Quantitatively characterizing benthic community-habitat relationships in soft-sediment, nearshore environments to yield useful results for management.

Effective management of benthic habitats is important for maintaining heathy and functional aquatic ecosystems. To provide managers with the best possible information, characterizing benthic habitats at the community level is essential; yet, acquiring the data sets needed to achieve this task is resource intensive and, at times, prohibitively expensive. Thus, thoughtful assessments of which data to collect and utilize in benthic habitat characterization studies are needed. Environmental data sets commonly used to characterize benthic habitats include a range of variables from water depth and sediment grain size to seabed features identified by sonar backscatter. The objective of this study was to identify the most useful environmental variables for characterizing infaunal benthic habitats and to determine how to best utilize these variables in analyses (e.g., by comparing continuous vs. categorical explanatory variables). The modeling approach used multivariate regression tree and redundancy analysis along with a critical cross-validation step for model evaluation. Results indicated that models with more than ~7 environmental predictors overfitted the data sets analyzed and that categorizing continuous predictors into categorical ones influenced the proportion of infaunal community variation explained by each model. Habitats identified and characterized on the basis of sonar backscatter explained more of the infaunal community variation than any model that used a combination of other environmental variables (e.g., water depth & sediment grain size) or those constructed using categorical habitat classes from existing classification schemes. We therefore recommend maximizing the potential of sonar-derived variables for characterizing infaunal benthic habitats in nearshore, soft-sediment ecosystems.

Best practices for the management of local-regional recurrent chordoma: a position paper by the Chordoma Global Consensus Group.

Chordomas are rare, malignant bone tumors of the skull-base and axial skeleton. Until recently, there was no consensus among experts regarding appropriate clinical management of chordoma, resulting in inconsistent care and suboptimal outcomes for many patients. To address this shortcoming, the European Society of Medical Oncology (ESMO) and the Chordoma Foundation, the global chordoma patient advocacy group, convened a multi-disciplinary group of chordoma specialists to define by consensus evidence-based best practices for the optimal approach to chordoma. In January 2015, the first recommendations of this group were published, covering the management of primary and metastatic chordomas. Additional evidence and further discussion were needed to develop recommendations about the management of local-regional failures. Thus, ESMO and CF convened a second consensus group meeting in November 2015 to address the treatment of locally relapsed chordoma. This meeting involved over 60 specialists from Europe, the United States and Japan with expertise in treatment of patients with chordoma. The consensus achieved during that meeting is the subject of the present publication and complements the recommendations of the first position paper.

An open, non-comparative, multicentre evaluation of performance and safety using an antimicrobial exudate transfer dressing on diabetic foot ulcers: a case series.

OBJECTIVE: To evaluate the performance and safety of Mepilex Transfer Ag (MTAg) in the treatment of infected diabetic foot ulcers (DFU). METHOD: Patients with locally infected DFU were treated with the test dressing for up to 4 weeks, with a further 12 weeks of follow-up in a non-comparative study. Changes to wound infection and wound size as well as the condition of the peri-wound skin from baseline were assessed. Wound pain during dressing change was measured using a visual analogue scale (VAS). The investigators and patients documented their opinions on their overall experience of the test dressing and on key performance parameters. RESULTS: Following treatment with the test dressing, the signs and symptoms of local wound infection present in the target DFU were substantially reduced compared with baseline. Following the posttreatment evaluation, the majority of the DFU exhibited no signs of infection. and mean wound size was reduced by 50%. Wound size also continued to steadily decrease during follow-up. At the end of treatment five DFUs were completely healed and a further six healed by the end of the follow-up period. Concomitantly, over the course of the study, wound exudate levels were reduced and there was a significant improvement in the condition of the peri-wound area. Wound pain at dressing change was low throughout; generally patients felt no anxiety during the dressing change procedure. The patients considered it a comfortable dressing that remained in place and allowed ease of movement during wear. The investigating clinicians were highly satisfied with the overall performance, especially with respect to its ease of application and removal, conformability and flexibility. CONCLUSION: This study has demonstrated the potential of the dressing to provide topical antimicrobial activity directly to an infected DFU, suggesting prompt treatment of an infected DFU with this topical antimicrobial could aid wound complications. DECLARATION OF INTEREST: The authors have no conflict of interest to declare.

Liver fluke control on sheep farms in Northern Ireland: A survey of changing management practices in relation to disease prevalence and perceived triclabendazole resistance.

Reports of resistance to triclabendazole (TCBZ) among fluke populations have increased in recent years. Allied to this, there has been a rise in the prevalence of the disease, which has been linked to climate change. Results from questionnaire surveys conducted in Northern Ireland (NI) in 2005 (covering the years 1999-2004) and 2011 (covering the years 2008-2011) have provided an opportunity to examine the extent to which fluke control practices have changed over a prolonged time-frame, in light of these changes. A number of differences were highlighted. There was a significant shift away from the use of TCBZ over time, with it being replaced largely by closantel. The timing of treatments had moved earlier in the year, perhaps in response to climate change (and an altered pattern of disease). In relation to the frequency of drug treatments, there were no major changes in the overall pattern of drug treatments between the two survey points, although on both occasions approximately one-third of flock owners gave more than 3 treatments per year to ewes. In lowland areas in 2011, flock owners were rotating drug classes more often (each year and at each treatment) than in 2005, whereas in upland areas, flock owners were rotating less often and more were not rotating at all. Between 2005 and 2011, the percentage of flock owners giving quarantine treatments to bought-in stock had halved, to a very low level (approximately 10%). Using data from a complementary TCBZ resistance survey (Hanna et al., 2015), it has been shown that the way in which data are selected and which efficacy formula is applied can influence the calculation of drug efficiency and impact on diagnosis of resistance.

EURAMOS-1, an international randomised study for osteosarcoma: results from pre-randomisation treatment.

BACKGROUND: Four international study groups undertook a large study in resectable osteosarcoma, which included two randomised controlled trials, to determine the effect on survival of changing post-operative chemotherapy based on histological response. PATIENTS AND METHODS: Patients with resectable osteosarcoma aged ≤40 years were treated with the MAP regimen, comprising pre-operatively of two 5-week cycles of cisplatin 120 mg/m(2), doxorubicin 75 mg/m(2), methotrexate 12 g/m(2) × 2 (MAP) and post-operatively two further cycles of MAP and two cycles of just MA. Patients were randomised after surgery. Those with ≥10% viable tumour in the resected specimen received MAP or MAP with ifosfamide and etoposide. Those with <10% viable tumour were allocated to MAP or MAP followed by pegylated interferon. Longitudinal evaluation of quality of life was undertaken. RESULTS: Recruitment was completed to the largest osteosarcoma study to date in 75 months. Commencing March 2005, 2260 patients were registered from 326 centres across 17 countries. About 1334 of 2260 registered patients (59%) were randomised. Pre-operative chemotherapy was completed according to protocol in 94%. Grade 3-4 neutropenia affected 83% of cycles and 59% were complicated by infection. There were three (0.13%) deaths related to pre-operative chemotherapy. At definitive surgery, 50% of patients had at least 90% necrosis in the resected specimen. CONCLUSIONS: New models of collaboration are required to successfully conduct trials to improve outcomes of patients with rare cancers; EURAMOS-1 demonstrates achievability. Considerable regulatory, financial and operational challenges must be overcome to develop similar studies in the future. The trial is registered as NCT00134030 and ISRCTN 67613327.

Locally aggressive fibrous dysplasia.

Although fibrous dysplasia (FD) is a benign fibro-osseous lesion, locally aggressive behaviour has rarely been described but is poorly characterised. In this study, we document clinical, radiological and pathological (including molecular genetics) findings in three cases of locally aggressive FD, two of which involved the ribs. Lesions in these cases, one of which was a recurrent lesion, were followed up for 2-7 years. All of the lesions showed typical histological features of FD but were characterised by extension through the bone cortex into the extra-osseous soft tissue. The lesions did not exhibit overexpression/amplification of CDK4 and MDM2; in two of the cases, a GNAS mutation was identified. Our findings confirm that FD can rarely exhibit locally aggressive behaviour with extension beyond the bone compartment into the surrounding soft tissue; these lesions can be distinguished from low-grade intramedullary osteosarcoma by lack of amplification/overexpression of CDK4 and MDM2 and the presence of a GNAS mutation.

Long-term follow-up of Van Nes rotationplasty in patients with congenital proximal focal femoral deficiency.

Van Nes rotationplasty may be used for patients with congenital proximal focal femoral deficiency (PFFD). The lower limb is rotated to use the ankle and foot as a functional knee joint within a prosthesis. A small series of cases was investigated to determine the long-term outcome. At a mean of 21.5 years (11 to 45) after their rotationplasty, a total of 12 prosthetic patients completed the Short-Form (SF)-36, Faces Pain Scale-Revised, Harris hip score, Oswestry back pain score and Prosthetic Evaluation Questionnaires, as did 12 age- and gender-matched normal control participants. A physical examination and gait analysis, computerised dynamic posturography (CDP), and timed 'Up & Go' testing was also completed. Wilcoxon Signed rank test was used to compare each PFFD patient with a matched control participant with false discovery rate of 5%. There were no differences between the groups in overall health and well-being on the SF-36. Significant differences were seen in gait parameters in the PFFD group. Using CDP, the PFFD group had reduced symmetry in stance, and reduced end point and maximum excursions. Patients who had undergone Van Nes rotationplasty had a high level of function and quality of life at long-term follow-up, but presented with significant differences in gait and posture compared with the control group.

MicroRNA profiling of peripheral nerve sheath tumours identifies miR-29c as a tumour suppressor gene involved in tumour progression.

BACKGROUND: Neurofibromatosis type 1 is one of the most common familial diseases, the hallmark of which is the development of multiple neurofibromas. These are benign nerve sheath tumours, which can transform into malignant peripheral nerve sheath tumours (MPNST). METHODS: The aim of this study was to identify differentially expressed microRNA (miRNA) in neurofibromas and MPNST obtained from patients with neurofibromatosis type 1 using microarray analysis. Differential expression was validated by reverse transcription quantitative-PCR, and functional studies were performed after transfection of miRNA oligonucleotide mimics into MPNST cells. RESULTS: Sixteen miRNA were significantly differentially expressed in MPNST compared with NF, and of these fourteen were downregulated in MPNST: these included miR-30e*, miR-29c*, miR-29c, miR-340*, miR-30c, miR-139-5p, miR-195, miR-151-5p, miR-342-5p, miR-146a, miR-150, miR-223, let-7 a and let-7 g with a false discovery rate of q=8.48E-03 for the least significant miRNA. In contrast, miR-210 and miR-339-5p were upregulated in MPNST compared with neurofibromas. Prediction softwares/algorithms identified a list of genes targeted by miR-29c including extracellular matrix genes and matrix metalloproteinase (MMP)-2, all of which are reported to be involved in cell migration and invasion. Functional studies in a MPNST cell line, sNF96.2, using a mimic of the mature miR-29c showed reduced invasion, whereas there was no change in proliferation. Zymography of the manipulated cells showed that MMP2 activity was also reduced when miR-29c expression was forced in sNF96.2. CONCLUSION: We provide evidence that reduction of miR-29c has a pivotal role in the progression of nerve sheath tumours and results by increasing the invasive/migratory properties of nerve sheath tumours.

Standardisation of a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole in Fasciola hepatica.

A sheep trial was performed to standardise a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole (TCBZ) in Fasciola hepatica). The CRT employs the BIO K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) to test for the presence of F. hepatica coproantigens in a faecal sample. If it is coproantigen-positive, the CRT protocol recommends that faecal samples are re-tested for coproantigens at 14 days post-treatment (dpt), with negative testing at this point indicating TCBZ success. Initial work aimed to confirm the sensitivity of the BIO K201 ELISA for Fasciola infection and investigate whether coproantigens represent a robust reduction marker of TCBZ efficacy. Thirty-eight, indoor-reared sheep were artificially infected with F. hepatica isolates known to be susceptible (Cullompton) and resistant (Sligo) to TCBZ action, respectively. Treatment was administered at 12 weeks post-infection (wpi), with 2 sheep groups, infected with each isolate, culled at 2 and 4 weeks post-treatment (wpt), respectively. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period. Additional work focused on the effect of temperature on faecal sample collection and storage. Faecal samples collected from sheep positive for F. hepatica infection were sub-sampled and left at room temperature. Individual sub-samples were tested by ELISA on consecutive days and these readings compared to the original test result on the day of collection. In addition, ELISA values were compared between faecal sub-samples prepared on the day of sampling and post storage at -20°C. Also, an immunocytochemical study was performed to determine the tissue site of origin of the coproantigen protein in the fluke. Results showed that the BIO K201 ELISA was sensitive for Fasciola coproantigens, with coproantigens detectable from 5 wpi onwards. The suitability of coproantigens as a diagnostic marker of TCBZ efficacy was supported by the absence and presence of coproantigens in TCBZ-treated Cullompton (TCBZ-susceptible) and Sligo (TCBZ-resistant) F. hepatica infections at 2 and 4 wpt, respectively. Study results suggest that low to moderate temperature has little, if any, impact on coproantigen stability in faecal samples, but that higher temperatures may have. Immunolabelling for the coproantigen showed that it was specific to the gastrodermal cells of both adult and juvenile flukes.

Comparison of two assays, a faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance to triclabendazole in Fasciola hepatica in sheep.

A sheep trial was performed to evaluate two diagnostic assays, a faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance of Fasciola hepatica to triclabendazole (TCBZ). The FECRT defines successful TCBZ treatment as a 95% or greater reduction in fluke faecal egg counts (FECs) at 14 days post-treatment (dpt). The CRT defines effective TCBZ treatment as faeces negative for Fasciola coproantigens at 14dpt, as measured by the commercial BIO K201 coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium). Forty-nine indoor-reared sheep were split into four trial groups and each sheep was infected with 200 metacercariae of 1 of 4 F. hepatica isolates, previously described as susceptible (Cullompton and Fairhurst) and resistant (Leon and Oberon) to TCBZ action, respectively. TCBZ treatment was administered at 12 weeks post-infection (wpi) to one sub-group in each infected sheep group, and these sheep were culled at 4 weeks post-treatment (wpt). Untreated sheep sub-groups, were culled at a parallel time-point, that is, at 16wpi. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period, sub-sampled and examined by a standardised egg sedimentation protocol and by the BIO K201 ELISA. Results supported the use of both the FECRT and the CRT for the diagnosis of resistance of F. hepatica to TCBZ. In addition, the study confirmed the TCBZ susceptibility of the Cullompton and Fairhurst F. hepatica isolates and the TCBZ resistance of the Oberon F. hepatica isolate. However, the Leon F. hepatica isolate was found to be susceptible, rather than resistant, to TCBZ action.

Multiple-site osteosarcomas of the jaw in a single patient. A true case of a metachronous lesion?

A case of multiple-site osteosarcomas in the jaw of a 56-year-old patient is reported. The disease occurred consecutively at three different sites (left maxilla, left mandible, right mandible) separated by time intervals of 12 and 18 months, respectively. Metachronous osteosarcomas of the long bones is a rare form of osteosarcoma and implies multiple lesions appearing at different times, each one behaving clinically as a primary lesion. The pathogenesis of this disease is unknown as it is unclear whether the lesions represent independent primary tumours or metastatic disease.

Elastofibroma dorsi: management, outcome and review of the literature.

Elastofibroma dorsi is an uncommon, benign, slow-growing soft-tissue tumour of uncertain aetiology. It classically presents as an ill-defined mass at the inferior pole of the scapula with symptoms which include swelling, discomfort, snapping, stiffness and occasionally pain. We report the symptoms, function and outcome after treatment of 21 elastofibromas in 15 patients. All were diagnosed by MRI and early in the series four also underwent CT-guided biopsy to confirm the diagnosis. In all, 18 tumours were excised and three were observed. After excision, the mean visual analogue score for pain decreased from 4.6 (0 to 10) pre-operatively to 2.4 (0 to 8) post-operatively (p = 0.04). The mean shoulder function, at a mean follow-up of 4.2 years (3 months to 16 years), was 78.1% (30 to 100) using the Stanmore percentage of normal shoulder assessment scoring system. The mean range of forward flexion improved from 135 degrees (70 degrees to 180 degrees ) to 166 degrees (100 degrees to 180 degrees ) after excision (p = 0.005). In four patients a post-operative haematoma formed; one required evacuation. Three patients developed a post-operative seroma requiring needle aspiration and one developed a superficial infection which was treated with antibiotics. Our findings support previous reports suggesting that a pre-operative tissue diagnosis is not necessary in most cases since the lesion can be confidently diagnosed by MRI, when interpreted in the light of appropriate clinical findings. Surgical excision in symptomatic patients, is helpful. It has been suggested that elastofibroma is caused by a local tissue reaction and is not a true neoplastic process. A strong association has been noted between elastofibroma and repetitive use of the shoulder, which is supported by our findings.

Fasciola hepatica: histological changes in the reproductive structures of triclabendazole (TCBZ)-sensitive and TCBZ-resistant flukes after treatment in vivo with TCBZ and the related benzimidazole derivative, Compound Alpha.

Twenty-four shed-reared lambs were each infected orally with 250 metacercariae of Fasciola hepatica, using either the triclabendazole (TCBZ)-sensitive Cullompton isolate or the TCBZ-resistant Sligo isolate. Twelve weeks after infection the lambs were treated with TCBZ (10mg/kg) or with the experimental fasciolicide, Compound Alpha (Cpd alpha), a benzimidazole derivative of TCBZ (15mg/kg). The lambs were euthanised 48, 72 and 96h after TCBZ treatment, or 24, 48 and 72h after Cpd alpha treatment, and flukes were collected from the liver and/or gall bladder of each animal. Untreated animals harbouring 12-week infections were euthanized 24h after administration of anthelmintic to the treatment groups, and the untreated flukes provided control material. A semi-quantitative assessment of the degree of histological change induced by the two drugs after different times of exposure was achieved by scoring the intensity of three well-defined lesions that developed in the testes and uteri of a representative sample of flukes from each lamb. In general, it was found that in those tissues where active meiosis and/or mitosis occurred (testis, ovary, and vitelline follicles), there was progressive loss of cell content due to apparent failure of cell division to keep pace with expulsion of the mature or effete products. Further, actively dividing cell types tended to become individualised, rounded and condensed, characteristic of apoptotic cell death. Protein synthetic activity was apparently inhibited in the Mehlis' secretory cells. In the uterus, where successful formation of shelled eggs represents the culmination of a complex sequence of cytokinetic, cytological and synthetic activity involving the vitelline follicles, the ovary and the Mehlis' gland, histological evidence indicating failure of ovigenesis was evident from 24h post-treatment onwards. The development of these lesions may be related to the known anti-tubulin activity of the benzimidazole class of anthelmintics, to the induction of apoptosis in cells where mitosis or meiosis has aborted due to failure of spindle formation, and to drug-induced inhibition of protein synthesis. The semi-quantitative findings indicated that Cpd alpha is slightly less efficacious than TCBZ itself in causing histological damage to the reproductive structures of TCBZ-sensitive flukes, and that, like TCBZ, it caused no histological damage in flukes of the TCBZ-resistant isolate. This study illustrates the potential utility of histological techniques for conveniently screening representative samples of flukes in field trials designed to validate instances of drug resistance or to test the efficacy of new products against known drug-resistant and drug-susceptible fluke isolates. It also provides reference criteria for drug-induced histopathological changes in fluke reproductive structures which may aid interpretation of TEM findings.

Outcome of intralesional curettage for low-grade chondrosarcoma of long bones.

BACKGROUND: Different treatment strategies for low-grade chondrosarcomas are reported in the literature with variable outcomes. The aim of this study was to assess the oncological and functional outcomes associated with intralesional curettage and cementation of the lesion as a treatment strategy. PATIENTS AND METHODS: We performed a retrospective review of 39 consecutive patients with intramedullary low-grade chondrosarcoma of long bones treated by intralesional curettage and cementation at our institution between 1999 and 2005. RESULTS: There were 10 males and 29 females with a mean age of 55.5 years (32-82), and a mean follow-up of 5.1 years (3-8.7). Local recurrence occurred in two patients (5%) within the first two years following index surgery. Both were treated by re-curettage and cementation of the resultant defects. A second local recurrence developed a year later in one of these two patients, for which a further curettage followed by local liquid nitrogen treatment was performed. Overall, there were no cases of post-operative complications or metastases. The patients were assessed using the Musculoskeletal Tumour Society scoring system (MSTS) to determine limb function. The average score achieved was 94% (79-100%). CONCLUSION: Intralesional curettage is an effective treatment strategy for low-grade intramedullary chondrosarcoma of long bones, with excellent oncological and functional results. Careful case selection with stringent clinical and radiographic follow-up is recommended.

Below-knee amputation through a joint-sparing proximal tibial replacement for recurrent tumour.

We report a case which highlights the progression of osteofibrous dysplasia to adamantinoma and questions whether intralesional curettage is the appropriate treatment. The role of a joint-sparing massive endoprosthesis using cortical fixation is demonstrated and we describe a unique biomedical design which resulted in the manufacture of an end cap to allow amputation through a custom-made proximal tibial replacement, rather than an above-knee amputation following recurrence.

Surface changes in adult Fasciola hepatica following treatment in vivo with the experimental fasciolicide, compound alpha.

Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were dosed with 15 mg/kg of compound alpha at 12 weeks postinfection. Adult flukes were recovered from the bile ducts at 24, 48, and 72 h post-treatment (p.t.). Changes to the surface morphology of the flukes were assessed using scanning electron microscopy (SEM). Flukes were still active at 24 h p.t. and displayed limited areas of disruption, which were restricted to the oral cone region. At 48 h p.t., a reduced level of motility was observed in approximately 50% of the flukes recovered. Swelling of the tegument was more widespread and was accompanied by blebbing and partial loss of the tegumental covering of the spines. By 72 h p.t., the reduction in motility was greater, and approximately one quarter of the flukes recovered were inactive. In the majority of the flukes examined, the midbody region was marked by a discoloration of the flukes' tissues. This was seen to be due to the loss of the tegumental syncytium. Sloughing extended into the tail region in some specimens and, in the more badly-affected specimens, the basal lamina was breached to expose the underlying musculature. Elsewhere on the body, the tegument that remained was relatively normal, although areas of swelling and blebbing were present. Overall, the results provided information on the time-scale of changes to the surface morphology of the fluke that underpin the efficacy of compound alpha.

Potential therapeutic targets for chordoma: PI3K/AKT/TSC1/TSC2/mTOR pathway.

Chordomas are radio- and chemo-resistant tumours and metastasise in as many as 40% of patients. The aim of this study was to identify potential molecular targets for the treatment of chordoma. In view of the reported association of chordoma and tuberous sclerosis complex syndrome, and the available therapeutic agents against molecules in the PI3K/AKT/TSC1/TSC2/mTOR pathway, a tissue microarray of 50 chordoma cases was analysed for expression of active molecules involved in this signalling pathway by immunohistochemistry and a selected number by western blot analysis. Chordomas were positive for p-AKT (92%), p-TSC2 (96%), p-mTOR (27%), total mTOR (75%), p-p70S6K (62%), p-RPS6 (22%), p-4E-BP1 (96%) and eIF-4E (98%). Phosphatase and tensin homologue deleted on chromosome 10 expression was lost in 16% of cases. Mutations failed to be identified in PI3KCA and RHEB1 in the 23 cases for which genomic DNA was available. Fluorescence in situ hybridisation analysis for mTOR and RPS6 loci showed that 11 of 33 and 21 of 44 tumours had loss of one copy of the respective genes, results which correlated with the loss of the relevant total proteins. Fluorescence in situ hybridisation analysis for loci containing TSC1 and TSC2 revealed that all cases analysed harboured two copies of the respective genes. On the basis of p-mTOR and or p-p70S6K expression there is evidence indicating that 65% of the chordomas studied may be responsive to mTOR inhibitors, rapamycin or its analogues, and that patients may benefit from combined therapy including drugs that inhibit AKT.

Ultrastructural changes to the tegumental system and the gastrodermal cells in adult Fasciola hepatica following in vivo treatment with the experimental fasciolicide, compound alpha.

Sheep infected with the triclabendazole-susceptible, Cullompton isolate of Fasciola hepatica were dosed with 15 mg/kg of compound alpha at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24, 48 and 72 h post-treatment (p.t.). Ultrastructural changes to the flukes were assessed using transmission electron microscopy (TEM), with a view to gathering information on the mechanism(s) of action for compound alpha and on the possible route of its entry into F. hepatica. The tegumental syncytium was more severely affected than the gut at all time-points p.t. with compound alpha, suggesting a predominantly trans-tegumental route of uptake. Disruption to the tegumental system became increasingly severe over time. A stress response was observed at 24 h p.t. and took the form of blebbing and increases in the production and transport of secretory bodies. By 72 h p.t., extensive tegumental loss and degeneration of the tegumental cell bodies had occurred. Degeneration of subtegumental tissues and internal flooding were also observed. Changes in the gastrodermal cells were slow to develop: reduced secretory activity was evident at 72 h p.t.. There was progressive disruption to the somatic muscle layers, with disorganization of the muscle blocks and loss of muscle fibres.

An evaluation of the efficacy of compound alpha and triclabendazole against two isolates of Fasciola hepatica.

Seventy indoor-reared sheep were divided into 10 groups to test the efficacy of the experimental fasciolicide, compound alpha (15mg/kg) against triclabendazole (TCBZ)-resistant and TCBZ-susceptible F. hepatica infections. Activity against the Sligo TCBZ-resistant isolate was tested at three time points post-infection (p.i.): 3 days, 4 weeks and 12 weeks (Groups 1-3, respectively). A parallel trial was carried out using TCBZ (10mg/kg) (Groups 5-7): this provided a direct comparison between the efficacies of the two drugs. Group 4 served as an untreated Sligo control. Groups 8 and 9 were setup to test the efficacy of TCBZ and compound alpha against 12-week-old and 4-week-old TCBZ-susceptible, Cullompton infections, respectively. Group 10 served as an untreated Cullompton control. Sheep were sacrificed at 16 weeks p.i. and efficacies were determined. All remaining flukes were collected and measured, before being processed for whole-mount staining to assess the condition of their reproductive structures (testis, vitellaria, ovary and uterus). A second study was carried out to test the activity of compound alpha (15mg/kg) against mature 12-week-old TCBZ-susceptible F. hepatica infections in sheep. Eighteen sheep were divided into two groups, A and B. Group A was treated and Group B served as an untreated control group. Efficacy was determined by reduction in faecal egg counts. The results showed that, whilst compound alpha was very active against adult TCBZ-susceptible flukes, producing a 100% reduction in faecal egg counts, it only caused a 62.5% reduction in fluke burden against juvenile flukes. Moreover, compound alpha was not effective against any stage of infection with TCBZ-resistant F. hepatica in sheep. Data from the trial also revealed biological differences between the two isolates. Thus, Sligo flukes were smaller in size and produced fewer eggs than the Cullompton flukes and their cysts were less infective to sheep. However, they reached the bile ducts more quickly and their eggs appeared in the faeces >2 weeks earlier.

BIM and other BCL-2 family proteins exhibit cross-species conservation of function between zebrafish and mammals.

Here we investigate the function of zebrafish Bcl-2 family proteins and demonstrate important conservation of function across zebrafish and mammalian systems. We have isolated a zebrafish ortholog of mammalian BIM and show that it is the most toxic of the zebrafish BH3-only genes examined, sharing this characteristic with the mammalian BIM gene. The zebrafish bad gene shows a complete lack of embryonic lethality, but like mammalian BAD, its pro-apoptotic activity is regulated through phosphorylation of critical serines. We also found that the pattern of mitochondrial dysfunction observed by zebrafish BH3 domain peptides in a mammalian cytochrome c release assay recapitulates the pattern of embryonic lethality induced by the respective mRNA injections in vivo. In contrast to zebrafish Bim, Bid exhibited only weak binding to zebrafish Bcl-2 and moderate-to-weak overall lethality in zebrafish embryos and isolated mitochondria. Given that zebrafish Bcl-2 binds strongly to mammalian BID and BIM peptides and proteins, the protein identified as the zebrafish Bid ortholog has different properties than mammalian BID. Overall, our results demonstrate the high degree of functional conservation between zebrafish and mammalian Bcl-2 family proteins, thus validating the zebrafish as a model system to further dissect the molecular mechanisms that regulate apoptosis in future forward genetic and chemical modifier screens.