A method for fatigue testing of equine McIII subchondral bone under a simulated fast workout training programme.

BACKGROUND: Standard fatigue testing of bone uses a single load and frequency applied until failure. However, in situ, the subchondral bone of Thoroughbred racehorses is subjected to a combination (or a spectrum) of loads and frequencies during training and racing. OBJECTIVE: To investigate the use of a fatigue testing method for equine third metacarpal (McIII) subchondral bone under a spectrum of loading conditions which a racehorse is likely to experience during a fast workout. STUDY DESIGN: In vitro biomechanical experimental study. METHODS: McIII subchondral bone specimens (n = 12) of racehorses were harvested from left and right medial condyles. A novel fatigue loading protocol was developed based upon a standard sequence of gaits during a typical fast workout protocol. This loading pattern, or loading loop, was repeated until the failure of the specimen. RESULTS: The mean ± standard deviation for all specimens for total time-to-failure was 76,393 ± 64,243 s (equivalent to 18.3 ± 15.7 fast workouts). Ten of twelve specimens withstood at least one complete loop equivalent to a fast workout. All specimens failed during simulated gallop loading. MAIN LIMITATIONS: The resting time between loops was much shorter than in vivo resting time and specimens were unconfined during compressive testing. CONCLUSIONS: This novel fatigue loading protocol more closely mimics in vivo fatigue loading of McIII subchondral bone and demonstrates the importance of the highest speeds in the development of subchondral bone injury.

Production of BioSNG from waste derived syngas: Pilot plant operation and preliminary assessment.

Bio-substitute natural gas (or BioSNG) produced from gasification of waste fuels and subsequent methanation of the product gas could play a crucial role in the decarbonisation of heating and transportation, and could be a vital part of the energy mix in the coming decades. The BioSNG demonstration plant described in this paper seeks to prove the technical feasibility of the thermal gasification of waste to renewable gas, through a preliminary experimental programme to take an existing stream of syngas, methanate it and show that it can be upgraded to gas grid quality requirements. The syngas used in the project is a waste-derived syngas from a two-stage fluidised bed - plasma pilot facility, which is then converted and upgraded in a new, dedicated conversion and clean up plant. Extensive trials were undertaken on methanation and gas upgrading units for over 60 h of continuous operation. The fundamentals of a once-through methanation process train have been established on the demonstration facility and a model built to extend the analysis over different operational parameters. Over the trials, methane outputs of greater than 50 kWth output were routinely produced from three methanation reactors in series, with a total CO conversion exceeding 90% at pressures as low as atmospheric, in line with kinetic model predictions. Retention of CO2 as well as adequate partial pressure of H2O in the process stream was important for process control. The plant provided demonstration of the efficacy of a PSA system for separation of CO2 (99% removal efficiency) as well as the potential to remove a proportion of residual H2, N2 and CO, although this was associated with appreciable CH4 slip. The process can be optimized primarily by reducing inlet temperature to methanation reactors, controlling syngas composition and using adequate steam to carbon ratio, depending on the type of waste. This information can be used to inform the design and economics of subsequent planned commercial plants that could significantly increase the potential of renewable gas in the UK and elsewhere in Europe, providing a low cost route to low carbon heat and transport.

A cranial intercondylar arthroscopic approach to the caudal medial femorotibial joint of the horse.

REASON FOR PERFORMING STUDY: Current noninvasive techniques for imaging the soft tissue structures of the stifle have limitations. Arthroscopy is commonly used for the investigation and treatment of stifle pain. Cranial and caudal arthroscopic approaches to the femorotibial joints are used. However, complete examination of the axial aspect of the medial femorotibial joint (MFTJ) is not possible currently. OBJECTIVE: To develop a cranial approach to the caudal pouch of the MFTJ and to assess whether it would allow a more complete examination of the compartment and facilitate the caudomedial approach. METHOD: The regional anatomy was reviewed and the technique developed on cadavers. A series of nonrecovery surgeries were performed to evaluate the procedure, which was then used in 7 clinical cases. Advantages compared to existing techniques and complications encountered were recorded. RESULTS: Successful entry into the caudal pouch of the MFTJ was achieved in 20 of 22 cadaver legs, 8 of 8 joints of nonrecovery surgery horses and 6 of 7 clinical cases operated. The caudal ligament of the medial meniscus could be visualised, along with other axial structures of the caudal joint pouch. The technique was used to facilitate a caudomedial approach and allowed better triangulation within the joint space. Complications were minor and included puncture of the caudal joint capsule and scoring of the axial medial femoral condyle. CONCLUSIONS AND POTENTIAL RELEVANCE: It is possible to access the caudal pouch of the MFTJ arthroscopically using a cranial intercondylar approach. The technique has advantages when compared to existing techniques and is associated with few significant complications. A cranial approach to the caudal pouch of the MFTJ could complement existing techniques and be useful clinically.

The role of evaluation in the development of a service for children with life-limiting conditions in the community.

BACKGROUND: Much of the care for children and young people with life-limiting conditions is now delivered in the home and new services have developed to support families in this setting. It is essential to monitor and evaluate whether these services are meeting the needs of families. AIMS: To evaluate a new rural community palliative care service for children according to the perceptions of families and service providers, to make changes suggested by families and to re-evaluate 1 year later. METHOD: In 2005, 2 years after the onset of the service, 24 families were sent postal questionnaires, including the Measure of Process of Care (MPOC-UK). Changes suggested by families were then implemented. In 2006, all of the families receiving care from the service (n=27) were given the option of completing the questionnaire independently or with the support of an impartial researcher. Two families also completed qualitative interviews about their experience of the service with an impartial researcher. In both years, the service providers, (n=12 and n=15, respectively) were asked to complete the Measure of Process of Care for Service Providers (MPOC-SP). The service providers were the clinicians providing direct care (paediatrician, community nurses, dietician, psychologist, occupational therapist, physiotherapist, and speech and language therapist). RESULTS: Seven (29%) of families completed the survey in 2005. Families rated 'respectful and supportive care' as the highest domain in the MPOC-UK and 'providing general information' as the lowest. Particular emphasis was placed on improving provision of information during the following year. Fourteen (52%) families completed the survey in 2006. Scores increased across all domains in the second survey. The largest increase was 'providing general information'. CONCLUSION: The results from both of the MPOC tools were extremely useful in helping providers to identify aspects of the service in need of improvement and hence implement valued changes.

Characterisation of the two most abundant genes in the Haemonchus contortus expressed sequence tag dataset.

Analysis of the Haemonchus contortus Expressed sequence tag (EST) dataset revealed that almost 10% of all ESTs (1719 ESTs) belong to a family of related genes. Close analysis of the ESTs suggests that these represent two genes (called here Hc-nim-1 and Hc-nim-2) with multiple alleles of each. These genes show significant similarity to two genes from Caenorhabditis elegans, F54D5.3 (Wormbase accession WBGene00010049, corresponding protein WP:CE28033) and F54D5.4 (WBGene00010050, WP:CE03409) of unknown function. Reverse transcriptase coupled-PCR showed that both genes are transcribed from the L4 stage onwards and are transcribed in both male and female adult worms. A partial bacterial recombinant of the Hc-NIM-1 protein was made and used to raise antiserum in rabbits which recognised a 19 kDa antigen in the water soluble protein fraction of adult worms. By immunohistochemistry, the Hc-NIM-1 protein was localised in the hypodermis of the pharyngeal region of adult worms but not posterior in the hypodermis surrounding the reproductive tract. To investigate the function of this novel protein family we conducted a RNA interference experiment for the homologuous proteins in C. elegans. No visible phenotype was detected after simultaneous RNAi treatment for both Ce-F54D5.3 and Ce-F54D5.4.

International collaborative study to establish the 3rd International Standard for Streptokinase.

An international collaborative study was organized to calibrate a replacement for the current (2nd) International Standard (IS) for Streptokinase, stocks of which are almost exhausted. Two candidate preparations were assayed against the 2nd IS in a study involving 16 laboratories in 12 countries: preparation 88/824 (coded B), and preparation 00/464 (C and D, coded duplicates). Laboratories could use two methods provided, either a fibrin clot lysis assay or a solution chromogenic method, or an in-house method. Laboratories were encouraged to perform more than one method if possible. With the exception of one laboratory which gave outlying results for preparation 00/464, there was good agreement within and between laboratories and no significant differences between potencies using the different methods employed. This study demonstrates that a solution chromogenic assay is an acceptable format for potency determination of the streptokinase preparations in this study and fibrin is not necessary. It has now been agreed that a solution chromogenic plasminogen activation assay replace the current euglobulin reference method for streptokinase activity determination in the European Pharmacopoeia. Study participants, SSC of the International Society on Thrombosis and Haemostasis and the Expert Committee on Biological Standardization (ECBS) at the World Health Organization approved preparation 00/464 (C,D in the study) as the 3rd IS for Streptokinase with a potency of 1030 IU per ampoule.

A proposed reference method for plasminogen activators that enables calculation of enzyme activities in SI units.

A method has been developed for accurately and precisely measuring the activity of a range of plasminogen activators (PAs) used as thrombolytic agents, including streptokinase, tissue plasminogen activator (tPA) and variants, and urokinase (uPA), both single and two chain forms. Plasminogen activation is monitored in a transparent, solid fibrin matrix but uses chromogenic substrate hydrolysis, rather than changes in fibrin, to quantitate the activity of PAs. The method has been tested in two recent international collaborative studies involving tPA and streptokinase where it has been shown to perform very well. Furthermore, the method is based on sound enzymological principles and once correction for the competitive inhibition of fibrin(ogen) is made, the generation of plasmin can be determined in molar terms and hence the activity of PAs can be expressed and compared in SI units (rate of increase in molar concentration of plasmin) as well as International Units. The assay is also arranged in such a way to reflect the behavior of PAs in vivo during thrombolytic therapy and it is shown that the specific activity of streptokinase and tPA in this system reflects plasmin generation capacity of these thrombolytics for doses given in infusions for treatment of myocardial infarction. The method would make a suitable reference method for PAs and provides a rigorous means of studying and modeling the enzymology of fibrinolysis and will be helpful in the rational design of third generation thrombolytic agents.

Expressed sequence tag survey of gene expression in the scab mite Psoroptes ovis--allergens, proteases and free-radical scavengers.

Psoroptes ovis, the causative agent of sheep scab, is an important ectoparasitic mite infecting sheep, goats and cattle. Infection is characterized by an extensive dermatitis, scab formation and intense itching. Initial focal lesions spread outwards, coalesce and may extend over the whole body. The host response to infestation has all the characteristics of an immediate-type hypersensitivity reaction but the mite antigens and allergens which initiate this response are almost completely undefined. Here, 507 randomly selected cDNAs derived from a mixed population of P. ovis were sequenced and the resultant nucleotide sequences subjected to Cluster analysis and Blast searches. This analysis yielded 280 clusters of which 49 had > 1 sequence with 24 showing significant Blast X homology to another protein in the databases. There were 231 sequences which appeared on one occasion and 109 of these showed significant Blast X homology to other sequences in the databases. This analysis identified homologues of 9 different types of allergens which have been characterized in other allergic conditions such as responses to house dust mites. It also identified a number of cysteine proteases which may contribute to lesion development as well as several free-radical scavenging enzymes which may protect the mite from host immune effector responses.

The sacroiliac joints: evaluation using nuclear scintigraphy. Part 1: The normal horse.

REASONS FOR PERFORMING STUDY: Sacroiliac (SI) disease is recognised as a cause of poor hindlimb action but differential diagnosis is often difficult. HYPOTHESES: That in clinically normal horses there would be a significant difference in the ratio of radiopharmaceutical uptake (RU) between the fifth lumbar vertebra (L5) and each tuber sacrale (TS) and between L5 and each SI joint; and that these ratios would alter with age, but ratios would be bilaterally symmetrical. METHODS: Dorsal scintigraphic images of the SI region of 15 horses, selected randomly from the clinic database, were analysed by 2 of the authors, comparing noncorrected and motion-corrected images. To determine scintigraphic anatomy, the scintigraphic images of 10 Thoroughbred horses were superimposed over a ventrodorsal radiographic image of an isolated pelvis. Dorsal scintigraphic images of 40 clinically normal horses age 3-16 years were evaluated using subjective examination, profile analysis and quantification using regions of interest. RESULTS: The tubera sacrale were seen as 2 well-defined oval regions immediately to the left and right of the midline, abaxial to which were larger, approximately oval areas with less RU, representing uptake in the SI joints. The definition between the SI region and the TS was more obvious in younger horses. Nonmotion-corrected images were often not of diagnostic quality or could be misinterpreted as abnormal. There were significant differences in RU between the TS and SI joints compared to L5, and decreased RU in the tubera sacrale with increasing age, but no change of uptake in the SI joint region and no effect of gender on RU. There was a high degree of left-right symmetry of the TS and SI joint regions. CONCLUSIONS AND POTENTIAL RELEVANCE: The scintigraphic images of horses with suspected sacroiliac joint disease should be compared with images of normal horses of comparable age. In normal horses, there was a high degree of symmetry; therefore, marked left-right asymmetry is likely to be abnormal.

Markers close to the dopamine D5 receptor gene (DRD5) show significant association with schizophrenia but not bipolar disorder.

Following the description of linkage of markers at chromosome 4p16 to bipolar disorder in several families [Blackwood et al., 1996], and the association of the alleles of a polymorphism closely linked to D5 dopamine receptor gene with schizophrenia [Williams et al., 1997], we have looked for linkage disequilibrium between a series of microsatellite markers from this region and major psychoses including schizophrenia, bipolar disorder, and unipolar major depressive disorder. A significant increase in the frequency of the 148 bp allele of DRD5 (P = 0.024) and the 244 bp allele of D4S615 (P = 0.001) was found in patients with schizophrenia (n = 158 DRD5; n = 133 D4S615), compared with patients with bipolar disorder (n = 270 DRD5; n = 107 D4S615), or controls without psychiatric illness (n = 437 DRD5; n = 309 D4S615). The frequency of the 148 bp allele of DRD5 was also increased in schizophrenia over unipolar major depressive disorder (n = 65). D4S615 was not typed in unipolar disorder. The estimated odds ratios confirmed that the 148 bp allele of DRD5 and the 244 bp allele of D4S615 conferred increased risk of schizophrenia. Estimated Haplotype (EH) analysis of 174 controls and 128 patients with schizophrenia who were typed for both markers confirmed the strong associations with these alleles but did not show evidence that the markers were in linkage disequilibrium with each other even though they lie approximately 150 kb apart. The data are consistent with an association between markers close to the D5 dopamine receptor and schizophrenia, but not bipolar disorder or unipolar major depression.

A 6.9-Mb high-resolution BAC/PAC contig of human 4p15.3-p16.1, a candidate region for bipolar affective disorder.

Bipolar affective disorder (BPAD) is a complex disease with a significant genetic component and a population lifetime risk of 1%. Our previous work identified a region of human chromosome 4p that showed significant linkage to BPAD in a large pedigree. Here, we report the construction of an accurate, high-resolution physical map of 6.9 Mb of human chromosome 4p15.3-p16.1, which includes an 11-cM (5.8 Mb) critical region for BPAD. The map consists of 460 PAC and BAC clones ordered by a combination of STS content analysis and restriction fragment fingerprinting, with a single approximately 300-kb gap remaining. A total of 289 new and existing markers from a wide range of sources have been localized on the contig, giving an average marker resolution of 1 marker/23 kb. The STSs include 57 ESTs, 9 of which represent known genes. This contig is an essential preliminary to the identification of candidate genes that predispose to bipolar affective disorder, to the completion of the sequence of the region, and to the development of a high-density SNP map.

A collaborative study to establish the 2nd International Standard for Fibrinogen, Plasma.

An International Collaborative Study involving 12 laboratories in 7 different countries was undertaken in order to replace the 1st International Standard (IS) for Fibrinogen, Plasma (89/644). The candidate replacement standard was the ampouled and freeze-dried residue of solvent/detergent treated plasma and was calibrated as coded duplicates (A and B) versus the 1st IS Fibrinogen, Plasma by automated Clauss assay and by a recommended clot collection (gravimetric) assay. This latter method had been used to calibrate the 1st IS Fibrinogen, Plasma. Comparing the ratios of the potency estimates of sample A to sample B (the coded duplicates), all of the laboratories obtained a ratio within 5% of the expected value of 1.0 by automated Clauss assay, which suggests that the laboratories were able to perform this assay well. Scrutiny of the data obtained from the gravimetric assays revealed that in almost all cases the results were invalid. The results of these assays are included in this report but clearly should be treated with caution and indeed produced significantly lower mean estimates of potency than the other assay methods. The overall geometric mean of all estimates of potency of the proposed 2nd IS Fibrinogen, Plasma (98/612) is 2.19 mg/ampoule by the automated Clauss assay. These data have been presented to the Fibrinogen Sub-Committee of the Standardisation and Scientific Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) (Washington, DC, August 1999), which recommended the establishment of 98/612 as the 2nd IS Fibrinogen, Plasma. This report has been presented to the Expert Committee on Biological Standardisation of the World Health Organisation (ECBS-WHO) at their 1999 session and 98/612 was established as the 2nd IS Fibrinogen, Plasma with a potency of 2.2 mg/ampoule.

The haemostatic balance -- Astrup revisited.

Prof. Tage Astrup first elaborated the notion that blood fluidity involved a balance between the tendency of blood to clot and for such clots to lyse. It would seem that, at that time, this haemostatic balance involved the notion that forming fibrin orchestrated its own destruction by stimulating fibrinolytic activity. In this review, we have clarified the detail of this balance and developed the thesis that Astrup's far-sighted balance notions involve a variety of control mechanisms. These involve the notion that thrombin, being at first sight a procoagulant, can also, in conjunction with thrombomodulin, act as a stimulus of anticoagulant activity by the generation of activated protein C. The thrombin-activatable fibrinolytic inhibitor (TAFI) is also involved in this balance since the generation of thrombin provokes the neutralisation of fibrinolysis by the TAFI pathway. The kallikrein/factor XII/urokinase pathway is discussed indicating yet another aspect of balance between the generation of coagulation and fibrinolysis. The overall theme of this review, apart from an insight into various aspects of the haemostatic balance, is that blood has a strong tendency to clot when tissue is damaged, and the intact vasculature requires major anticoagulant systems to prevent clots adhering to and stabilising in the vasculature.

High and constant plasma levels of tissue plasminogen activator and PEG-hirudin can be achieved by subcutaneous delivery.

Intramural thrombosis is a consistent finding in the arteries of patients who die following coronary angioplasty. This thrombosis is thought to have a role in restenosis, which is a common complication of coronary angioplasty. It has been hypothesised that antithrombotics such as hirudin or tissue-type plasminogen activator (tPA), may be therapeutically useful following angioplasty. This report describes the bioavailability of both agents following subcutaneous (sc) injection in cholesterol-fed rabbits. Intravenously delivered tPA has a half-life of 3-5 minutes. The half-life of intravenously administered hirudin is less than one hour in many species. In order to prolong the duration of action recombinant hirudin was conjugated to polyethylene glycol (PEG). Polyethylene glycol conjugated recombinant hirudin (PEG-rH) (0.7 mg/kg) antigen and activity were measurable after just 1 hr, reaching a maximum (663 and 884 ng/ml respectively) at 12 hours. Significant levels were present in rabbit plasma 24 hours after injection. Subcutaneously delivered recombinant (r-tPA) (1 mg/kg) was present in significant amounts 1 hr after injection, reaching a maximum (92 IU/ml) at 2 hours. Levels of tPA at 9 hours were approximately 80x normal circulating levels. High and constant levels of functional activity of both PEG-rH and r-tPA in rabbit plasma are achieved by subcutaneous delivery.

An explanation and the clinical significance of the failure of microcolumn tests to detect weak ABO and other antibodies.

Shear forces are proposed to explain the failure of antiglobulin and 'neutral' (no antiglobulin) microcolumn tests at 37 degrees C to detect weak ABO incompatibilities and other weak antibodies, clearly detectable by spin-tube methods. These shear forces can be minimized in a microcolumn test using a biphasic centrifugation phase. Although this biphasic test is not suitable for routine use, it may be of use as an investigational method for reference laboratories. This failure of microcolumn test to detect weak ABO incompatibilities is of little clinical significance as the antibodies are dubiously active at 37 degrees C.

A locus for bipolar affective disorder on chromosome 4p.

The main clinical feature of bipolar affective disorder is a change of mood to depression or elation. Unipolar disorder, also termed major depressive disorder, describes the occurrence of depression alone without episodes of elevated mood. Little is understood about the underlying causes of these common and severe illnesses which have estimated lifetime prevalences in the region of 0.8% for bipolar and 6% for unipolar disorder. Strong support for a genetic aetiology is found in the familial nature of the condition, the increased concordance of monozygotic over dizygotic twins and adoption studies showing increased rates of illness in children of affected parents. However, linkage studies have met with mixed success. An initial report of linkage on the short arm of chromosome 11 (ref. 4) was revised and remains unreplicated. Reports proposing cosegregation of genes found on the X chromosome with bipolar illness have not been supported by others. More recently bipolar disorder has been reported to be linked with markers on chromosomes 18, 21, 16 and a region on the X chromosome different from those previously suggested. We have carried out a linkage study in twelve bipolar families. In a single family a genome search employing 193 markers indicated linkage on chromosome 4p where the marker D4S394 generated a two-point lod score of 4.1 under a dominant model of inheritance. Three point analyses with neighbouring markers gave a maximum lod score of 4.8. Eleven other bipolar families were typed using D4S394 and in all families combined there was evidence of linkage with heterogeneity with a maximum two-point lod score of 4.1 (theta = 0, alpha = 0.35).