Causes of mortality and pathological lesions observed post-mortem in red squirrels (Sciurus vulgaris) in Great Britain.

BACKGROUND: The red squirrel population in Great Britain has declined dramatically in recent decades, principally due to squirrelpox. Concern exists that red squirrels may become extinct nationally and, as there has been limited research in to diseases other than squirrelpox, this study aimed to identify additional causes of mortality. RESULTS: Post-mortem examinations on 163 red squirrels found dead on Isle of Wight (IoW) England, in Scotland and at other locations in Great Britain showed that 41.7% (n = 68) were killed by road traffic and 9.2% (n = 15) by predators, principally domestic cats and dogs. The overall male/female ratio was 1.08/1. Fleas were recorded on 34.9% of IoW squirrels and on 43.8% of Scottish squirrels but sucking lice and ixodid ticks were only seen on Scottish squirrels. Bacterial infections were significant, particularly in association with respiratory disease (n = 16); two squirrels died of Bordetella bronchiseptica bronchopneumonia. Cases of fatal exudative dermatitis (n = 5) associated with a lukM-positive clone of Staphylococcus aureus occurred only on the IoW. Toxoplasmosis (n = 12) was also confined to IoW where it was responsible for almost one tenth (9.5%) of all deaths. Hepatozoonosis was common, especially in IoW squirrels, but was not considered a primary cause of mortality. Hepatic capillariasis affected four IoW squirrels and one from Scotland. Fungal infections included oral candidiasis, adiaspiromycosis and pulmonary phaeohyphomycosis. Neoplastic conditions diagnosed were: pulmonary carcinoma, gastric spindle cell tumour, renal papillary adenoma and trichoepithelioma. Epidermal hyperplasia of unknown aetiology was seen in squirrels showing crusty lesions of the ear pinnae on IoW (n = 3) and Brownsea Island (n = 1), associated in two cases with cutaneous wart-like growths. Miscellaneous diagnoses included chylothorax, electrocution, intussusception, suspected cholecalciferol rodenticide poisoning and foetal death and mummification. No cases of squirrelpox were diagnosed. CONCLUSIONS: Red squirrels in Britain suffer premature or unnatural mortality due to a number of conditions in addition to squirrelpox, many of which result, directly or indirectly, from human activities: road traffic trauma, pet predation, toxoplasmosis, trap injuries, rodenticide poisoning and electrocution accounted for 61% of all recorded mortality in this study. Red squirrels are also affected by several diseases of unknown aetiology which merit further research.

Expression of Snail2 in long bone osteosarcomas correlates with tumour malignancy.

Snail2 is a marker of malignancy in epithelial tumours; however, in sarcomas, it is not known if this protein is present. Here we examine the expression of Snail2 in one type of sarcoma, osteosarcoma, and explore its relationship to tumour grade, subtype and anatomical location in cases of long bone and cranial bone osteosarcoma. Long bone osteosarcomas typically have a much greater metastatic capability and a poorer prognosis. We find that Snail2 is expressed in the three main subtypes of long bone osteosarcoma--osteoblastic, chondroblastic and fibroblastic. Regression analysis showed that Snail 2 expression was statistically correlated with tumour grade (p = 0.014) in all of these subtypes. Snail2 was only expressed in high-grade cranial bone osteosarcomas, suggesting a link between Snail2 expression and metastasis. This is the first time Snail2 has been associated with any sarcoma, and this study shows that Snail2 may be a useful prognostic marker for this disease.

Assessing the most clinically and cost effective method of closing skin following surgery.

BACKGROUND: Topical skin adhesives (TSAs) and short-term absorbable sutures (STASs) are not as widely used in surgery as nonabsorbable or slowly absorbed sutures. AIM: To assess the effectiveness and cost of skin closure techniques for surgical wounds. METHOD: Data from clinical trials was used to assess the effectiveness of different skin closure methods. NHS costs were determined from questionnaires sent to nurses to identify treatment elements for individual patients. RESULTS: The study found TSAs and STASs were as effective as traditional surgical wound closure methods. DISCUSSION AND CONCLUSION: Increasing the use of TSAs and STASs in routine surgery could lead to considerable cost savings for the NHS without compromising clinical effectiveness or safety.

Inactivation of CYP2D6 by methylenedioxymethamphetamine in different recombinant expression systems.

Recombinantly expressed CYP450 systems (rCYPs) are often used to screen for irreversible/quasi-irreversible enzyme inhibitors during drug development. The concentration- and time-dependent inactivation of CYP2D6 by methylenedioxymethamphetamine (MDMA) was compared in three different rCYP2D6 systems (yeast microsomes, Supersomestrade mark and Bactosomestrade mark) under the conditions of the most commonly used protocols in assessing mechanism-based inactivation (MBI). MDMA (2-20microM) was pre-incubated with enzyme for 0, 2.5 and 5min followed by a five-fold dilution and further incubation with dextromethorpan (DEX) (50microM). The formation of dextrorphan (DOR) from DEX was used as a specific marker of CYP2D6 activity. Concentration- and time-dependent inactivation of CYP2D6 by MDMA was observed with each rCYP system. However, the apparent kinetic parameters for MBI (k(inact), the maximum inactivation rate constant and K(I), the inhibitor concentration associated with half maximal rate of inactivation) were significantly greater (p<0.05) for Bactosomestrade mark (0.95+/-0.33min(-1), 42.9+/-20.1microM) than those found using yeast microsomes (0.28+/-0.04min(-1), 2.86+/-1.18microM) and Supersomestrade mark (0.38+/-0.05min(-1), 3.66+/-0.10microM). After correction for depletion of MDMA during pre-incubation, k(inact) and K(I) values determined using Bactosomestrade mark decreased significantly but remained higher than for the other rCYP systems (p<0.05). Substantial metabolism of DOR after its formation from DEX was also observed using Supersomestrade mark and Bactosomestrade mark. Sub-optimal study design when investigating MBI may compromise the quantitative characterization of inhibitory characteristics using some rCYP systems.

Kinetics of the time-dependent inactivation of CYP2D6 in cryopreserved human hepatocytes by methylenedioxymethamphetamine (MDMA).

Methylenedioxymethamphetamine (MDMA) was investigated in cryopreserved human hepatocytes as a time-dependent inactivator (TDI) of CYP2D6 using dextromethorphan (DEX) as a probe substrate. Inhibition kinetic parameters k(inact), the maximal rate of inactivation, and K(I), the inhibitor concentration at half the maximal activation rate, were determined. Time- and concentration-dependent inhibition were confirmed, and the influence of different elements of study design (e.g. cell number, stability of hepatocytes, dilution after preincubation) on estimated kinetic parameters were evaluated. Dilution factors (DF) of 1.2, 5 or total removal of inhibitor (by washing cells after preincubation, WR) resulted in k(inact) and K(I) (+/-S.E.) values of 0.02+/-0.002 min(-1) and 0.88+/-0.31 microM, 0.01+/-0.001 min(-1) and 1.23+/-0.70 microM, and 0.01+/-0.001 min(-1) and 2.10+/-1.32 microM, respectively; indicating that insufficient dilution may lead to overestimation of CYP2D6 inactivation. Accounting for MDMA depletion during the preincubation, corrected K(I) values were significantly lower (0.11+/-0.05 microM, 0.15+/-0.09 microM, 0.24+/-0.16 microM for DF of 1.2, 5, and WR, respectively). Inactivation efficiency in hepatocytes, as measured by k(inact)/K(I), was 10-fold less than that previously reported in human liver microsomes or recombinantly expressed systems. Possible causes for the observed differences between in vitro systems warrant further investigation. These may include differences in metabolic consumption of MDMA in each system, non-specific binding and presence of active efflux in hepatocytes.

The impact of experimental design on assessing mechanism-based inactivation of CYP2D6 by MDMA (Ecstasy).

MDMA (3-4-methylenedioxymethamphetamine, commonly known as Ecstasy) is a potent mechanism-based inhibitor (MBI) of cytochrome P450 2D6 (CYP2D6), causing quasi-irreversible inhibition of the enzyme in vitro. An evaluation of the in vivo implications of this phenomenon depends on the accuracy of the estimates of the parameters that define the inhibition in vitro, namely k(inact) (the maximal inhibition rate) and KI (the inactivation constant). These values are determined in two steps, pre-incubation of the enzyme with the inhibitor (enzyme inactivation), followed by dilution and further incubation to measure residual enzyme activity with a probe substrate. The aim of this study was to assess the impact of different dilutions and probe substrate concentrations on the estimates of k(inact) and KI using recombinantly expressed CYP2D6. Enzyme activity was measured by the conversion of dextromethorphan (DEX) to dextrorphan (DOR). Dilution factors of 1.25, 2, 5, 10, 25 and 50 (DEX at 30 microM) gave mean (+/-SE) values of k(inact) (min-1) of 0.20+/-0.06, 0.21+/-0.05, 0.31+/-0.06, 0.37+/-0.11, 0.51+/-0.10 and 0.58+/-0.08, respectively, and KI (microM) values (after correction for non-specific microsomal binding) of 2.22+/-1.90, 2.80+/-1.34, 5.78+/-2.07, 6.36+/-2.93, 3.99+/-1.57 and 4.86+/-1.37, respectively. Accordingly, high (e.g. 50 fold) and low (e.g. 1.25 fold) dilutions were associated with statistically significant differences in kinetic values (p <0.05). Varying DEX concentration (10-100 microM) was not associated with significant changes in k(inact) and KI values when a five-fold dilution was used (with the exception of a lower KI at 10 microM DEX). High dilution was also shown to reduce non-specific microsomal binding of MDMA. The changes in the two kinetic parameters were dependent on the experimental procedure and shown to be unlikely to have a material influence on the maximum inhibition of CYP2D6 expected in vivo after typical recreational doses of MDMA (50-100 mg), since the potency of inhibition was high. The different values of the kinetic parameters were predicted to have a marginal influence on the time for recovery of enzyme activity following re-synthesis of CYP2D6.

Metabolism of dextrorphan by CYP2D6 in different recombinantly expressed systems and its implications for the in vitro assessment of dextromethorphan metabolism.

Cytochrome P450 2D6 (CYP2D6) mediated formation of dextrorphan (DOR) from dextromethorphan (DEX) is widely used as a marker to assess the activity of this enzyme both in vitro and in vivo. The sequential metabolism of DOR during in vitro studies, particularly using recombinant systems (rCYPs) expressing human CYP2D6, is assumed to be negligible. The extent of metabolism was investigated for a range of DEX and DOR concentrations in microsomal preparations from three different rCYPs expressing human CYP2D6 (yeast, Supersomes and Bactosomes) containing 10 pmol of the enzyme. Bactosomes and Supersomes, but not yeast rCYP microsomes, were capable of metabolising DOR to 3-hydroxymorphinan (HYM). Two novel CYP2D6 related metabolites were identified in Bactosomes, and assigned as single hydroxylations in the phenyl rings of DOR and HYM using ion-trap mass spectrometry. Therefore, in rCYP systems with high turn over rate (e.g. Bactosomes) DOR may not be considered as an end product particularly at low concentrations of DEX; leading to an underestimation of true metabolic rate. The results also put further emphasis on the necessity of optimising study conditions when switching between rCYP sources.

Preclinical anticancer activity of the potent, oral Src inhibitor AZD0530.

AZD0530, an orally available Src inhibitor, demonstrated potent antimigratory and anti-invasive effects in vitro, and inhibited metastasis in a murine model of bladder cancer. Antiproliferative activity of AZD0530 in vitro varied between cell lines (IC(50) 0.2 ->10µM). AZD0530 inhibited tumor growth in 4/10 xenograft models tested and dynamically inhibited in vivo phosphorylation of Src substrates paxillin and FAK in both growth-inhibition-resistant and -sensitive xenografts. The activity of AZD0530 in NBT-II bladder cancer cells in vitro was consistent with inhibition of cell migration and stabilization of cell-cell adhesion. These data suggest a dominant anti-invasive pharmacology for AZD0530 that may limit tumor progression in a range of cancers. AZD0530 is currently in Phase II clinical trials.