Standard operating procedure. Interview by Fred Bazzoli.
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Administrative Personnel↗
Biomedical subjects
R Skinner
Publications and source records attributed to R Skinner.
The anticoagulant activation of antithrombin by heparin.
Antithrombin, a plasma serpin, is relatively inactive as an inhibitor of the coagulation proteases until it binds to the heparan side chains that line the microvasculature. The binding specifically occurs to a core pentasaccharide present both in the heparans and in their therapeutic derivative heparin. The accompanying conformational change of antithrombin is revealed in a 2.9-A structure of a dimer of latent and active antithrombins, each in complex with the high-affinity pentasaccharide. Inhibitory activation results from a shift in the main sheet of the molecule from a partially six-stranded to a five-stranded form, with extrusion of the reactive center loop to give a more exposed orientation. There is a tilting and elongation of helix D with the formation of a 2-turn helix P between the C and D helices. Concomitant conformational changes at the heparin binding site explain both the initial tight binding of antithrombin to the heparans and the subsequent release of the antithrombin-protease complex into the circulation. The pentasaccharide binds by hydrogen bonding of its sulfates and carboxylates to Arg-129 and Lys-125 in the D-helix, to Arg-46 and Arg-47 in the A-helix, to Lys-114 and Glu-113 in the P-helix, and to Lys-11 and Arg-13 in a cleft formed by the amino terminus. This clear definition of the binding site will provide a structural basis for developing heparin analogues that are more specific toward their intended target antithrombin and therefore less likely to exhibit side effects.
Anticoagulants↗
Preparative induction and characterization of L-antithrombin: a structural homologue of latent plasminogen activator inhibitor-1.
The inhibitory mechanism of the serpin family of serine protease inhibitors is characterized by a remarkable degree of conformational flexibility. Various conformational states have been elucidated by X-ray crystallography and indicate that the inhibitory loop, the central A-beta-sheet, and the outside edge of the C-beta-sheet are particularly mobile. However, no crystal structure of a serpin-enzyme complex is yet available, and the likely nature of the protease-complexed serpin remains for biochemical and biophysical researchers to examine. Here, we show that the biochemical induction of the latent state of antithrombin is slow relative to polymer formation, and infer that this may reflect structural features that are important for the regulation of the initial docking and subsequent locking of serpins with cognate proteases. L-Antithrombin was induced by incubation of native antithrombin at 60 degrees C for 10 h in the presence of citrate to prevent polymerization. L-Antithrombin was more stable to denaturation by both heat and urea than native antithrombin. Whereas native antithrombin formed binary complexes with synthetic peptide homologues of the inhibitory loop, biochemically induced L-antithrombin did not, indicating that the inhibitory loop of L-antithrombin is probably fully inserted into the A-beta-sheet as in the crystal structure. This was confirmed by limited proteolysis studies which demonstrated that the inhibitory loop of L-antithrombin could not be cleaved by five proteases which do cleave the loop of native antithrombin. The limited proteolysis studies also indicated that the "gate" region (residues 236-248) of the biochemically induced L-antithrombin was in a conformation substantially different from that of the native antithrombin. This again is similar to L-antithrombin in the crystal structure in which the gate has "opened" away from the body of the molecule by a rotation of 24 degrees to facilitate the relocation of strand 1C from its ordered position in the C-beta-sheet to a disordered surface loop. At 60 degrees C in the absence of citrate, antithrombin (and other serpins) rapidly polymerizes. In the presence of citrate, the formation of L-antithrombin is slow and increases with time, indicating that the inhibition of polymer formation by citrate allows the time necessary for the much slower formation of the L form. We therefore suggest that L-antithrombin formation is a two-step process: an initial rapid conformational change, probably including partial incorporation of the reactive loop into the A-sheet (as in the active molecule in the crystal structure) and displacement of s1C from the C-beta-sheet which supports polymer formation, and a much slower transition to complete loop insertion within the A-beta-sheet. It is likely that both the first rapid transitional step and the structural features that impose resistance to the second more extensive conformational change reflect the optimization of the unique inhibitory function in the serpins.
Amino Acid Sequence↗
Improved diffraction of antithrombin crystals grown in microgravity.
Crystals of antithrombin were grown both on earth and in microgravity aboard US Space Shuttle Flight STS-67. The quality of crystals grown in both environments was highly variable and many could not be indexed. The microgravity crystals, however, generally diffracted better, as demonstrated by a novel procedure that estimates the resolution of the Bragg scatter from single diffraction images, without requiring knowledge of the cell dimensions of the crystal. Whereas the best earth-grown crystals never diffracted beyond 3 angstroms resolution, the best microgravity crystal diffracted to 2.6 angstroms. The improvement, demonstrated here by a comparison of 23 microgravity and 12 earth-grown crystals, is attributed to better ordered crystal growth in microgravity, although other factors may have contributed also.
Antithrombin III↗
Heparin-dependent modification of the reactive center arginine of antithrombin and consequent increase in heparin binding affinity.
Antithrombin, the principal plasma inhibitor of coagulation proteinases, circulates in a form with low inhibitory activity due to partial insertion of its reactive site loop into the A-beta-sheet of the molecule. Recent crystallographic structures reveal the structural changes that occur when antithrombin is activated by the heparin pentasaccharide, with the exception of the final changes, which take place at the reactive center itself. Here we show that the side chain of the P1 Arg of alpha-antithrombin is only accessible to modification by the enzyme peptidylarginine deiminase on addition of the heparin pentasaccharide, thereby inactivating the inhibitor, whereas the natural P1 His variant, antithrombin Glasgow, is unaffected, indicating that only the P1 Arg becomes accessible. Furthermore, the deimination of P1 Arg converts antithrombin to a form with 4-fold higher affinity for the heparin pentasaccharide, similar to the affinity found for the P1 His variant, due to a lowered dissociation rate constant for the antithrombin-pentasaccharide complex. The results support the proposal that antithrombin circulates in a constrained conformation, which when released, in this study by perturbation of the bonding of P1 Arg to the body of the molecule, allows the reactive site loop to take up the active inhibitory conformation with exposure of the P1 Arg.
Antithrombin III↗
The 2.6 A structure of antithrombin indicates a conformational change at the heparin binding site.
The crystal structure of a dimeric form of intact antithrombin has been solved to 2.6 A, representing the highest-resolution structure of an active, inhibitory serpin to date. The crystals were grown under microgravity conditions on Space Shuttle mission STS-67. The overall confidence in the structure, determined earlier from lower resolution data, is increased and new insights into the structure-function relationship are gained. Clear and continuous electron density is present for the reactive centre loop region P12 to P14 inserting into the top of the A-beta-sheet. Areas of the extended amino terminus, unique to antithrombin and important in the binding of the glycosaminoglycan heparin, can now be traced further than in the earlier structures. As in the earlier studies, the crystals contain one active and one latent molecule per asymmetric unit. Better definition of the electron density surrounding the D-helix and of the residues implicated in the binding of the heparin pentasaccharide (Arg47, Lys114, Lys125, Arg129) provides an insight into the change of affinity of binding that accompanies the change in conformation. In particular, the observed hydrogen bonding of these residues to the body of the molecule in the latent form explains the mechanism for the release of newly formed antithrombin-protease complexes into the circulation for catabolic removal.
Antithrombin III↗
Late reversibility of chronic ifosfamide-associated nephrotoxicity in a child.
Although reversibility of acute ifosfamide-induced nephrotoxicity is well documented, there is a paucity of data concerning the long-term outcome of chronic renal toxicity, and full recovery from established damage has not been reported. A 4-year-old boy presented with hypophosphatemic rickets 9 months after completion of combination chemotherapy (including ifosfamide) for prostatic rhabdomyosarcoma. Further investigation confirmed glomerular and generalised tubular dysfunction with a Fanconi syndrome. However, serial investigation over the next 4 years revealed complete and sustained recovery of this chronic nephrotoxicity.
Antineoplastic Agents, Alkylating↗
The influence of ifosfamide scheduling on acute nephrotoxicity in children.
Nephrotoxicity is a significant problem in children after treatment with ifosfamide. Acute changes in renal function were compared in 16 children receiving 9 g m(-2) of ifosfamide as a 72-h continuous infusion on one occasion and, on another course, divided into three 1-h infusions on consecutive days. Subclinical acute nephrotoxicity was demonstrated with both schedules, but there were no significant differences in severity.
Adolescent↗
How to organise the paediatric MRCP (UK) part II clinical examination.
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Attitude to Health↗
Intralesional fluorouracil/epinephrine injectable gel for treatment of condylomata acuminata. A phase 3 clinical study.
BACKGROUND AND DESIGN: A new intralesional sustained-release chemotherapy is under development as a treatment for condylomata acuminata; it is administered as an injectable gel that consists of fluorouracil and epinephrine with a purified bovine collagen as the gellant (fluorouracil/epinephrine gel). In this randomized, double-blind study, we evaluated the safety and efficacy of this intralesional treatment in 401 patients, using 2 active drug formulations (fluorouracil/epinephrine gel and fluorouracil gel alone) and a placebo. Each lesion was injected once a week for up to 6 weeks, and patients were followed up for 3 months. RESULTS: A total of 359 patients with 1926 condylomata underwent evaluation. For all lesions treated with fluorouracil/epinephrine gel, the complete response (CR) rate was 77%. For all patients treated with fluorouracil/epinephrine gel, the CR rate was 61%. The fluorouracil/epinephrine gel was significantly more effective (P < .002) in treating condylomata than the fluorouracil gel without epinephrine (CR rate, 43%); both were superior to placebo (CR rate, 5%). At 3 months after completion of treatment, recurrence rates in patients with CRs were as follows: fluorouracil/epinephrine gel group, 50%; fluorouracil gel group, 58%. No clinically significant drug-related systemic reactions occurred. Finally, the type and severity of local tissue reactions of patients with a positive pretreatment collagen skin test result (6/401 [1.5%]) were similar to those of patients with a negative collagen skin test result. CONCLUSION: The fluorouracil/epinephrine injectable gel is a safe and effective treatment for condylomata acuminata.
Adolescent↗
Structural mobility of antithrombin and its modulation by heparin.
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Antithrombin III↗
Risk factors for ifosfamide nephrotoxicity in children.
BACKGROUND: Risk factors for long-term nephrotoxicity after ifosfamide for childhood cancers are not fully known. We have studied patient-related and treatment-related risk factors for chronic ifosfamide nephrotoxicity. METHODS: A group of 23 children who had received ifosfamide at age 2.1-16.2 years (median 6.9) for various cancers were assessed for nephrotoxicity, at 1-28 (2) months after the end of treatment, by renal function testing, laboratory values, and a grading score (none, mild, moderate, severe). No patient had received cisplatin or undergone nephrectomy. 13 children were reassessed at 10-26 (23) months; eight had died and two were not evaluable. The median total ifosfamide dose was 100.8 (9.0-160.4) g/m2 over a median of 15 courses every 3 weeks as a 48-72 h continuous intravenous infusion (in 22 cases), with mesna and hydration. FINDINGS: Glomerular filtration rate was below normal in ten (45%) of 22 evaluable children; their rate was 61-85 mL/min per 1.73 m2. Proximal tubular toxicity led to hypophosphataemic rickets and/or renal tubular acidosis in six children, and distal tubular toxicity caused nephrogenic diabetes insipidus in one. Of the risk factors analysed by multiple regression, only total ifosfamide dose was associated with proximal tubular toxicity. Only two of ten evaluable patients who received under 100 g/m2 developed moderate nephrotoxicity, whereas six of ten who received over this dose had moderate or severe nephrotoxicity. INTERPRETATION: High total ifosfamide dose was the only risk factor we identified. Although inter-patient variability was high, cumulative doses of 100 g/m2 or higher should be avoided in children with cancer.
Adolescent↗
Specificity of pH and osmolality of early morning urine sample in assessing distal renal tubular function in children: results in healthy children.
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Adolescent↗
Ifosfamide nephrotoxicity: limited influence of metabolism and mode of administration during repeated therapy in paediatrics.
This study investigated the relationship between both acute and chronic nephrotoxic effects of ifosfamide (IFO) and its metabolism. 15 paediatric patients (4 girls) were investigated. Each received 6-9 g/m2 IFO over 15 days, repeated every 3 weeks for up to 16 courses. The pharmacokinetics and metabolism of IFO were measured during its administration, either as a continuous 72 h infusion or as three bolus doses of 3 g/m2 on consecutive days. In 8 patients, the metabolism of IFO was investigated during one early course and one late course to determine the magnitude of any changes following repeated administration. Acute measures of renal toxicity were not correlated with any of the IFO pharmacokinetic or metabolic parameters in the same course, whether the drug was administered as a bolus or by continuous infusion. Chronic renal toxicity, determined 1 month (n = 13) or 6 months (n = 8) after treatment, did not correlate with any of the IFO pharmacokinetic or metabolic parameters in any individual course of treatment. The overall degree of nephrotoxicity, however, was correlated with the changes in metabolism between late and early courses (n = 8). There was a negative correlation between the change in area under the curve of the dechloroethylated metabolites of IFO and the overall nephrotoxicity at 1 month or 6 months after treatment (both r2 = 0.66, P = 0.014). The results imply that patients in whom metabolism via dechloroethylation decreases are at a greater risk of chronic nephrotoxicity. This is contrary to the hypothesis that the systemic production of chloroacetaldehyde is the mechanism by which IFO causes nephrotoxicity. The importance of acute and chronic changes in renal function for long-term outcome remains to be determined.
Acute Disease↗
Pineal tumours in the north of England 1968-93.
The records of 38 patients under 25 years of age presenting with pineal tumours between 1968 and 1993, identified from the Northern Region Children's and Young Adults' Malignant Disease Registry, were analysed retrospectively with regards to clinical presentation, diagnostic approach, treatment strategy, and outcome. The overall five year survival was 45%. Fifteen patients had a histological diagnosis: six with germinomas, three with teratomas, three with astrocytomas, and three with pinealoblastomas. One patient had a definitive diagnosis of teratoma made on the basis of raised tumour markers (alpha fetoprotein). Treatment consisted of surgery (87%) (ventriculoperitoneal or atrial shunt and/or biopsy), and/or radiotherapy (82%), and/or chemotherapy (26%). Those patients with a tissue diagnosis appeared to have a more favourable outcome, especially after 1976 when treatment was determined by tumour type (five year survival for those with a tissue diagnosis was 91% v 51% for those without, 95% confidence intervals 74 to 100% and 26 to 75%). This study suggests that tissue diagnosis allows more appropriate treatment to be delivered for children with pineal tumours resulting in improved survival. Referral to a centre with neurosurgery, radiotherapy, neuropathology, and paediatric oncology collaboration is essential.
Adolescent↗
Disseminated BCG infection in severe combined immunodeficiency presenting with severe anaemia and associated with gross hypersplenism after bone marrow transplantation.
An infant with severe combined immunodeficiency (SCID) is described, who presented with severe anaemia and hepatosplenomegaly due to disseminated Bacillus Calmette-Guérin (BCG) infection involving the bone marrow, liver and spleen. After BMT, huge splenic enlargement occurred, presumably due to proliferation of engrafted donor lymphocytes, leading to severe hypersplenism. Peripheral blood cell consumption was resolved by splenectomy, but gradual loss of the marrow graft followed.
Anemia↗
The measles/rubella immunisation campaign in Scotland.
This report describes why the 1994 measles/rubella immunisation campaign was necessary, reviews its implementation, and provides information on its impact in Scotland where 60,504 children were immunised. Data on suspected adverse reactions to the measles/rubella vaccine are also presented. A large UK measles epidemic was predicted by independent researchers, and action in response was planned in accordance with the recommendations of the Joint Committee on Vaccination and Immunisation. As a result, the epidemic predicted for England and Wales was averted and the epidemic which had occurred in the West of Scotland was prevented from spreading to the East. Laboratory-confirmed cases of measles and rubella are now rare in all parts of the UK, especially in the age group targeted by the campaign. Most reported adverse reactions were mild and self-limiting: only one child per 6,700 vaccinated in the UK was reported to have suffered any adverse reaction. Immediate, serious reactions were either allergic or were described as convulsions (most associated with immediate syncope); all of these children recovered fully. Later onset neurological problems were reported at rates no higher than expected from normal population incidence.
Adolescent↗
Osteogenic sarcoma of the neural arch.
Osteogenic sarcoma arising in the spine, and in particular the neural arch, is rare. We present the case of a 4-year-old girl with an osteogenic sarcoma arising in the left transverse process of the second lumbar vertebra. The child received chemotherapy and delayed surgical excision was performed following a favourable response. The radiological features and differential diagnosis are discussed.
Child, Preschool↗