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Biomedical subjects

S J Holt

Publications and source records attributed to S J Holt.

At least 19 recordsLinked to original sources
PubMed2002

A radiation perspective for treating loin pain in pregnancy by double-pigtail stents.

OBJECTIVE: To review the risks of placing double-pigtail stents during pregnancy in women presenting with loin pain associated with hydronephrosis. PATIENTS AND METHODS: A consecutive series of women presenting with loin pain and hydronephrosis in pregnancy were treated with double-pigtail ureteric stents. A flexible cystoscope was used to identify the ureteric orifice and to pass a guidewire into the renal pelvis under fluoroscopic guidance. Stents were placed using the exchange technique over a stiff guidewire. Procedures were carried out under none or limited sedoanalgesia. Screening times and radiation dose were recorded. Data were collected for the average uterine radiation dose from a variety of radiological procedures. Previous publications were reviewed to determine the lethal, teratogenic and carcinogenic risk to the developing fetus from radiation exposure. RESULTS: Seven patients referred with symptomatic hydronephrosis during pregnancy were treated. The screening time during placement was minimized. The mean (range) uterus (i.e. fetal) dose was 0.40 (0.03-0.79) mGy. Most radiological procedures involve uterine doses of < 20 mGy, far below the 100 mGy that may result in fetal damage during periods of maximum radiosensitivity. CONCLUSION: Minimized radiation exposure from a range of uroradiological procedures in pregnant women has limited fetal risk. The use of fluoroscopy for symptomatic hydronephrosis during pregnancy allows ureteric stents to be placed safely and reliably. The average excess risk to the fetus from this procedure is 1 in 43 000 of cancer induction and 1 in 100 000 of heritable disease, i.e. very small when compared with the natural incidence. Pregnancy should not exclude the use of appropriate diagnostic radiation exposure and urologists may conduct appropriate diagnostic and therapeutic procedures, taking care to limit X-ray exposure without fear of risk to the developing fetus.

Adult↗
PubMed2001

Work of breathing associated with pressure support ventilation in two different ventilators.

The purpose of this study was to compare the work of breathing during pressure support ventilation (PSV) with positive end expiratory pressure (PEEP) utilizing the Siemens SV300 (SV300) and Dräger Evita 4 (EV4) ventilators. Our hypothesis was that patients' work of breathing (WOB(P)) would be unchanged in PSV utilizing flow triggering (FT) in both the SV300 and EV4. We compared two ventilators using six healthy, intubated, sedated, spontaneously breathing pigs weighing approximately 10 kg each. WOB(P) (j/L) and ventilator work of breathing (WOB(V)) (j/L) were measured using a portable monitor which utilizes an esophageal balloon and flow transducer. Each breath was further analyzed for duration of inspiratory effort and negative deflection of pressure needed to trigger PSV. Animals were studied with the SV300 and EV4 on a pressure support of 5 cmH(2)O and PEEP settings of 0 and 5 cmH(2)O. Data were analyzed using the Wilcoxon signed rank test with significance set at P <or= 0.05 WOB(P) was 90% (PS 5, PEEP 0) and 52% (PS 5, PEEP 5) lower on the SV300 compared to the EV4. WOB(V) was 94% (PS 5, PEEP 0) and 39% (PS 5, PEEP 5) higher on the SV300 when compared to the EV4. The change in airway pressure (delta p) from baseline and most negative deflection of pressure were greater with the EV4 as compared to the SV300, although delta pressure was not found to be statistically significant on PS of 5 cmH(2)O and PEEP of 5 cmH(2)O. The SV300 also had shorter duration of inspiratory effort from initiation of breath to most negative deflection of pressure and to maximum flow than the EV4. In conclusion, these results suggest there are significant differences in WOB(P) between the SV300 and EV4 ventilators. Response time of the ventilators may explain the differences in duration of inspiratory effort and the patient's work of breathing and thus may have an impact on weaning time for ventilated patients.

Animals↗
PubMed2001

Outpatient ureteric procedures: a new method for retrograde ureteropyelography and ureteric stent placement.

OBJECTIVE: To evaluate a new method for retrograde ureteropyelography and retrograde ureteric stent placement. PATIENTS AND METHODS: Procedures were undertaken using a flexible cystoscope and digital C-arm fluoroscopy in outpatients under sedoanalgesia. The flexible cystoscope was used to identify the ureteric orifice and a straight 0.9 mm hydrophilic guidewire inserted and passed into the renal pelvis under fluoroscopic guidance. A 4 F general-purpose catheter was then passed over the wire and ureteropyelography performed. To place the stent the hydrophilic guidewire was exchanged for an ultra-stiff wire, over which the stent was passed directly. RESULTS: Over a 47-month period, 723 procedures were carried out in 472 patients. The clinical indications were ureteric obstruction in 229 (32%), stone disease in 165 (23%), unexplained hydronephrosis in 150 (21%), haematuria in 94 (13%) and others in 85 (12%). Of the 723 procedures, 643 (89%) were technically successful. Failure was most commonly caused by failure to cannulate the ureteric orifice (51, 7%). Just over half the procedures (366, 51%) involved stent placement or replacement. Immediate complications occurred in 17 patients (3%). Of those who were questioned, 94% (282 of 300) reported the procedure to be acceptable. CONCLUSION: Retrograde ureterography and ureteric stent placement may be satisfactorily undertaken with the patient under sedoanalgesia on an outpatient basis. This technique can reduce costs, hospital admissions, general anaesthetic use, demands on theatre time and complication rates.

Adolescent↗
PubMed2001

An evaluation of Automode, a computer-controlled ventilator mode, with the Siemens Servo 300A ventilator, using a porcine model.

BACKGROUND: Weaning of mechanical ventilation in patients optimally includes meeting their needs by making frequent ventilator adjustments. The Siemens Servo 300A mechanical ventilator is designed to allow the ventilator to be interactive with the patient's needs by making breath-by-breath adjustments in both control and support modes. We undertook the following experiment to validate that the Automode algorithm responded appropriately using a pediatric animal model when apnea occurred and if there was any impact on work of breathing. METHODS: We ventilated 6 sedated spontaneously-breathing piglets using Automode in pressure-regulated volume control/volume support (PRVC/VS) mode, pressure control/pressure support (PC/PS) mode, and volume control/volume support (VC/VS) mode. Data were collected using both a computerized respiratory monitor and data acquisition system that recorded and analyzed individual animal breaths for response time, effort of triggering, and work of breathing. Data collection began with the animals breathing spontaneously in each support mode, followed by the administration of a short-acting neuromuscular blocker (succinylcholine) to induce apnea, thus allowing the ventilator to switch between modes automatically. Data collection was continued before, during, and after apnea to observe the duration of inspiratory effort, trigger response time, and any significant pressure or flow variances of the Automode feature. In addition, patient work of breathing (WOB(P)) and ventilator work of breathing (WOB(V)) were measured before and after each phase. RESULTS: We found no instances of failure of Automode to follow the predetermined algorithms. There was a difference in both the amount of change in pressure and most negative deflection of pressure by each animal during triggering in the post-paralysis phase (p < 0.05). Response time for individual breaths was shorter from initiation of breath to most negative deflection of pressure during the post-paralysis phase (p < 0.05). Maximum flow reached was lower in the post-paralysis phase for VC/VS and PC/PS (p < 0.05). We also found WOB(P) decreased and WOB(V) increased in the post-paralysis phase for all modes tested. CONCLUSIONS: The Automode algorithm performed as expected in this animal experiment. We conclude that differences in response time and negative deflection of pressure, as an indication of animal effort, and maximum flow reached were due to continued weakness from the neuro-muscular blocker. However, the ventilator continued to trigger despite decreased effort by the animal.

Algorithms↗
PubMed2000

Comparison of work of breathing between two neonatal ventilators utilizing a neonatal pig model.

OBJECTIVE: The purpose of this study was to determine whether variations in the delivery systems of continuous positive airway pressure between two ventilators would lead to differences in patient work of breathing (WOBp). DESIGN: Comparison of two neonatal ventilators with a neonatal pig model. SETTING: Animal laboratory. SUBJECTS: Thirty healthy, intubated, sedated, spontaneously breathing neonatal piglets weighing 1.0-2.0 kg. INTERVENTIONS: Patient work of breathing (WOBp) (gm cm/kg) was measured by using measurements based on an esophageal balloon and a flow transducer. Each breath was analyzed for ventilator response times (in msecs) and negative deflection of pressure. Each animal was studied with the Siemens SV300 and Drager Babylog 8000, on continuous positive airway pressure settings of 0, 3, and 5 cm H2O. Data were analyzed by using Wilcoxon's Signed Rank Test with significance of p 1 cm H2O negative pressure before flow was available with the Babylog. CONCLUSIONS: In intubated patients, maximum energy expenditure occurs at the initiation of ventilator breaths. WOBp in neonatal pigs was significantly increased. The response time of the ventilators may explain the differences in initiation of flow times and patient work. These differences may have important implications for energy kinetics, weight gain, and duration of mechanical ventilation in preterm neonates.

Journal Article↗
PubMed1996

Growth hormone-responsive DT-diaphorase-mediated bioreduction of tetrazolium salts.

Microculture tetrazolium assays (MTAs) rely upon the bioreduction of tetrazolium salts to their intensely coloured formazans. Although these assays are being extensively used, the intracellular mechanisms responsible for the formazan production are not known. MTAs currently provide the basis for uniquely precise in vitro bioassays for human growth hormone (hGH) which use the Nb2 cells. We have compared two contrasting tetrazolium salts, namely 3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) and 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-++ +sulfophenyl) tetrazolium, inner salt (MTS), in this system. An intermediate electron acceptor (IEA) is obligatory for the MTS- but not the MTT-bioassay. We report that inhibitors of DT-diaphorase abolished MTS- but not MTT-formazan production. We conclude that substitution of MTT with MTS/menadione resulted in formazan production via a different electron transfer pathway which is exclusively mediated by DT-diaphorase.

Animals↗
PubMed1996

The use of intermediate electron acceptors to enhance MTT bioreduction in a microculture tetrazolium assay for human growth hormone.

We contrast the effects of three intermediate electron acceptors (IEAs) on the highly quantitative ESTA bioassay for human growth hormone. This is a microculture tetrazolium assay based upon the in vitro reduction of the tetrazolium salt MTT, by Nb2 cells which have been activated with hGH. Each of the IEAs influenced MTT-formazan production in a distinctive manner. The two quinonoids, namely menadione and co-enzyme Q0 markedly increased the MTT-formazan produced by hormone activated Nb2 cells and thereby amplified the response of our bioassay for human growth hormone (hGH). The exceptionally low bioassay baseline which is characteristic of the unstimulated Nb2 cells when only MTT is added was retained in the presence of CoQ0, but was greatly increased by menadione. Phenazine methosulphate, which is the most widely used redox intermediary in microculture tetrazolium assays, also increased the baseline, but had only a minimal additional effect on MTT reduction by activated Nb2 cells. We conclude that CoQ0 is the preferred IEA for this ESTA bioassay for hGH.

Animals↗
PubMed1995

Two newly established cell lines derived from the same colonic adenocarcinoma exhibit differences in EGF-receptor ligand and adhesion molecule expression.

Two morphologically distinct cell lines, GP2d and GP5d, derived from the same adenocarcinoma of the colon, have been established and characterised. Both clones have the same genetic changes, consistent with the usual pattern of tumour progression in colon cancer. The cells also have an inverted duplication of bands 10q11 to 10q21, but Southern blot analysis failed to identify any translocations involving the ret protooncogene, which maps to this region. GP2d grew by spreading from the edges of microcolonies to form a confluent layer of cells. GP5d grew in discrete islands of cells forming multi-layered colonies. These differing patterns of growth correlated with variation in expression or cellular distribution of alpha 2-integrin, desmoplakin and e-cadherin. Only GP2d responded to exogenously added epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha) or insulin with an increase in cell numbers, even though both cell lines possessed similar numbers of EGF receptors. Analysis of EGF receptor ligand expression showed that GP5d cells expressed relatively more TGF alpha mRNA than did GP2d; in contrast, amphiregulin mRNA, which was abundant in GP2d, was virtually undetectable in GP5d. Even though GP5d failed to exhibit a growth response to EGF, it underwent a marked epithelial-mesenchymal transition when treated with EGF, indicating separation of growth and morphological responses to EGF.

Adenocarcinoma↗
PubMed1995

Modulation of the receptor binding affinity of amphiregulin by modification of its carboxyl terminal tail.

Amphiregulin (AR), a heparin-binding, epidermal growth factor (EGF) receptor ligand has homology with EGF but exhibits a lower affinity for the EGF receptor than EGF. As the mature form of AR is truncated at the C terminus and lacks a conserved leucine residue known to be essential for high affinity binding of EGF to the EGF receptor, wild-type AR (AR1-84), a C-terminally extended AR construct incorporating six residues from the predicted coding sequence of AR (AR1-90) and a similarly extended construct with a Met86 to Leu substitution (AR1-90(leu86)) were expressed as recombinant proteins in yeast, purified by heparin affinity and C18 reverse phase chromatography and their relative biological activities determined. The growth factors were tested in mitogenesis and EGF receptor autophosphorylation assays and their relative order of potencies was found to be leu86 > met86 > wt. The AR1-90(leu86) construct was found to be 50- to 100-fold more active than wild type AR1-84 consistent with previously reported studies of the role of the equivalent C-terminal leucine in EGF or TGF alpha. Significantly, the C-terminally extended form of AR, AR1-90, which utilized six residues from the predicted coding sequence, was 10-times more active than wild type AR1-84. This difference in activity of the C-terminally extended form of AR may be of biological significance since differential proteolytic processing of the AR precursor in vivo could result in production of multiple forms of the growth factor with differing affinities for the EGF receptor and hence differing biological potencies.

Amphiregulin↗
PubMed1995

Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS.

Microculture tetrazolium assays are being widely exploited to investigate the mechanisms of both cell activation and cell damage. They are colorimetric assays which are based upon the bioreduction of a tetrazolium salt to an intensely coloured formazan. We contrast the responses obtainable with two new tetrazolium salts, MTS and XTT, when used on the rat lymphoma cell line (Nb2 cells), which has been activated by human growth hormone. These tetrazolium salts, unlike the more commonly used MTT, form soluble formazans upon bioreduction by the activated cells. This has the advantage that it eliminates the error-prone solubilisation step which is required for the microculture tetrazolium assays which employ MTT. Bioreduction of XTT and MTS usually requires addition of an intermediate electron acceptor, phenazine methosulphate (PMS). We found that the XTT/PMS, but not the MTS/PMS, reagent mixture was unstable. Nucleation and crystal formation in the XTT/PMS reagent mixture, prepared in DPBS, could occur within 1-3 min. This resulted in a decline in XTT-formazan production and manifested itself in the microculture tetrazolium assay as both poor within-assay precision and serious assay drift. Several features of the system suggested that the formation of charge-transfer complexes between XTT and PMS accounted for this instability. No such instability was encountered when MTS and PMS were mixed. We demonstrate that MTS/PMS provides microculture tetrazolium assays for hGH which are free from these serious artefacts and which are uniquely precise. In conclusion we therefore advocate the use of MTS in preference to XTT for the new generation of microculture tetrazolium assays.

Animals↗
PubMed1995

Ligand-induced translocation of epidermal growth factor receptor to the nucleus of NR6/HER fibroblasts is serum dependent.

Ligand-induced translocation of epidermal growth factor receptors (EGF-R) to the nucleus of NR6/HER fibroblasts has been studied by immunoelectron microscopy. Following treatment of NR6/HER cells with epidermal growth factor (EGF) for 1 h, there was a decrease in EGF-R labeling at the plasma membrane and a corresponding increase in EGF-R in the nucleus. This was preceded by a rapid and sustained increase in nuclear phosphotyrosine content, detectable within 2 min of EGF treatment. EGF-R translocation into the nucleus was completely prevented by 18 h serum starvation prior to treatment with EGF. These results indicate that translocation of EGF-R to the nucleus is a controlled process and they suggest that EGF-R may directly influence nuclear function.

Animals↗
PubMed1995

The development of an eluted stain bioassay (ESTA) for human growth hormone.

The basic characteristics of MTT-formazan production by both quiescent Nb2 cells and those activated by fetal calf serum or human growth hormone (hGH) are described. These characteristics are exploited for the development of an MTT-ESTA bioassay for purified preparations of lactogens such as growth hormone. The resulting in vitro bioassay is sensitive and precise, with a detection limit of about 0.05 mU hGH/l (19 ng/l) and a within-assay imprecision of 2.5% in the presence of 0.3 mU hGH/l (114 ng/l). When utilizing quiescent Nb2 cells for bioassays, large magnitudes of response are observed. The major component of the response is clearly derived from metabolic activation of the cells, rather than increased cell proliferation. The response was abolished by anti-human growth hormone. Delayed addition of the latter demonstrated that the presence of the hormone is required for the entire 96 h of the recommended bioassay incubation period to obtain the maximum response. At high doses, the dose-response relationship reaches a prolonged plateau which covers 4 orders of magnitude of incremental hormone concentrations. A decline in response is observed at the highest dose tested, 10(6) mU hGH/l (385 mg/l). This auto-inhibition is consistent with recent reports of a reduction in response due to stoichiometric blockade of sequential receptor dimerisation which is crucial for activation of both somatogenic and lactogenic receptors by hGH.

Animals↗
PubMed1995

A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function.

Microculture tetrazolium assays (MTAs) are being widely applied to probe the relationships between cell survival, growth, and differentiation and also to investigate associations between compromised cell metabolism, oxidative stress, and programmed cell death as occurs in apoptosis. MTAs rely upon the cellular reduction of tetrazolium salts to their intensely coloured formazans. The resulting colorimetric assays form the basis of exceptionally precise systems which are technically amenable and capable of a high throughput of samples. As a consequence, MTAs are being used to monitor responses to both extracellular activators and toxic agents in disciplines as diverse as radiobiology and endocrinology. We review the chemistry and histochemical applications of tetrazolium salts and subsequently discuss the criteria for their use in MTAs. These assays are one of the latest examples of the application of the tetrazolium/formazan system to cell biology. We outline current views on the mechanisms of the bioreduction of tetrazolium salts. These probably combine to reflect the integrated pyridine nucleotide dependent redox state of the cell. We try to illustrate how an understanding of these mechanisms helps to avoid some of the pitfalls of the MTA systems. There is now for example, extensive evidence that changes in cell culture environments, such as glucose supply or pH of the medium, influence the reduction of tetrazolium salts and thereby introduce artefacts into MTAs. Finally, we provide examples of situations in which MTAs can be used to complement other more established experimental systems. They then act as unique probes with which to investigate changes in the redox state of the cell. These changes are associated with regulation of cell growth, proliferation and differentiation and conversely, the different pathways leading to cell death.

Animals↗
PubMed1994

Epidermal growth factor induced tyrosine phosphorylation of nuclear proteins associated with translocation of epidermal growth factor receptor into the nucleus.

Treatment of human squamous carcinoma cells (HN5 cells) with epidermal growth factor (EGF) caused a time-dependent increase in tyrosine phosphorylation of six nuclear proteins of molecular mass 166, 140, 117, 95, 86 and 79 kDa. The major tyrosine phosphorylated protein was indistinguishable from the plasma membrane form of the epidermal growth factor receptor and was shown by enzyme linked immunosorbent assay (ELISA) to be translocated into the nucleus from extra-nuclear sites upon ligand stimulation. Using immunoelectron microscopy of both isolated nuclei and whole cells, epidermal growth factor receptor (EGF-R) was found to be associated with the chromatin and, to a lesser extent, with the inner surface of the nuclear membrane. Tyrosine phosphorylation of proteins other than EGF-R was particularly notable in the nucleoli. These observations suggest that EGF-R may exert some of its physiological functions by directly inducing tyrosine phosphorylation of specific nuclear proteins. Translocation of EGF-R to the nucleus may provide a vital link between plasma membrane signalling and gene activation.

Amino Acid Sequence↗
PubMed1994

A study on the effects of low haemoglobin on postnatal women.

OBJECTIVE: to assess the effect of low haemoglobin (Hb) on the mental and physical health of postnatal women. DESIGN: survey conducted between May 1991 and February 1992. SETTING: maternity unit in district general hospital in the UK. PARTICIPANTS: 1010 postnatal women who had delivered a live baby, did not stay in hospital for seven or more days postnatally, whose baby was not admitted to the neonatal unit and who did not have a current psychiatric disorder. MEASUREMENTS: Hb levels at 'booking', 34 weeks gestation, three days and six weeks post delivery; the Edinburgh Postnatal Depression Scale (EPDS) and a self-completion questionnaire at ten days, four weeks and six weeks post delivery. FINDINGS: women with a low Hb are more likely to be under 25 years of age, primiparous, be anaemic at 34 weeks gestation, not to have had a normal delivery, have had a blood loss greater than 250 ml and to have had heavy lochia postnatally. Low Hb levels were not associated with high EPDS scores. Low Hb levels were related to reports of low energy, breathlessness, faintness/dizziness, painful perineal sutures and tingling in fingers and toes at ten days post delivery. Whilst tiredness persisted to six weeks post delivery the other symptoms disappeared. IMPLICATIONS FOR PRACTICE: in order to reduce the incidence of postnatal anaemia a review of policies for testing is recommended so that the incidence of physical problems in the first six weeks can be reduced. There is a need for further research into the causes and potential alleviation of tiredness.

Adolescent↗
PubMed1989

Interaction of intracerebroventricular insulin and glucose in the regulation of the activity of sympathetic efferent nerves to brown adipose tissue in lean and obese Zucker rats.

The effects of injection of insulin and glucose into the third cerebral ventricle on the firing rate of the sympathetic efferent nerves to intercapsular brown adipose tissue was investigated in anaesthetized lean and obese Zucker rats. Injection of insulin resulted in a dose-dependent (70-480 pmol) inhibition of nerve firing rate, whereas in combination with glucose (140 pmol of insulin and 139 nmol of glucose), insulin strongly potentiated the increase in firing rate seen with glucose alone. Although basal levels of nerve firing rates were lower in the obese rat, responses to insulin, glucose, and insulin plus glucose were qualitatively similar to those seen in the lean rat. These results are consistent with the hypothesis that insulin acts in the central nervous system as a physiological signal in the control of thermogenesis after feeding, and that this effector system is intact in the obese rat.

Action Potentials↗
PubMed1989

Tumour necrosis factor-alpha and lymphotoxin have opposite effects on sympathetic efferent nerves to brown adipose tissue by direct action in the central nervous system.

Intracerebral injection of recombinant human tumour necrosis factor-alpha (rTNF alpha) decreased, whereas lymphotoxin increased the activity of the sympathetic efferent nerves to interscapular brown adipose tissue (IBAT). The changes in firing rates were accompanied by a significant dose-dependent change in IBAT and rectal temperatures (which were decreased with rTNF alpha and increased with lymphotoxin treatment). These results indicate that rTNF alpha and lymphotoxin act directly in the CNS to modulate the sympathetic outflow to IBAT. The opposite effects of the two peptides also suggest that in the CNS, separate receptor systems exist.

Action Potentials↗