Cairo consensus on the IVF laboratory environment and air quality: report of an expert meeting.

This proceedings report presents the outcomes from an international Expert Meeting to establish a consensus on the recommended technical and operational requirements for air quality within modern assisted reproduction technology (ART) laboratories. Topics considered included design and construction of the facility, as well as its heating, ventilation and air conditioning system; control of particulates, micro-organisms (bacteria, fungi and viruses) and volatile organic compounds (VOCs) within critical areas; safe cleaning practices; operational practices to optimize air quality while minimizing physicochemical risks to gametes and embryos (temperature control versus air flow); and appropriate infection-control practices that minimize exposure to VOC. More than 50 consensus points were established under the general headings of assessing site suitability, basic design criteria for new construction, and laboratory commissioning and ongoing VOC management. These consensus points should be considered as aspirational benchmarks for existing ART laboratories, and as guidelines for the construction of new ART laboratories.

Wound closure in patients with DFU: a cost-effectiveness analysis of two cellular/tissue-derived products.

OBJECTIVE: Determine the cost-effectiveness of extracellular matrix (ECM) relative to human fibroblast-derived dermal substitute (HFDS) on diabetic foot ulcer (DFU) wound closure. METHOD: Outcomes data were obtained from a 12-week, randomised, clinical trial of adults aged 18 years or older diagnosed with type 1 or type 2 diabetes with a DFU. Patients were treated with either ECM or HFDS treatment. A two-state Markov model (healed and unhealed) with a 1-week cycle length was developed using wound-closure rates from the trial to estimate the number of closed-wound weeks and the expected DFU cost per patient. Results were recorded over 12 weeks to estimate the number of closed-wound weeks per treatment and the average cost to achieve epithelialisation (primary outcome). The perspective of the analysis was that of the payer, specifically the Centers for Medicare and Medicaid Services. No cost discounting was performed because of the short duration of the study. RESULTS: The study consisted of 26 patients, with 13 in each group. In the ECM group, 10 wounds closed (77%), with an average closure time of 36 days; 11 wounds closed in the HFDS group (85%), with an average closure time of 41 days. There was no significant difference between these results (p=0.73). Over 12 weeks, the expected cost per DFU was $2522 (£1634) for ECM and $3889 (£2524) for HFDS. Patients treated with HFDS incurred total treatment costs that were approximately 54% higher than those treated with ECM. Sensitivity analyses revealed that the total cost of care for two applications of HFDS was more costly than eight applications of ECM by approximately $500 (£325). CONCLUSION: In patients with DFU, ECM yielded similar clinical outcomes to HFDS but at a lower cost. Health-care providers should consider ECM as a cost-saving alternative to HFDS. DECLARATION OF INTEREST: A.M. Gilligan, and C.R. Waycaster, are employees of Smith & Nephew Inc.. This study was funded by Smith & Nephew Inc.. A.L. Landsman, reports no conflicts of interest.

Patient-reported outcomes with selpercatinib treatment in patients with RET-driven cancers in the phase I/II LIBRETTO-001 trial.

BACKGROUND: This post-hoc retrospective study describes long-term patient-reported outcomes (PROs) for REarranged during Transfection (RET)-altered non-small-cell lung cancer (NSCLC), medullary thyroid cancer (MTC), non-MTC thyroid cancer (TC), and tumor agnostic (TA) patients (Data cut-off: January 2023) from the LIBRETTO-001 trial. PATIENTS AND METHODS: Patients completed the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire-Core 30 (QLQ-C30). Patients with MTC also completed a modified version of the Systemic Therapy-Induced Diarrhea Assessment Tool (mSTIDAT). The proportion of patients with improved, stable, or worsened status after baseline was reported. PROs were summarized at 3 years (cycle 37) post-baseline for the NSCLC and MTC cohorts, and at 2 years (cycle 25) post-baseline for the TC and TA cohorts. Time-to-event outcomes (time to first improvement or worsening and duration of improvement) were reported. RESULTS: The baseline assessment was completed by 200 (63.3%), 209 (70.8%), 50 (76.9%), and 38 (73.1%) patients in the NSCLC, MTC, TC, and TA cohorts, respectively. The total compliance rate was 80%, 82%, 70%, and 85%, respectively. Approximately 75% (NSCLC), 81% (MTC), 75% (TC), and 40% (TA) of patients across all cohorts reported improved or stable QLQ-C30 scores at year 3 (NSCLC and MTC) or year 2 (TC and TA) with continuous selpercatinib use. Across cohorts, the median time to first improvement ranged from 2.0 to 19.4 months, the median duration of improvement ranged from 1.9 to 28.2 months, and the median time to first worsening ranged from 5.6 to 44.2 months. The total compliance rate for the mSTIDAT was 83.7% and the proportion of patients with MTC who reported diarrhea on the mSTIDAT was reduced from 80.8% at baseline to 35.6% at year 3. CONCLUSIONS: A majority of patients with RET-driven cancers improved or remained stable on most QLQ-C30 domains, demonstrating favorable health-related quality of life as measured by the QLQ-C30 during long-term treatment with selpercatinib.

Automated Interictal Epileptiform Discharge Detection from Scalp EEG Using Scalable Time-series Classification Approaches.

Deep learning for automated interictal epileptiform discharge (IED) detection has been topical with many published papers in recent years. All existing works viewed EEG signals as time-series and developed specific models for IED classification; however, general time-series classification (TSC) methods were not considered. Moreover, none of these methods were evaluated on any public datasets, making direct comparisons challenging. This paper explored two state-of-the-art convolutional-based TSC algorithms, InceptionTime and Minirocket, on IED detection. We fine-tuned and cross-evaluated them on a public (Temple University Events - TUEV) and two private datasets and provided ready metrics for benchmarking future work. We observed that the optimal parameters correlated with the clinical duration of an IED and achieved the best area under precision-recall curve (AUPRC) of 0.98 and F1 of 0.80 on the private datasets, respectively. The AUPRC and F1 on the TUEV dataset were 0.99 and 0.97, respectively. While algorithms trained on the private sets maintained their performance when tested on the TUEV data, those trained on TUEV could not generalize well to the private data. These results emerge from differences in the class distributions across datasets and indicate a need for public datasets with a better diversity of IED waveforms, background activities and artifacts to facilitate standardization and benchmarking of algorithms.

Functional pancreatic beta-cell line from SV40 T-antigen transgenic mouse.

Cell line IgSV195, derived from a pancreatic tumor that arose in an SV40 T-antigen transgenic mouse, retains certain morphological and physiological characteristics of pancreatic beta-cells throughout in vitro and in vivo passage. Insulin secretion is stimulated by exposure of these cells to fetal bovine serum and a combination of 3-isobutyl-1-methylxanthine and glutamine but not by concentrations of glucose in the physiological range. Insulin processing appears to be intact. Neither class I nor class II major histocompatibility complex (MHC) antigens are routinely expressed at the cell surface; however, MHC class I--but not class II--encoded gene products are detected after treatment with recombinant interferon-gamma (IFN-gamma) alone or in combination with tumor necrosis factor. Cytolysis of IgSV195 cells by SV40 T-antigen-specific H-2b-restricted lymphocytes is similarly dependent on IFN-gamma pretreatment. These results emphasize that SV40 T-antigen transgenic mice are likely sources of cell lines that retain their differentiated function in vitro. The IgSV195 cell line provides an accessible model in which to investigate the control of gene expression and function of pancreatic beta-cells.

Differential subcellular distribution of bioactive human chorionic gonadotropin in pseudopregnant rat ovary.

The subcellular metabolism of internalized hCG was examined by monitoring the distribution of bioactive and immunoreactive hCG in subcellular fractions of pseudopregnant rat ovaries. Homogenates of ovaries from rats injected with 1.0 microgram (12.8 IU; bioassay) hCG were fractionated on self-generating Percoll gradients into three hCG-containing compartments: soluble proteins (cytosolic fraction), a combined plasma membrane/prelysosomal vesicle fraction, and lysosomes. The hCG level in each fraction was measured by RIA and in vitro bioassay. When necessary, receptor-bound hCG was dissociated at low pH before assay. Levels of cytosolic hCG were highest 1-3 h after injection, attaining peak immunoreactive concentrations of 18 ng/ovarian pair. Cytosolic hormone was not primarily derived from nonspecific trapping of serum or interstitial fluid, because after 1.0 microgram [125I]iodobovine gamma-globulin was injected into rats, cytosolic globulin levels (nanograms per ovarian pair) were approximately 7-10 times lower than those of hCG. Cytosolic hCG retained significant bioactivity for at least 10 h after hCG stimulation. Peak immunoreactive hCG levels associated with the plasma membrane/prelysosomal fraction were 82 ng/ovarian pair between 3 and 6 h after hCG injection, and hormone associated with that fraction retained the highest bioactivity of the three fractions examined. Peak lysosomal hCG levels reached 55 ng/ovarian pair 10 and 14 h after hCG stimulation, but lysosomal hCG was not bioactive. These results suggest that the lysosomal compartment is a major pathway for hCG inactivation. A nonlysosomal pathway for hCG inactivation may exist, because the cytosolic compartment contained partially inactivated hormone that did not appear to be of lysosomal origin. Cytosolic hCG may reflect hormone delivered to the cell cytoplasm or to the extracellular fluid that is either modified within prelysosomal vesicles or is degraded subsequently by nonlysosomal proteases.

Baseline blood pressure but not early computed tomography changes predicts major hemorrhage after streptokinase in acute ischemic stroke.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage is the most serious complication of thrombolytic therapy for stroke. We explored factors associated with this complication in the Australian Streptokinase Trial. METHODS: The initial CT scans (< or =4 hours after stroke) of 270 patients were reviewed retrospectively by an expert panel for early signs of ischemia and classified into the following 3 categories: no signs or < or =1/3 or >1/3 of the vascular territory. Hemorrhage on late CT scans was categorized as major or minor on the basis of location and mass effect. Stepwise, backward elimination, multivariate logistic regression analysis was used to identify risk factors for each hemorrhage category. RESULTS: Major hemorrhage occurred in 21% of streptokinase (SK) and 4% of placebo patients. Predictors of major hemorrhage were SK treatment (odds ratio [OR], 6.40; 95% CI, 2.50 to 16.36) and elevated systolic blood pressure before therapy (OR, 1.03; 95% CI, 1.01 to 1.05). Baseline systolic blood pressure >165 mm Hg in SK-treated patients resulted in a >25% risk of major secondary hemorrhage. Early ischemic CT changes, either < or =1/3 or >1/3, were not associated with major hemorrhage (OR, 1.58; 95% CI, 0.65 to 3.83; and OR, 1.11; 95% CI, 0.45 to 2.76, respectively). Minor hemorrhage occurred in 30% of the SK and 26% of the placebo group. Predictors of minor hemorrhage were male sex, severe stroke, early CT changes, and SK treatment. Ninety-one percent of patients with major hemorrhage deteriorated clinically compared with 23% with minor hemorrhage. CONCLUSIONS: SK increased the risk of both minor and major hemorrhage. Major hemorrhage was also more likely in patients with elevated baseline systolic blood pressure. However, early CT changes did not predict major hemorrhage. Results from this study highlight the importance of baseline systolic blood pressure as a potential cause of hemorrhage in patients undergoing thrombolysis.

Epidermal growth factor-induced increases in inositol trisphosphates, inositol tetrakisphosphates, and cytosolic Ca2+ in a human hepatocellular carcinoma-derived cell line.

A human hepatocellular carcinoma-derived cell line, PLC/PRF/5, was examined for its ability to respond to epidermal growth factor (EGF) exposure with increased phosphatidylinositol 4,5-bisphosphate hydrolysis. Upon addition of EGF (25 ng/ml), a rapid (10-15 s) but transient increase in Ins(1,4,5)P3 levels and large, prolonged (2 min) increases in Ins(1,3,4,5)P4 and Ins(1,3,4)P3 levels were detected. Increases in cytosolic Ca2+ were observed after a 10 to 20 s lag, reaching peak value at 1 min, and remaining elevated for 10 min. The initial burst of cytosolic Ca2+ occurred in the absence of extracellular Ca2+ and probably reflects mobilization of intracellular Ca2+ stores. In cells pretreated with EGTA, the sustained component of the Ca2+ response was not observed. Comparison of the inositol phosphate and Ca2+ responses of PLC/PRF/5 cells to responses reported in other cell types indicates that this cell line is a good model for EGF action in liver.

Cortical excitability and recovery curve analysis in generalized epilepsy.

Seven untreated patients with idiopathic generalized epilepsy were studied with recovery curve analysis using transcranial magnetic stimulation and compared with 16 controls. Patients had a shorter period of inhibition of the test response following a conditioning stimulus and demonstrated a period of increased facilitation of the test response at interstimulus intervals of 200 to 300 msec, which was similar to the mean interdischarge interval of spike-wave activity on EEG. This provides further evidence of cortical hyperexcitability in idiopathic generalized epilepsy.

Rat ovarian subcellular compartmentalization of luteinizing hormone.

To determine if human luteinizing hormone (hLH) follows the same subcellular route in the pseudo-pregnant rat ovary as human chorionic gonadotropin (hCG), the distribution of immunoreactive and bioactive hLH within cytosol, lysosomes, and a combined plasma membrane/prelysosomal vesicle fraction was examined. Immunoreactive levels were determined using a specific radioimmunoassay and bioactive levels were determined in an in vitro Leydig cell bioassay. Low cytosolic hLH levels were apparent the first few hours after hLH administration and may reflect at least some contamination by serum and interstitial fluid. Plasma membrane/prelysosomal vesicles attained the highest hLH concentration 1 h after hLH injection, after which hLH levels declined rapidly until barely detectable levels were observed at 18 h. The hormone in this fraction was receptor bound and exhibited the highest bioactivity of the three fractions examined. Lysosomal hLH concentrations were highest at 12 after hLH injection and were maintained at high levels through 18 h. Substantial amounts of lysosomal hLH appeared to be receptor bound but this hormone was not bioactive. In situ degradation of the oligosaccharides may have occurred while lysosomal hLH was receptor bound. Lysosomal degradation is the major pathway for hLH inactivation as has been described for hCG. However, hLH may be degraded within lysosomes more quickly than hCG. The differential subcellular distribution of immunoreactive and bioactive hLH provides strong support for hLH internalization and degradation within ovarian luteal cells.

Sex differences in presentation, severity, and management of stroke in a population-based study.

OBJECTIVES: Women may have poorer outcomes after stroke than men because of differences in their acute management. We examined sex differences in presentation, severity, in-hospital treatment, and early mortality in a cohort of first-ever-in-a-lifetime stroke patients. METHODS: Data were collected from May 1, 1996, to April 30, 1999, in the North East Melbourne Stroke Incidence Study. Stroke symptoms, prestroke medical history, in-hospital investigations, admission and discharge medications, initial stroke severity, and 28-day mortality were recorded. Multivariable regression was used to estimate sex differences in treatment, investigations, and 28-day mortality. RESULTS: A total of 1,316 patients were included. Women were older (mean age 76 +/- 0.6 vs 72 +/- 0.6, p < 0.01), had more severe strokes (median NIH Stroke Scale score 6 vs 5, p < 0.01), and more likely to experience loss of consciousness (31% vs 23%, p = 0.003) and incontinence (22% vs 11%, p = 0.01) than men. Women were less often on lipid-lowering therapy on admission. Echocardiography and carotid investigations were less frequently performed in women due to greater age and stroke severity. Women had greater 28-day mortality (32% vs 21%, p < 0.001) and stroke severity (44% vs 36%, p = 0.01) than men, but adjustment for age, comorbidities, and stroke severity (for mortality only) completely attenuated these associations. CONCLUSION: Sex differences seen in this study were mostly explained by women's older age, greater comorbidity, and stroke severity. The reasons for differences according to age may need further examination.

Culture and quality control of embryos.

Improvement of embryo quality during in-vitro culture can be achieved by understanding and controlling the requirements of gametes and embryos. The most obvious route is to alter culture media, but standardization could be influenced by diverse environmental factors. Abnormal embryos from patients with multiple failures probably do not benefit from standardization and require specialized therapy, that is if their physiology is not already irreversibly jeopardized during gametogenesis. This paper describes the adverse environmental factors present in laboratory air and released by common products used by laboratories. Assays and results of the air determinations in several laboratories are reported, as well as potential counter measures. The possibility of altering the immediate environment of the nucleus of the egg by ooplasmic transplantation is also considered, and the first attempts resulting in two ongoing pregnancies are reported.

EGF receptor down-regulation attenuates ligand-induced second messenger formation.

Epidermal growth factor (EGF)-induced increases in cytosolic Ca2+ and inositol polyphosphate production were compared in a human hepatocellular carcinoma-derived cell line, PLC/PRF/5, and in an EGF receptor-overexpressing subline, NPLC/PRF/5. Formation of these second messengers was correlated to EGF receptor display at the cell surface by monitoring ligand-induced EGF receptor down-regulation. Both cell lines exhibited a strikingly similar cytosolic Ca2+ increase upon exposure to EGF. The initial inositol phosphate responses were also similar in the two cell lines; inositol 1,4,5-trisphosphate increased within 10-15 s and returned to prestimulatory values after 2 min in both cell lines, while inositol tetrakisphosphate and inositol 1,3,4-trisphosphate were elevated after a 2-min exposure to EGF. At later times the responses were markedly different; NPLC/PRF/5 cells exhibited prolonged production of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate (maximum at 1-3 h) but PLC/PRF/5 cells showed decreased levels of these isomers after 10 min and a return to basal values by 1 h. Exposure of PLC/PRF/5 cells to EGF caused a progressive decrease in the amount of EGF receptor at the cell surface whereas such treatment did not change the surface receptor levels in NPLC/PRF/5 cells. Kinetic analysis of EGF receptor down-regulation showed that receptor internalization was rapid enough to account for the transient nature of the inositol phosphate response in PLC/PRF/5 cells. Thus, the divergent patterns of signaling exhibited by the two cell lines may reflect differences in the efficiency of EGF-induced down-regulation of surface receptors.

Divergent metabolism of human chorionic gonadotropin subunits within rat ovarian lysosomes.

Pseudopregnant rats were injected with either native human chorionic gonadotropin or with (125I)-human chorionic gonadotropin and their ovarian homogenates fractionated on Percoll density gradients. The levels of alpha and beta subunits within subcellular fractions were measured using radioimmunoassays specific for each subunit. Radioactivity measurements of fractions obtained from rats injected with (125I)-human chorionic gonadotropin were used as a separate index of alpha subunit distribution. The alpha subunit was primarily restricted to a combined plasma membrane/prelysosomal vesicle fraction. Immunoreactive beta subunit was present at high concentrations within both this plasma membrane/prelysosomal vesicle fraction and within lysosomes. The striking difference in alpha and beta subcellular distribution may arise from differential sensitivities to lysosomal enzymes.

Variation in EGF-induced EGF receptor downregulation in human hepatoma-derived cell lines expressing different amounts of EGF receptor.

The effect of epidermal growth factor (EGF) receptor overexpression on ligand-induced EGF receptor downregulation was examined using a hepatoma-derived cell line, PLC/PRF/5, which expresses normal amounts of the EGF receptor, and a subline, NPLC/PRF/5, which expresses 10-fold more receptors at its cell surface. PLC/PRF/5 cells efficiently downregulated surface receptor levels upon exposure to saturating and subsaturating concentrations of EGF; the rate of receptor downregulation corresponded to that of ligand-receptor internalization. Upon internalization, EGF receptors were degraded and receptor biosynthesis remained at basal levels. EGF surface receptor remained downregulated for as long as cells were exposed to EGF. By contrast, surface EGF receptor abundance in NPLC/PRF/5 cells decreased by only 5-15% after 1-4 h incubation with subsaturating doses of EGF and actually increased by 67% within 20 h. Exposure of these cells to saturating concentrations of EGF induced modest decreases in surface receptor abundance during the initial 12 h incubation, followed by a progressive decline to 30% of initial values by 24 h. Relative ligand-receptor internalization rates in NPLC/PRF/5 cells were lower than those in PLC/PRF/5, although their surface receptor population was even higher than that predicted by the decreased internalization rates. EGF receptor degradation in NPLC/PRF/5 cells was also inhibited; exposure to saturating levels of EGF for more than 16 h was necessary before significant degradation occurred. Receptor protein and mRNA biosynthesis in NPLC/PRF/5 were stimulated by 8 h exposure to EGF but when saturating concentrations of EGF were present for 16 h, receptor biosynthesis was inhibited. EGF receptor overexpression circumvents the downregulatory effect of EGF by decreasing the rate of receptor internalization, inhibiting degradation of the internalized receptor pool, and stimulating EGF receptor biosynthesis. Conversely, receptor downregulation becomes pronounced at late times when receptor degradation is high and biosynthesis is inhibited.

Ambient air and its potential effects on conception in vitro.

Incidences of chemical air contamination (CAC) are common in assisted reproductive technology, but not reported in peer review format. Justified fear of car and industrial emissions clearly exists among reproductive specialists, but standards for air contents and gaseous emission limits have not been reported. Here, we describe air sampling methods and assay systems which can be applied to any laboratory or laboratory item. It was found that unfiltered outside air may be cleaner than high efficiency particulate air filtration (HEPA) filtered laboratory air or air obtained from incubators, due to accumulation of volatile organic compounds derived from adjacent spaces or specific laboratory products such as compressed CO2, sterile Petri dishes and other materials or devices known to release gaseous emissions. Specific groups of products such as anaesthetic gases, refrigerants, cleaning agents, hydrocarbons and aromatic compounds such as benzene and toluene are described. The latter were shown to accumulate specifically in incubators. Isopropyl alcohol was the most dominant product found, though it was not used by the laboratory staff. Concentrations of this agent were low in incubator air, indicating that it was probably absorbed by the water in the pan or by culture medium. Measures to counter CAC are proposed, including the use of activated carbon filters and oxidizing material placed in the central air handling systems, in separate free-standing units or even inside the incubators.

The origin, effects and control of air pollution in laboratories used for human embryo culture.

Testing shows that most laboratories conducting human gamete and embryo culture have air quality and sources of contamination that exceed the levels measured in homes, businesses and schools. The sources of these contaminants have been shown to be either from activities outside the laboratory, or emitted from materials used in the facility, such as compressed gas, cleaning and sterilizing agents, plastic and stored materials. Both the laboratory structure and the air handling systems may affect the air composition. The significance of these findings is being validated by the accumulation of field case studies and now by assay procedures. Products given off by road sealant were shown to have accumulated in one of the examined laboratories, adjacent to a large re-surfaced parking area. Aldehydes such as acrolein, hexanal, decanal, pentanal and others were detected at elevated concentrations that were statistically significant. Since it is not appropriate to add potentially suspect chemicals to human embryos, we used a mouse-model to study the effect of acrolein. The growth of mouse embryos was significantly affected after acrolein was added at different concentrations to the culture environment. The physiological effect was noted at concentrations in the low ppm range. The testing end-point of embryo death must still be considered to be a crude basis for evaluating toxicological effects, since it involves addition of compounds to culture media and unprotected growth until the blastocyst stage. The findings may, however, support observations of decreased pregnancy rate following exposure of human embryos to aldehydes or other adverse conditions. With proper engineering and material selection, it is possible to reduce such contamination. The usefulness of this approach for controlling aldehydes has been demonstrated by decreasing levels in the laboratory to below those of the outside air.

Intra-arterial streptokinase in acute ischaemic stroke. A pilot study.

AIMS: To assess the safety and efficacy of the intra-arterial administration of streptokinase within 24 hours of acute ischaemic stroke. METHODS: Patients who presented to the Austin Hospital casualty department between 3 and 22 hours after an acute stroke were considered for the study. Eligible patients had pretreatment non-contrast computed tomographic scans of the brain to exclude haemorrhage. Streptokinase (250,000 units) was administered directly into the common carotid artery or the cervical portion of the internal carotid artery. MAIN OUTCOME MEASURES: The incidence of symptomatic and asymptomatic cerebral haemorrhage, haemorrhagic transformation of infarction, angiographic reperfusion, clinical outcome at seven to 10 days and the frequency of other complications. RESULTS: Thirteen patients were treated over a 16-month period. Major clinical improvement occurred in five patients (39%) at 48 hours. This was associated with angiographically demonstrated recanalisation of a middle cerebral artery occlusion in two patients and partial recanalisation in two others. Significant hypotension in two patients required therapy to be stopped. In five other cases mild hypotension developed but the streptokinase infusion was completed. Haemorrhagic transformation of the infarct occurred in four patients without clinical deterioration. CONCLUSION: Intra-arterial administration of streptokinase is safe in selected patients with acute ischaemic stroke. The theoretical benefit of an increased local thrombolytic effect and reduced systemic complications, compared with the use of higher intravenous doses, justifies a randomised clinical trial. If therapies such as this are to be successful, rapid referral to an appropriate centre is necessary.