Descriptive study of current therapeutic practices, clinical reproductive findings and incidence of pregnancy loss in intensively managed thoroughbred mares.

Therapeutic practices in equine reproductive medicine have dramatically evolved over the last 20 years but current usage is not described. The aims of this study were to provide a description of medication use and clinical findings of reproductive examinations alongside measures of reproductive efficiency in thoroughbreds. A prospective cohort study was conducted in the 2013 and 2014 breeding seasons. Mare and stallion details, information on veterinary interventions and findings of reproductive ultrasound scans were collected using questionnaires and entered into a custom-designed Microsoft Access database. Descriptive summary statistics were derived directly from the database and using Microsoft Excel. Information was collected from 2246 pregnancies in 1754 mares from 29 stud farms. Ovulatory induction agents were used in 91.8% of cases, oestrus induction agents in 38.4% and covering therapies in 62.7%. Intrauterine antimicrobials were used in 49.6% of mares. Single pregnancies accounted for 83.9% of pregnancies, twins for 15.3% and triplets for 0.7%. The overall incidence of pregnancy loss between days 15-42 was 6.4% (95% CI 5.4%, 7.4%) and 1.6% (95% CI 1.1%, 2.1%) between days 43-65. A further 1.3% of pregnancies were lost by October and 4.5% by birth (including stillbirths). Eighty-three percent of all pregnancies resulted in a live foal. In conclusion, there has been a considerable increase in the use of reproductive therapeutics over the last 12 years. Nonetheless, incidence of pregnancy loss and live foal percentages remain essentially unchanged. Risk factor studies are required to determine if the substantial increase in therapeutic usage is conferring positive benefits.

Comparison of adjunctive azithromycin and amoxicillin/metronidazole for patients with chronic periodontitis: preliminary randomized control trial.

BACKGROUND: There are insufficient guidelines for the use of adjunctive systemic antibiotics for patients with periodontal disease. The aim of this study was to compare clinical outcomes for patients with moderate-advanced chronic periodontitis treated with: scaling and root planing (SRP), SRP with amoxicillin and metronidazole (A+M), SRP with Azithromycin (Az). METHODS: Thirty-seven non-smokers with generalized moderate to advanced chronic periodontitis were divided into three treatment groups: SRP, A+M and Az. Patients received the medications after the last SRP session and were reviewed three months later. Changes in clinical parameters were compared between the groups. Separate analyses were executed for: 'all sites', 'molar sites', 'sites with different PPD severities' and 'number of sites with shallow, moderate and deep PPD'. RESULTS: The three groups exhibited improvements in most clinical parameters. At three months, A+M showed a higher reduction in PPD compared to Az in the 'all sites analysis'. Molars exhibited better reduction in BOP and PPD with A+M than SRP. Pocket depth of the 4-6 mm category reduced more in the A+M than SRP. A+M experienced a higher increase in the number of sites with PPD 1-3 mm than Az. CONCLUSIONS: Adjunctive systemic antibiotics in the initial phase of treatment may result in improved clinical outcomes.

A method for isolating and culturing placental cells from failed early equine pregnancies.

Early pregnancy loss occurs in 6-10% of equine pregnancies making it the main cause of reproductive wastage. Despite this, reasons for the losses are known in only 16% of cases. Lack of viable conceptus material has inhibited investigations of many potential genetic and pathological causes. We present a method for isolating and culturing placental cells from failed early equine pregnancies. Trophoblast cells from 18/30 (60%) failed equine pregnancies of gestational ages 14-65 days were successfully cultured in three different media, with the greatest growth achieved for cells cultured in AmnioChrome™ Plus. Genomic DNA of a suitable quality for molecular assays was also isolated from 29/30 of these cases. This method will enable future investigations determining pathologies causing EPL.

Preliminary Survey of Ectoparasites and Associated Pathogens from Norway Rats in New York City.

The Norway rat (Rattus norvegicus) is a reservoir of many zoonotic pathogens and lives in close proximity to humans in urban environments. Human infection with rodent-borne disease occurs either directly through contact with a rat or its excreta, or indirectly via arthropod vectors such as fleas and ticks. Here, we report on the diversity and abundance of ectoparasitic arthropod species and associated pathogenic bacteria from 133 Norway rats trapped over a 10-mo period in Manhattan, New York, NY. Norway rats were host to the tropical rat mite [Ornithonyssus bacoti (Hirst)], the spiny rat mite (Laelaps echidnina Berlese), Laelaps nuttalli Hirst, the spined rat louse [Polyplax spinulosa (Burmeister)], and the Oriental rat flea [(Xenopsylla cheopis) (Rothschild)], with an average of 1.7 species per individual. A flea index of 4.1 X. cheopis was determined, whereas previous studies in New York City reported 0.22 fleas per rat. Multiple species of pathogenic Bartonella were identified from Oriental rat fleas that were related to Bartonella tribocorum, Bartonella rochalimae, and Bartonella elizabethae. However, no evidence of Yersinia pestis or Rickettsia spp. infection was detected in fleas. The identification of multiple medically important ectoparasite species in New York City underscores the need for future efforts to fully characterize the diversity and distribution of ectoparasites on Norway rats, and assess the risk to humans of vector-borne disease transmission.

Does communication skills training make a difference to patients' experiences of consultations in oncology and palliative care services?

There is much evidence supporting the efficacy of communication skills training; however, very little of this evidence comes from patient feedback. The primary aim of this pilot study was to evaluate whether the advanced communications skills training improves patients' experience of consultations. Healthcare professionals working in oncology and palliative care services from the North East of England were invited to participate in this study. Interactions between healthcare professionals (n = 21) and patients (n = 1103) were evaluated using the Consultation and Relational Empathy (CARE) Measure, which is a brief questionnaire designed to assess the patients' perceptions of relational empathy in the consultation. Additional demographic variables, such as patient age, length of consultation, familiarity with healthcare professional and overall satisfaction with consultation, were also collected. Healthcare professionals were either part of the intervention group who attended a 3-day communication skills training course or part of the control group who were on the waiting list for training. No differences in the patients' ratings on the CARE measure were found between Time 1 (before training) and Time 2 (after training) for the intervention group. Possible explanations for the findings are explored and implications for communication skills training are discussed.

Incorporation of antigens from Mannheimia haemolytica culture supernatant, and recombinant bovine C3d into ISCOM matrix using neutravidin-biotin interaction.

The aim of this study was to incorporate antigens from Mannheimia haemolytica culture supernatant, and an immune modulatory molecule, recombinant bovine C3d (rBoC3d), into immune stimulating complexes (ISCOMs) using neutravidin-biotin interaction. Biotinylated ISCOM matrix was generated using a commercial kit. The biotinylated ISCOM matrix was incubated with neutravidin and then centrifuged in a sucrose density gradient. The rBoC3d was expressed as an in vivo biotinylated protein and with a c-Myc tag (EQKLISEEDL) engineered to facilitate detection. The neutravidin-coated ISCOM matrix was incubated with biotinylated antigens from M. haemolytica culture supernatants and rBoC3d. To test the association among the neutravidin-coated ISCOM matrix, biotinylated antigens and rBoC3d, an analytical sucrose density gradient (10-40%, w/w) was performed. The experimental formulations were run in SDS-PAGE gels under reducing conditions. For Western immunoblot analysis, polyclonal bovine antiavidin, monoclonal anti-c-Myc, monoclonal antileukotoxin, and anti-GS60 antibodies were used to detect the presence of neutravidin, rBoC3d, leukotoxin, and GS60 antigens, respectively. By taking advantage of the biotin-neutravidin interaction, not only leukotoxin but also the recombinant immunomodulatory molecule, rBoC3d, was incorporated into ISCOM particles.

Epidemiological studies of the non-specific effects of vaccines: II--methodological issues in the design and analysis of cohort studies.

We review sources of bias which can affect non-randomized cohort studies of non-specific effects of vaccines on child mortality. Using examples from the literature on non-specific effects, we describe different sources of selection and information bias, and, where possible, outline analysis strategies to mitigate or eliminate such biases.

Glucocorticoid resistance in T-lineage acute lymphoblastic leukaemia is associated with a proliferative metabolism.

Glucocorticoids (GCs) are among the most important drugs for acute lymphoblastic leukaemia (ALL), yet despite their clinical importance, the exact mechanisms involved in GC cytotoxicity and the development of resistance remain uncertain. We examined the baseline profile of a panel of T-ALL cell lines to determine factors that contribute to GC resistance without prior drug selection. Transcriptional profiling indicated GC resistance in T-ALL is associated with a proliferative phenotype involving upregulation of glycolysis, oxidative phosphorylation, cholesterol biosynthesis and glutamate metabolism, increased growth rates and activation of PI3K/AKT/mTOR and MYC signalling pathways. Importantly, the presence of these transcriptional signatures in primary ALL specimens significantly predicted patient outcome. We conclude that in lymphocytes the activation of bioenergetic pathways required for proliferation may suppress the apoptotic potential and offset the metabolic crisis initiated by GC signalling. It is likely that the link between GC resistance and proliferation in T-ALL has not been fully appreciated to date because such effects would be masked in the context of current multiagent therapies. The data also provide the first evidence that altered expression of wild-type MLL may contribute to GC-resistant phenotypes. Our findings warrant the continued development of selective metabolic inhibitors for the treatment of ALL.

Genome-wide pharmacogenetic investigation of a hepatic adverse event without clinical signs of immunopathology suggests an underlying immune pathogenesis.

One of the major goals of pharmacogenetics is to elucidate mechanisms and identify patients at increased risk of adverse events (AEs). To date, however, there have been only a few successful examples of this type of approach. In this paper, we describe a retrospective case-control pharmacogenetic study of an AE of unknown mechanism, characterized by elevated levels of serum alanine aminotransferase (ALAT) during long-term treatment with the oral direct thrombin inhibitor ximelagatran. The study was based on 74 cases and 130 treated controls and included both a genome-wide tag single nucleotide polymorphism and large-scale candidate gene analysis. A strong genetic association between elevated ALAT and the MHC alleles DRB1(*)07 and DQA1(*)02 was discovered and replicated, suggesting a possible immune pathogenesis. Consistent with this hypothesis, immunological studies suggest that ximelagatran may have the ability to act as a contact sensitizer, and hence be able to stimulate an adaptive immune response.

Authenticity and drug resistance in a panel of acute lymphoblastic leukaemia cell lines.

Cell lines are important models for drug resistance in acute lymphoblastic leukaemia (ALL), but are often criticised as being unrepresentative of primary disease. There are also doubts regarding the authenticity of many lines. We have characterised a panel of ALL cell lines for growth and drug resistance and compared data with that published for primary patient specimens. In contrast to the convention that cell lines are highly proliferative, those established in our laboratory grow at rates similar to estimates of leukaemic cells in vivo (doubling time 53-442 h). Authenticity was confirmed by genetic fingerprinting, which also demonstrated the potential stability of long-term cultures. In vitro glucocorticoid resistance correlated well with that measured ex vivo, but all lines were significantly more sensitive to vincristine than primary specimens. Sensitivity to methotrexate was inversely correlated to that of glucocorticoids and L-asparaginase, indicating possible reciprocity in resistance mechanisms. A cell line identified as highly methotrexate resistant (IC50 > 8000-fold higher than other lines) was derived from a patient receiving escalating doses of the drug, indicating in vivo selection of resistance as a cause of relapse. Many of these lines are suitable as models to study naturally occurring resistance phenotypes in paediatric ALL.

Altered glucose metabolism in childhood pre-B acute lymphoblastic leukaemia.

The cells of solid tumours are known to have an altered metabolism, with high rates of glucose uptake and glycolysis, which results in the excessive production of lactate. To date there has been no definitive research documenting metabolic changes in acute lymphoblastic leukaemia (ALL) cells. In order to investigate whether ALL cells have an altered metabolism, we initially compared the transcriptional profiles of 22 specimens from paediatric patients diagnosed with ALL to five CD34+ specimens isolated from bone marrow, which was verified in an independent cohort of 101 specimens. Profiling revealed the upregulation of genes facilitating glycolysis in the ALL specimens compared to the CD34+ specimens, while those involved in the tricarboxylic acid cycle were downregulated. Functional studies supported the microarray findings threefold: (1) higher expression of the glucose transport protein glucose transporter 1 in ALL compared to CD34+ specimens, (2) the excessive production of lactate in ALL cell lines and (3) sensitivity of ALL cell lines to the glycolysis inhibitor 2-deoxy-D-glucose. While metabolic alterations have been well documented in solid tumours, this is the first study to provide direct evidence for the existence of metabolic changes in the leukaemic cells of ALL patients. The finding offers new options for targeted therapy for ALL patients.

Mum knows best? Psychological status in an oncology sample.

OBJECTIVE: To examine the influence of age, gender and diagnosis upon agreement of children's psychological status between children with cancer and their parents. METHODS: Total 51 families (49 mothers, 45 fathers and 51 children) completed the Strengths and Difficulties Questionnaire. Children's ages ranged from 11 to 16 years, with a mean age of 13.4 years. Eleven of the children had central nervous system (CNS) disease. RESULTS: Age, gender and diagnosis did not impact upon level of agreement between parents and children. There were no significant differences between father/child and mother/child reports of children's psychological status. CONCLUSIONS: Both mothers and fathers are perceptive to their child's psychological status, and it is therefore satisfactory to consider their opinions about their children. Children in the age range 11-16 years are able to express their views with as much accuracy as parents irrespective of their gender and diagnosis and so should be included in assessment.

Association between antenatal cytokine production and the development of atopy and asthma at age 6 years.

BACKGROUND: Various lines of evidence suggest that antenatal factors are important in determining susceptibility to atopy and asthma. One possible mechanism is cytokines, production of which in the placenta is high throughout gestation and which protect placental integrity via control of local immunological homoeostasis. We investigated antenatal cytokine concentrations in a prospective birth cohort, intensively monitored for atopy and asthma outcomes at age 6 years. METHODS: Cryopreserved cord-blood serum samples from 407 children were assayed for interleukins 4, 5, 6, 10, 12, and 13, interferon gamma, and tumour necrosis factor alpha (TNFalpha). Associations between family, antenatal, and perinatal factors, cord-blood cytokine concentrations, and atopy or asthma outcomes were analysed by logistic regression. Causal effects of cytokines on outcomes were estimated by propensity scores based on family, antenatal, and perinatal factors. FINDINGS: Detectable cord-blood concentrations of interleukin 4 and interferon gamma were each associated with lower risk of physician-diagnosed asthma (adjusted odds ratios 0.60 [95% CI 0.37-0.99] and 0.60 [0.37-0.97] respectively), current asthma (0.59 [0.33-1.00] and 0.39 [0.22-0.71]), and current wheeze (0.55 [0.32-0.93] and 0.52 [0.31-0.90]) and atopy (sensitisation to some inhalant allergens) outcomes at 6 years. High concentrations of TNFalpha were associated with lower risk of atopy but not with asthma risk. These associations were broadly unaltered by propensity-score adjustment. Maternal smoking was associated with higher risk of both wheeze at 6 years and lower concentrations of interleukin 4 and interferon gamma in cord blood. INTERPRETATION: The mechanism underlying attenuated T-helper-1/T-helper-2 cytokine production in high-risk children also apparently operates in control of cytokine production in the fetoplacental unit. The finding that this mechanism is dysregulated by maternal smoking suggests it is a target for antenatal environmental factors relevant to asthma aetiology.

Jumpers and fallers: a comparison of the distribution of skeletal injury.

AIM: The aim of this study was to compare the distribution of injuries between patients who have fallen and those who have jumped from a height, and to relate the mechanism of injury to the fractures sustained. MATERIALS AND METHODS: Three hundred and ninety-nine patients, admitted via Helicopter Emergency Medical Service (HEMS), classified as either having fallen or jumped from a height were included in the study. The radiographs from the primary survey, together with radiographs of specific injury sites were analysed. The distribution of injury was compared in the two groups. RESULTS: Of the 399 patients, 342 were fallers and 57 were jumpers. Jumpers had a higher Injury Severity Score (ISS), death rate and number of fractures per person. Jumpers sustained more rib fractures (particularly on the right), pelvic and lower limb fractures but fewer skull fractures. CONCLUSION: Jumpers tend to sustain different injuries to fallers. It is proposed that jumpers have a tendency to land feet-first and then try to break their falls on their dominant side, sustaining more right-sided rib fractures in the process. The patterns of injury that have emerged from this study have important implications for evaluating skeletal injuries in those who jump or fall from a height.

Electrophoresis of cerebrospinal fluid.

Under normal circumstances, cerebrospinal fluid (CSF) is a clear and colorless fluid that is formed in the ventricles of the brain. It is in close proximity to the surface of both the brain and spinal cord, and, as a result, the analysis of CSF proteins and other constituents in samples taken by lumbar puncture have long been used as an aid in the diagnosis of neurological disorders. Various electrophoretic methods have been used, including agar gel (1), polyacrylamide (2), two-dimensional (3), and isoelectric focusing (4), with the aim of detecting profiles that are diagnostic, especially in the case of proteins. These techniques have been labor-intensive, time-consuming, and, at best, only semiquantitative. This chapter describes how the technique of capillary electrophoresis (CE) in free solution (FSCE) can be used to provide a very fast, sensitive, and reproducible method for the analysis of CSF constituents, using only nanoliter volumes of sample (5). Furthermore, on line detection of the separated constituents, using UV absorption, allows accurate quantitation.

The analysis of aqueous humor constituents using capillary zone electrophoresis.

One of the difficulties encountered in the study of aqueous humor is the relatively small volume generally available for analysis. The purpose of this study was to investigate the potential use of capillary zone electrophoresis (CE) for the analysis of nanolitre quantities of this fluid. Twelve samples of aqueous humor were obtained from patients undergoing cataract surgery and a further three samples were from non cataract post mortem subjects within 6 hr of death. CE was carried out in an uncoated fused silica glass capillary, 75 mu internal diameter and 100 cm long using a run buffer of 40 mM borate pH 9.4 containing 0.4 g l-1 methylcellulose. Detection of the separated zones was by ultra violet absorption at 200 nm. Preliminary identification of peaks was achieved by enzymatic hydrolysis and spiking with purified analytes. A number of very well resolved peaks were obtained from both cataract and post mortem samples using nanolitre quantities of unmodified fluid. Additional peaks were noted in the post mortem samples, most of which were likely to be due to a partial breakdown of the blood aqueous humor barrier. The profiles obtained were not significantly affected by various drugs routinely administered during cataract surgery. This preliminary study has demonstrated the potential value of CE in the analysis of aqueous humor in health and disease.

PRO_SELECT: combining structure-based drug design and combinatorial chemistry for rapid lead discovery. 1. Technology.

This paper describes a novel methodology, PRO_SELECT, which combines elements of structure-based drug design and combinatorial chemistry to create a new paradigm for accelerated lead discovery. Starting with a synthetically accessible template positioned in the active site of the target of interest, PRO_SELECT employs database searching to generate lists of potential substituents for each substituent position on the template. These substituents are selected on the basis of their being able to couple to the template using known synthetic routes and their possession of the correct functionality to interact with specified residues in the active site. The lists of potential substituents are then screened computationally against the active site using rapid algorithms. An empirical scoring function, correlated to binding free energy, is used to rank the substituents at each position. The highest scoring substituents at each position can then be examined using a variety of techniques and a final selection is made. Combinatorial enumeration of the final lists generates a library of synthetically accessible molecules, which may then be prioritized for synthesis and assay. The results obtained using PRO_SELECT to design thrombin inhibitors are briefly discussed.