This increase in the levels of transmembrane TNF-alpha on the activated human monocytoid cell line mono mac 6 was associated with a similar (6.7-fold) increase in TNF-alpha-mediated cytotoxicity toward the human adenocarcinoma cell line Colo 205, which is sensitive only to the transmembrane form of TNF-alpha. Mono mac 6 cells, expressing transmembrane TNF-alpha, were found to be killing the Colo 205 target cells through apoptosis. This cytotoxicity could be neutralized by pre-incubating the mono mac 6 cells with either sTNF-alphaR or polyclonal anti-TNF-alpha serum.

Animals↗
PubMed1999

The effect of sevoflurane and isoflurane anesthesia on interictal spike activity among patients with refractory epilepsy.

UNLABELLED: The electrophysiologic effects of sevoflurane are not well characterized in humans. Among patients with refractory epilepsy, this study compared 1) electroencephalographic (EEG) interictal spike activity during wakefulness and sevoflurane anesthesia, and 2) electrocorticographically (ECoG) recorded interictal spike activity during sevoflurane and isoflurane anesthesia. We studied 12 patients undergoing insertion of subdural electrodes. Before commencing anesthesia, awake (baseline) EEG recordings were obtained. After inhaled induction, EEG interictal spike activity was evaluated during stable, normocapnic, and hypocapnic (Paco2 = 28-30 mm Hg), sevoflurane anesthesia administered at 1.5 times the minimum alveolar anesthetic concentration (1.5 MAC). Immediately after surgery, ECoG recordings were obtained from subdural electrodes during 1) 1.5 MAC isoflurane, 2) 0.3 MAC isoflurane, and 3) 1.5 MAC sevoflurane anesthesia. EEG spike frequency increased in all patients during sevoflurane anesthesia compared with awake recordings (P = 0.002). Compared with 0.3 MAC isoflurane anesthesia, ECoG interictal spike frequency was higher in all patients during 1.5 MAC sevoflurane anesthesia (P = 0.004) and in 8 of 10 patients during 1.5 MAC isoflurane anesthesia (P = 0.016). Under sufficiently rigorous conditions, both sevoflurane and isoflurane can provoke interictal spike activity at near burst-suppression doses. This property is more prominent with sevoflurane than isoflurane. IMPLICATIONS: The results of this study suggest that the capacity to modulate neuroexcitability is a dose-dependent feature of volatile anesthetics that is manifested most prominently at near burst-suppression doses (i.e., 1.5 times the minimum alveolar anesthetic concentration) and is minimal or absent at low doses.

Adolescent↗
PubMed1998

Propofol and hyperventilation for the treatment of increased intracranial pressure in rabbits.

UNLABELLED: Using a rabbit model of intracranial hypertension, we studied (a) the additive effect of propofol and hyperventilation (HV) on increased intracranial pressure (ICP), (b) the ICP-lowering effect of additive therapy (i.e., propofol plus HV versus HV plus propofol), and (c) whether combined therapy induced cerebral ischemia. Twenty-three New Zealand White rabbits were studied, of which seven were control animals. The animals were anesthetized with midazolam and fentanyl. An extradural balloon was used to increase ICP to 26+/-2 mm Hg. Elevation of ICP resulted in a >50% reduction in ipsilateral somatosensory evoked potential (SEP) amplitude. The rabbits were then randomized to be treated with propofol followed by HV (Group 1, n = 8) or HV then propofol (Group 2, n = 8). With HV, PaCO2 was reduced from 41+/-2 mm Hg to 27+/-3 mm Hg. In Group 1 rabbits, the ICP decreased with treatment (from 26+/-2 to 12+/-2 mm Hg). The reduction in ICP was significantly greater (P = 0.008) than that in Group 2 rabbits (from 26+/-2 to 16+/-5 mm Hg). In both groups, propofol and HV had an additive effect in reducing ICP. Further, when propofol was used as the initial treatment, there was a significant increase in SEP amplitude that was not apparent in Group 2 rabbits. In conclusion, we found propofol and HV to be additive in the treatment of increased ICP. In animals treated with propofol followed by HV, there was a greater decrease in ICP than in rabbits treated initially with HV and then propofol. IMPLICATIONS: We have demonstrated that propofol has a greater effect on increased intracranial pressure than hyperventilation when used as the initial treatment and that the two treatments are additive. Should control of intracranial pressure be required after the administration of propofol, then hyperventilation may be added to the treatment.

Anesthesia↗
PubMed1998

Effect of nitrous oxide on cerebral blood flow velocity after induction of hypocapnia.

Hyperventilation may reverse increases in cerebral blood flow velocity caused by inhalation of nitrous oxide (N2O). This study sought to determine whether inhalation of 50% nitrous oxide after the induction of hyperventilation increases cerebral blood flow velocity as measured by transcranial Doppler ultrasonography. Seven volunteers breathed air/O2 through a modified Circle system at normocapnia followed by air/O2 with hyperventilation, and then N2O/O2 with hyperventilation. Expired gas concentrations were measured in the expiratory limb of the circuit distal to a one-way valve. Hyperventilation reduced end-tidal carbon dioxide from 38+/-1 mmHg to 26+/-1 mmHg. Hypocapnia was maintained during inhalation of N2O (EtCO2=28+/-1 mmHg). Mean cerebral blood flow velocity decreased 34% with hyperventilation (38+/-4 cm/second versus 59+/-9 cm/second, p < 0.05) and returned to baseline with the addition of nitrous oxide (58+/-7 cm/second), despite persistent hypocapnia. The addition of nitrous oxide to the inspired gas mixture after induction of hypocapnia reversed reductions in cerebral blood flow velocity associated with hyperventilation. Potential benefits of induced hypocapnia in patients with intracranial pathology may be offset by the administration of N2O.

Adult↗
PubMed1997

Separation of tumor necrosis factor alpha isoforms by two-dimensional polyacrylamide gel electrophoresis.

The mouse macrophage cell-line RAW264.7, stimulated with lipopolysaccharide, was used as a model for the study of the production of tumor necrosis factor (TNF) isoforms. TNF is synthesised initially as a 26 kDa transmembrane precursor, which is then processed enzymatically by a protease to release a mature molecule of 17 kDa. Dose-dependent production of transmembrane TNF was assessed by fractionation of cell membranes and Western blot analysis followed by autoradiography and densitometry. Isoforms of both the precursor and mature molecules were separated using two-dimensional (2-D) electrophoresis with immobilised pH gradient 3-10 linear gels as the first dimension. After radiolabelling of cells with 35S, both cell-associated and supernate-associated TNF isoforms were immunoprecipitated. A large number of protein spots were visualised on the 2-D gel map, for both the transmembrane and mature TNF species, more than have been detected previously using one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The likelihood that these putative isoforms were the result of differential glycosylation was tested by preincubating the cells with tunicamycin. This had the effect of reducing the number of protein spots, notably the higher molecular weight species. There were a number of precursor TNF isoforms that were unchanged upon tunicamycin treatment and these presumably reflect protein modifications other than glycosylation.

Animals↗
PubMed1997

Comparison of the Bullard and Macintosh laryngoscopes for endotracheal intubation of patients with a potential cervical spine injury.

BACKGROUND: In the emergency trauma situation, in-line stabilization (ILS) of the cervical spine is used to reduce head and neck extension during laryngoscopy. The Bullard laryngoscope may result in less cervical spine movement than the Macintosh laryngoscope. The aim of this study was to compare cervical spine extension (measured radiographically) and time to intubation with the Bullard and Macintosh laryngoscopes during a simulated emergency with cervical spine precautions taken. METHODS: Twenty-nine patients requiring general anesthesia and endotracheal intubation were studied. Patients were placed on a rigid board and anesthesia was induced. Laryngoscopy was performed on four occasions: with the Bullard and Macintosh laryngoscopes both with and without manual ILS. Cricoid pressure was applied with ILS. To determine cervical spine extension, radiographs were exposed before and during laryngoscopy. Times to intubation and grade view of the larynx were also compared. RESULTS: Cervical spine extension (occiput-C5) was greatest with the Macintosh laryngoscope (25.9 degrees +/- 2.8 degrees). Extension was reduced when using the Macintosh laryngoscope with ILS (12.9 +/- 2.1 degrees) and the Bullard laryngoscope without stabilization (12.6 +/- 1.8 degrees; P < 0.05). Times to intubation were similar for the Macintosh laryngoscope with ILS (20.3 +/- 12.8 s) and for the Bullard without ILS (25.6 +/- 10.4 s). Manual ILS with the Bullard laryngoscope results in further reduction in cervical spine extension (5.6 +/- 1.5 degrees) but prolongs time to intubation (40.3 +/- 19.5 s; P < 0.05). CONCLUSIONS: Cervical spine extension and time to intubation are similar for the Macintosh laryngoscope with ILS and the Bullard laryngoscope without ILS. However, time to intubation is significantly prolonged when the Bullard laryngoscope is used in a simulated emergency with cervical spine precautions taken. This suggests that the Bullard laryngoscope may be a useful adjunct to intubation of patients with potential cervical spine injury when time to intubation is not critical.

Adult↗
PubMed1992

Comparison of the use of the laryngeal mask and face mask by inexperienced personnel.

Ten junior doctors with no postgraduate anaesthetic experience attempted to ventilate the lungs of 50 anaesthetized patients, using either a laryngeal mask or a Guedel airway and face mask. Success was defined as the production of two successive tidal volumes exceeding 800 ml within 40 s. The failure rate was significantly greater using the laryngeal mask compared with the face mask (P < 0.05) and the average time was significantly longer with the laryngeal mask than with the face mask (P < 0.01). The results from this investigation suggest the laryngeal mask airway cannot be recommended as a resuscitation device for use by inexperienced operators.

Cardiopulmonary Resuscitation↗
PubMed1991

Severe late postsplenectomy infection.

In all, 1490 patients underwent splenectomy in Western Australia between 1971 and 1983, giving 7825 person years exposure. Thirty-three patients developed severe late postsplenectomy infection (septicaemia, meningitis or pneumococcal pneumonia requiring hospitalization) and three developed overwhelming postsplenectomy infection. The incidence and mortality rates of severe late postsplenectomy infection were 0.42 and 0.08 per 100 person years exposure respectively and for overwhelming postsplenectomy infection the incidence and mortality rates were 0.04 per 100 person years exposure. There were 628 splenectomies after trauma, giving 3922 person years exposure. Eight patients developed severe late postsplenectomy infection of whom one had overwhelming postsplenectomy infection. Following trauma, the incidence of severe late postsplenectomy infection was 0.21 per 100 person years exposure, with the incidence and mortality rates of overwhelming postsplenectomy infection being 0.03 per 100 person years exposure. Patients undergoing splenectomy have a 12.6-fold increased risk of developing late septicaemia compared with the general population. Splenectomy following trauma gives an 8.6-fold increased risk of late septicaemia. The majority of severe late postsplenectomy infections did not occur within the first 2 years and 42 per cent of severe late postsplenectomy infections occurred greater than 5 years after splenectomy. The low incidence of severe late postsplenectomy infection and overwhelming postsplenectomy infection makes statistical evaluation of the effectiveness of prophylactic antibiotics, vaccination and splenic repair most difficult.

Adolescent↗
PubMed1988

Plasma steroid, relaxin and dihydro-keto-prostaglandin F-2 alpha changes in the minipig in relation to myometrial electrical and mechanical activity in the pre-partum period.

Recordings of electromyographic and mechanical activities of the uterus of intact and ovariectomized minipigs during the last days of pregnancy, combined with a frequent blood sampling regimen, indicated that the evolution of electrical activity was dependent upon the clearance both of progesterone and relaxin from the plasma. Increases in dihydro-keto-prostaglandin-F-2 alpha (DHKF-2 alpha) occurred only after the initial decline in progesterone, and were positively correlated with increases in electrical activity only after circulating relaxin titres had fallen. These results demonstrate that relaxin provides for the inhibition of the myometrium during the period when progesterone titres decline. The temporal relationship of the decline of progesterone and the rise in DHKF-2 alpha suggests that uterine PGF-2 alpha may not initiate luteolysis in the sow at term.

Animals↗
PubMed1986

Relaxin and progesterone are myometrial inhibitors in the ovariectomized non-pregnant mini-pig.

Intravenous bolus injections of pig relaxin which produced short-lived peaks of the hormone equivalent in concentration to those observed at term promptly rendered the uterus almost totally quiescent and the inhibition persisted for about 2.5 h. During this time the uterus remained responsive to oxytocin. The main effect of relaxin was to reduce the frequency of intrauterine pressure cycles rather than the amplitude. In contrast progesterone, which also inhibited myometrial activity, took between 6 and 24 h to exert its maximum effect by reducing both amplitude and frequency of IUP cycles and it also abolished the responsiveness of the myometrium to oxytocin. Its actions were reversible but recovery took between 54 and 140 h. Oestradiol benzoate had no significant effect on myometrial activity in 21 out of 26 treatments. At 24 h after the 5 remaining treatments, however, myometrial activity was virtually zero. No evidence was obtained of a biphasic effect of oestradiol on myometrial activity as reported for the rat and ewe. This work demonstrates that purified pig relaxin is an active myometrial inhibitor in the oestrogen-treated ovariectomized non-pregnant pig in vivo.

Animals↗
PubMed1985

A comparison of 5-hydroxytryptamine receptors mediating contraction in rabbit aorta and dog saphenous vein: evidence for different receptor types obtained by use of selective agonists and antagonists.

Using recently available selective agonists and antagonists we have examined further our postulate (Apperley et al., 1980) that 5-hydroxytryptamine (5-HT) mediates contraction of dog saphenous vein via a different 5-HT receptor type from that in the rabbit aorta. In the rabbit isolated aorta, ketanserin and spiperone were potent, specific, competitively-acting antagonists of the contractile effects of 5-HT. In contrast, in the dog isolated saphenous vein neither ketanserin nor spiperone caused any rightward displacement of concentration-response curves to 5-HT although the maximum response was reduced by about 10%. In the rabbit aorta 5-carboxamidotryptamine (5-CONH2-T) was a weak agonist whilst the 5-N,N-dimethyl and 5-N-ethyl derivatives were even weaker or inactive. The contractile effect of 5-CONH2-T in the rabbit aorta was potently and competitively antagonized by ketanserin. In contrast, in the dog saphenous vein 5-CONH2-T and its 5-N,N-dimethyl and 5-N-ethyl derivatives were all potent agonists. The contractile effect of 5-CONH2-T was not markedly affected by ketanserin. The profile of action of ketanserin and spiperone in the rabbit aorta is consistent with the view that 5-HT2 receptors mediate contraction in this preparation. However, the 5-HT receptor mediating contraction in the dog saphenous vein appears to be '5-HT1-like', sharing a number of characteristics with the 5-HT1 recognition site identified from [3H]-5-HT ligand binding studies in brain tissue.

Animals↗
PubMed1983

5-Hydroxytryptamine-induced relaxation of isolated mammalian smooth muscle.

5-HT caused concentration-dependent relaxation of methoxamine contracted cat isolated saphenous vein and atropine pretreated guinea-pig isolated ileum contracted with histamine. The concentration required to cause 50% of its maximum effect was 7.4 X 10(-7) and 1.4 X 10(-6) mol/l respectively. alpha-Methyl 5-HT was at least 200 times weaker than 5-HT in this respect. The relaxant action of 5-HT in both preparations was not antagonised by atropine, indomethacin, cimetidine, propranolol or tetrodotoxin. In both preparations the relaxant effect was not specifically antagonised by cyproheptadine but was specifically and competitively antagonised by methysergide (pA2 values of 6.75 and 7.37 in cat saphenous vein and guinea-pig ileum, respectively). The results suggest that the 5-HT mediated relaxation in both preparations is mediated directly via a similar 5-HT receptor. The weak antagonistic action of methysergide and lack of relaxant effects with alpha-methyl 5-HT is consistent with the view that this receptor is not a 'D' or 'M' 5-HT-receptor.

Animals↗
PubMed1983

Prejunctional effects of 5-hydroxytryptamine on noradrenergic nerves in the cardiovascular system.

Recent studies indicate that some peripheral noradrenergic nerves have specific 5-hydroxytryptamine (5-HT) receptors on their terminals that can mediate inhibition of release of transmitter after neuronal stimulation. It is now also known that 5-HT will cause norepinephrine release from noradrenergic nerve terminals in the isolated rabbit heart by a receptor mechanism. The evidence for the existence of these two types of prejunctional 5-HT receptor is discussed together with findings to date on their distribution and possible functional importance in the whole animal. In addition 5-HT has weak sympathomimetic actions, which result from its ability to interact with noradrenergic removal processes. The displaced transmitter can then interact with adrenoceptors situated pre- or postjunctionally.

Animals↗