Pre-targeting of polymeric nanomaterials to balance tumour accumulation and clearance.

Pre-targeting of bispecific antibodies is probed to enhance tumour retention while limiting clearance of administered multifunctional branched PEGylated nanomedicines. The temporal influence of pre-targeting on polymer interaction with tumour cells and tissue is explored using in vitro assays through to preclinical validation.

Blocking FcRn in humans reduces circulating IgG levels and inhibits IgG immune complex-mediated immune responses.

The neonatal crystallizable fragment receptor (FcRn) functions as an intracellular protection receptor for immunoglobulin G (IgG). Recently, several clinical studies have reported the lowering of circulating monomeric IgG levels through FcRn blockade for the potential treatment of autoimmune diseases. Many autoimmune diseases, however, are derived from the effects of IgG immune complexes (ICs). We generated, characterized, and assessed the effects of SYNT001, a FcRn-blocking monoclonal antibody, in mice, nonhuman primates (NHPs), and humans. SYNT001 decreased all IgG subtypes and IgG ICs in the circulation of humans, as we show in a first-in-human phase 1, single ascending dose study. In addition, IgG IC induction of inflammatory pathways was dependent on FcRn and inhibited by SYNT001. These studies expand the role of FcRn in humans by showing that it controls not only IgG protection from catabolism but also inflammatory pathways associated with IgG ICs involved in a variety of autoimmune diseases.

Inhibition of prolyl hydroxylase domain proteins selectively enhances venous thrombus neovascularisation.

BACKGROUND: Hypoxia within acute venous thrombi is thought to drive resolution through stabilisation of hypoxia inducible factor 1 alpha (HIF1α). Prolyl hydroxylase domain (PHD) isoforms are critical regulators of HIF1α stability. Non-selective inhibition of PHD isoforms with l-mimosine has been shown to increase HIF1α stabilisation and promote thrombus resolution. OBJECTIVE: The aim of this study was to investigate the therapeutic potential of PHD inhibition in venous thrombus resolution. METHODS: Thrombosis was induced in the inferior vena cava of mice using a combination of flow restriction and endothelial activation. Gene and protein expression of PHD isoforms in the resolving thrombus was measured by RT-PCR and immunohistochemistry. Thrombus resolution was quantified in mice treated with pan PHD inhibitors AKB-4924 and JNJ-42041935 or inducible all-cell Phd2 knockouts by micro-computed tomography, 3D high frequency ultrasound or endpoint histology. RESULTS: Resolving venous thrombi demonstrated significant temporal gene expression profiles for PHD2 and PHD3 (P < 0.05), but not for PHD1. PHD isoform protein expression was localised to early and late inflammatory cell infiltrates. Treatment with selective pan PHD inhibitors, AKB-4924 and JNJ-42041935, enhanced thrombus neovascularisation (P < 0.05), but had no significant effect on overall thrombus resolution. Thrombus resolution or its markers, macrophage accumulation and neovascularisation, did not differ significantly in inducible all-cell homozygous Phd2 knockouts compared with littermate controls (P > 0.05). CONCLUSIONS: This data suggests that PHD-mediated thrombus neovascularisation has a limited role in the resolution of venous thrombi. Directly targeting angiogenesis alone may not be a viable therapeutic strategy to enhance venous thrombus resolution.

United Kingdom national paediatric bilateral project: Results of professional rating scales and parent questionnaires.

OBJECTIVES: This fourteen-centre project used professional rating scales and parent questionnaires to assess longitudinal outcomes in a large non-selected population of children receiving simultaneous and sequential bilateral cochlear implants. METHODS: This was an observational non-randomized service evaluation. Data were collected at four time points: before bilateral cochlear implants or before the sequential implant, one year, two years, and three years after. The measures reported are Categories of Auditory Performance II (CAPII), Speech Intelligibility Rating (SIR), Bilateral Listening Skills Profile (BLSP) and Parent Outcome Profile (POP). RESULTS: Thousand and one children aged from 8 months to almost 18 years were involved, although there were many missing data. In children receiving simultaneous implants after one, two, and three years respectively, median CAP scores were 4, 5, and 6; median SIR were 1, 2, and 3. Three years after receiving simultaneous bilateral cochlear implants, 61% of children were reported to understand conversation without lip-reading and 66% had intelligible speech if the listener concentrated hard. Auditory performance and speech intelligibility were significantly better in female children than males. Parents of children using sequential implants were generally positive about their child's well-being and behaviour since receiving the second device; those who were less positive about well-being changes also generally reported their children less willing to wear the second device. CONCLUSION: Data from 78% of paediatric cochlear implant centres in the United Kingdom provide a real-world picture of outcomes of children with bilateral implants in the UK. This large reference data set can be used to identify children in the lower quartile for targeted intervention.

United Kingdom national paediatric bilateral project: Demographics and results of localization and speech perception testing.

OBJECTIVES: To assess longitudinal outcomes in a large and varied population of children receiving bilateral cochlear implants both simultaneously and sequentially. METHODS: This observational non-randomized service evaluation collected localization and speech recognition in noise data from simultaneously and sequentially implanted children at four time points: before bilateral cochlear implants or before the sequential implant, 1 year, 2 years, and 3 years after bilateral implants. No inclusion criteria were applied, so children with additional difficulties, cochleovestibular anomalies, varying educational placements, 23 different home languages, a full range of outcomes and varying device use were included. RESULTS: 1001 children were included: 465 implanted simultaneously and 536 sequentially, representing just over 50% of children receiving bilateral implants in the UK in this period. In simultaneously implanted children the median age at implant was 2.1 years; 7% were implanted at less than 1 year of age. In sequentially implanted children the interval between implants ranged from 0.1 to 14.5 years. Children with simultaneous bilateral implants localized better than those with one implant. On average children receiving a second (sequential) cochlear implant showed improvement in localization and listening in background noise after 1 year of bilateral listening. The interval between sequential implants had no effect on localization improvement although a smaller interval gave more improvement in speech recognition in noise. Children with sequential implants on average were able to use their second device to obtain spatial release from masking after 2 years of bilateral listening. Although ranges were large, bilateral cochlear implants on average offered an improvement in localization and speech perception in noise over unilateral implants. CONCLUSION: These data represent the diverse population of children with bilateral cochlear implants in the UK from 2010 to 2012. Predictions of outcomes for individual patients are not possible from these data. However, there are no indications to preclude children with long inter-implant interval having the chance of a second cochlear implant.

Strategies for engaging with future radiation protection professionals: a public outreach case study.

It is evident that there is a nuclear skills shortage within the UK, and logically it can be assumed that the shortfall extends to the radiation protection arena. Plans for nuclear new-build and the decommissioning of existing nuclear sites will require many more people with radiological knowledge and practical competencies. This converts to a nuclear industry requirement in the order of 1000 new recruits per year over at least the next ten years, mainly as new apprentices and graduates. At the same time, the strong demand for persons with radiation protection know-how in the non-nuclear and health care sectors is unlikely to diminish. The task of filling this skills gap is a significant one and it will require a determined effort from many UK stakeholders. The Society for Radiological Protection (SRP) has adopted a strategy in recent years to help address this skills gap. The aim is to engage the interest of secondary school students in the science of radiation and inspire them to follow a career in radiation protection. This paper presents the reasoning behind this strategy and, in an 'outreach case study', describes the establishment of the annual SRP Schools Event. This event is becoming an important addition to the national efforts aimed at increasing the numbers of skilled UK radiation protection professionals over the forthcoming decades.

Pre-emptive, early, and delayed alendronate treatment in a rat model of knee osteoarthritis: effect on subchondral trabecular bone microarchitecture and cartilage degradation of the tibia, bone/cartilage turnover, and joint discomfort.

OBJECTIVE: Bisphosphonates are considered potential disease modifying osteoarthritis (OA) agents. The present study investigated the efficacy of pre-emptive, early, and delayed alendronate (ALN) treatment initiation on subchondral trabecular bone and cartilage in low-dose monosodium iodoacetate (MIA)-induced knee OA in rats. METHODS: Male rats received pre-emptive (n = 12, day 0-end of week 2), early (n = 12, end of week 2-end of week 6), or delayed (n = 12, end of week 6-end of week 10) ALN treatment (30 µg/kg/week). Pre-emptive ALN-treated rats were scanned using in vivo micro-computed tomography (micro-CT) after 2 weeks and then sacrificed, early ALN-treated rats were scanned after 2 and 6 weeks and sacrificed, and the delayed ALN-treated rats were scanned after 2, 6, and 10 weeks of OA induction and sacrificed. After sacrifice, bone histomorphometry and histology of the tibia and biomarker analyses were undertaken. Changes in hind limb weight-bearing were assessed from day -1 until day 14. RESULTS: MIA-induced pathological features similar to progressive human OA in the cartilage and subchondral bone. Pre-emptive ALN treatment preserved subchondral trabecular bone microarchitecture, prevented bone loss, decreased bone turnover and joint discomfort. Pre-emptive ALN treatment had moderate effects on cartilage degradation. Early and delayed ALN treatments prevented loss of trabeculae and decreased bone turnover, but had no significant effect on cartilage degradation. CONCLUSION: ALN prevented increased bone turnover and preserved the structural integrity of subchondral bone in experimental OA. The time point of treatment initiation is crucial for treating OA. Treating both the subchondral bone and cartilage in OA would be clinically more beneficial.

In situ fatty acid profile of femoral cancellous subchondral bone in osteoarthritic and fragility fracture females: implications for bone remodelling.

We report here differences in the fatty acid profile of cancellous bone matrix, including n-3, n-6, mono- and poly-unsaturated, as well as saturated fats, between femoral heads from female OA (n=8, aged 68-88years), fractured neck of femur (#NOF) (n=19, 67-88years) and autopsy controls (CTRL) (n=4, 85-97years). Femoral heads were collected from individuals undergoing orthopaedic surgery for OA or #NOF; the fatty acid profile of sub-samples from the superior principal compressive and superior principal tensile regions were determined by gas chromatography. A total of 42 individual fatty acids were detected at varying concentrations with significant differences between subchondral bone from OA subjects, subchondral bone from #NOF subjects and subchondral bone from CTRL subjects, as well as between the superior principal compressive and superior principal tensile regions (for saturated fats only). Subchondral bone from OA subjects had higher total n-6 (OA=10.89±3.17, #NOF=11.11±1.83, CTRL=8.32±2.05, p=0.008) and total n-3 (OA=1.34±0.38, #NOF=1.19±0.18, CTRL=1.15±0.48, p=0.011) percentages than subchondral bone from #NOF subjects and subchondral bone from CTRL subjects, and there was no difference in the n-6:n-3 ratio, nor within the percentage of n-9 fatty acids. Arachidonic acid (OA=0.42±0.16, #NOF=0.26±0.06, CTRL=0.28±0.06, p=0.01), and γ-linolenic acid (OA=0.11±0.03, #NOF=0.05±0.02, CTRL=0.04±0.02, p<0.001) were higher in subchondral bone from OA subjects than subchondral bone from #NOF subjects and subchondral bone from CTRL subjects. In conclusion, there is a wide diversity of fatty acids in cancellous bone matrix from the femoral heads of OA and #NOF, suggesting they may have regulatory effects on inflammatory processes, and their metabolites. This article is part of a Special Issue entitled "Osteoarthritis".

Techniques of assessing hypoxia at the bench and bedside.

Tissues require an adequate supply of oxygen in order to maintain normal cell function. Low oxygen tension (hypoxia) is characteristic of a number of conditions, including cancer, atherosclerosis, rheumatoid arthritis, critical limb ischaemia, peripheral vascular disease, and ischaemic heart disease. Tissue hypoxia is found in tumours, atherosclerotic plaque, and ischaemic myocardium. There is a growing interest in methods to detect and assess hypoxia, given that hypoxia is important in the progression of these diseases. Hypoxia can be assessed at the level of the whole organ, tissue, or cell, using both invasive and non-invasive methods, and by a range of immunohistochemical, biochemical, or imaging techniques. This review describes and critiques current methods of assessing hypoxia that are used at the bench and in clinical practice.

Controlled release recombinant human interferon-α2b for treating patients with chronic hepatitis C genotype 1: a phase 2a clinical trial.

Better convenience and tolerability and sustained therapeutic concentrations might improve interferon (IFN) treatment for chronic hepatitis C virus (HCV) infection. In an open-label, randomized study, controlled release free (chemically unmodified) recombinant human IFN-α(2b) in poly(ether-ester) microspheres (CR-rhIFN-α(2b)), was injected at doses of 160, 320, 480 or 640 µg every 2 weeks for 12 weeks with concomitant weight-based oral ribavirin in 32 treatment-naïve patients with chronic HCV genotype 1. Treatment was well tolerated, with 31 patients (97%) successfully completing the study. Full doses of CR-rhIFN-α(2b) were administered on 96% of scheduled occasions. Flu-like symptoms were generally mild and brief. Injection site reactions developed in 13 patients (41%), and neutropenia occurred in six of eight patients receiving 640 µg. In the 320, 480 and 640 µg groups, 62-75% of patients achieved a ≥2 log(10) HCV RNA reduction by 4 weeks and 88-100% by 12 weeks. For those groups, the pooled median time to ≥2 log(10) reduction was 11 days (95% confidence interval, 7-35 days). In those groups, viral reduction below the limit of detection was accomplished in 25% of patients by 4 weeks and in 62% by 12 weeks. The 160-µg dose was less potent. After CR-rhIFN-α(2b) injection, stable plateau levels of serum IFN-α(2b) were generally reached within 72 h. Treatment-emergent neutralizing antibodies to IFN-α(2b) were observed in one patient. No antibodies to host plant proteins were detected. CR-rhIFN-α(2b) with ribavirin cotherapy was well tolerated and displayed potent early antiviral activity in patients with chronic HCV genotype 1.

Legumain and cathepsin-L expression in human unstable carotid plaque.

OBJECTIVE: The cysteine protease, legumain, is thought to have a role in the processing and activation of proteases such as cathepsin-L, which have been implicated in plaque rupture. This study aimed to determine: if legumain activity is up-regulated in unstable areas of plaque; the effect of legumain over-expression on the activity of cathepsin-L and the effect of mutation of the legumain RGD sequence on its cellular location. METHODS AND RESULTS: Legumain was measured in human carotid plaque extracts (n=17) using a novel ELISA and modified activity assay. Unstable regions of plaque contained more than twice the amount of legumain protein (P<0.001) and activity (P<0.03) compared with stable regions of the same plaque. Over-expression of legumain in THP-1 macrophages using an adenoviral construct resulted in the processing of cathepsin-L from its 30kDa to its 25kDa form compared with controls. CONCLUSION: Unstable regions of plaque contain increased levels of active legumain. Over-expression of legumain in macrophages alters intracellular processing of cathepsin-L to its mature 25kDa form. This may be a means by which legumain could contribute to plaque instability.

Four-dimensional quantitative analysis of the gait of mutant mice using coarse-grained motion capture.

To analyze an abnormal gait pattern in mutant mice (Hugger), we conducted coarse-grained motion capture. Using a simple retroreflective marker-based approach, we could detect high-resolution mutant-specific gait patterns. The phenotypic gait patterns are caused by extreme vertical motion of limbs, revealing inefficient motor functions. To elucidate the inefficiency, we developed a musculoskeletal computer model of the mouse hindlimb based on X-ray CT data. By integrating motion data with the model, we determined mutant-specific musculotendon lengths, suggesting that three major muscles were involved in the abnormal gait. This approach worked well on laboratory mice, which were putatively too small to be motion capture subjects. Motion capture technology was originally developed for human study, and our approach may help fill neuroscience gaps between mouse and human behavioral phenotypes.

Adenovirus-mediated VEGF gene therapy enhances venous thrombus recanalization and resolution.

OBJECTIVE: Rapid thrombus recanalization reduces the incidence of post-thrombotic complications. This study aimed to discover whether adenovirus-mediated transfection of the vascular endothelial growth factor gene (ad.VEGF) enhanced thrombus recanalization and resolution. METHODS AND RESULTS: In rats, thrombi were directly injected with either ad.VEGF (n=40) or ad.GFP (n=37). Thrombi in SCID mice (n=12) were injected with human macrophages transfected with ad.VEGF or ad.GFP. Thrombi were analyzed at 1 to 14 days. GFP was found mainly in the vein wall and adventitia by 3 days, but was predominantly found in cells within the body of thrombus by day 7. VEGF levels peaked at 4 days (376+/-299 pg/mg protein). Ad.VEGF treatment reduced thrombus size by >50% (47.7+/-5.1 mm(2) to 22.0+/-4.0 mm(2), P=0.0003) and increased recanalization by >3-fold (3.9+/-0.69% to 13.6+/-4.1%, P=0.024) compared with controls. Ad.VEGF treatment increased macrophage recruitment into the thrombus by more than 50% (P=0.002). Ad.VEGF-transfected macrophages reduced thrombus size by 30% compared with controls (12.3+/-0.89 mm(2) to 8.7+/-1.4 mm(2), P=0.04) and enhanced vein lumen recanalization (3.39+/-0.34% to 5.07+/-0.57%, P=0.02). CONCLUSIONS: Treatment with ad.VEGF enhanced thrombus recanalization and resolution, probably as a consequence of an increase in macrophage recruitment.

Effect of statins on proteolytic activity in the wall of abdominal aortic aneurysms.

BACKGROUND: The aim of this study was to examine the effect of statin treatment on the activity of proteases in the wall of abdominal aortic aneurysms (AAAs). METHODS: The activities of matrix metalloproteinases (MMPs) 9 and 3, cathepsins B, H, K, L and S, and the cystatin C level were measured in extracts of AAA wall taken from 82 patients undergoing AAA repair; 21 patients were receiving statin treatment before surgery. All values were standardized against soluble protein (SP) concentration in the extract, and reported as median (interquartile range) or mean(s.e.m.). RESULTS: The two groups had similar demographics. Reduced activity of MMP-9 (43 (34-56) versus 80 (62-110) pg per mg SP; P < 0.001), cathepsin H (183 (117-366) versus 321 (172-644) nmol 4-methylcoumarin-7-amide released per mg SP; P = 0.016) and cathepsin L (102 (51-372) versus 287 (112-816) micromol 7-amino-4-trifluoromethylcoumarin released per mg SP; P = 0.020) was found in the statin-treated aortas compared with AAAs from patients not taking a statin. The statin-treated group had lower MMP-3 activity, but this did not reach statistical significance (P = 0.053). Cystatin C levels were higher in statin-treated aortas than in controls (41.3(3.1) versus 28.9(2.1) ng per mg SP; P = 0.003). CONCLUSION: Statins decreased the activity of proteases that have been implicated in aneurysm disease.

Microbial community structure and trichloroethylene degradation in groundwater.

Trichloroethylene (TCE) is a prevalent contaminant of groundwater that can be cometabolically degraded by indigenous microbes. Groundwater contaminated with TCE from a US Department of Energy site in Ohio was used to characterize the site-specific impact of phenol on the indigenous bacterial community for use as a possible remedial strategy. Incubations of 14C-TCE-spiked groundwater amended with phenol showed increased TCE mineralization compared with unamended groundwater. Community structure was determined using DNA directly extracted from groundwater samples. This DNA was then analyzed by amplified ribosomal DNA restriction analysis. Unique restriction fragment length polymorphisms defined operational taxonomic units that were sequenced to determine phylogeny. DNA sequence data indicated that known TCE-degrading bacteria including relatives of Variovorax and Burkholderia were present in site water. Diversity of the amplified microbial rDNA clone library was lower in phenol-amended communities than in unamended groundwater (i.e., having Shannon-Weaver diversity indices of 2.0 and 2.2, respectively). Microbial activity was higher in phenol-amended ground water as determined by measuring the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride. Thus phenol amendments to groundwater correlated with increased TCE mineralization, a decrease in diversity of the amplified microbial rDNA clone library, and increased microbial activity.

Signal transduction in larval trematodes: putative systems associated with regulating larval motility and behaviour.

The multi-host lifestyle of parasitic trematodes necessitates their ability to communicate with their external environment in order to invade and navigate within their hosts' internal environment. Through recent EST and genome sequencing efforts, it has become clear that members of the Trematoda possess many of the elaborate signal transduction systems that have been delineated in other invertebrate model systems like Drosophila melanogaster and Caenorhabditis elegans. Gene homologues representing several well-described signal receptor families including receptor tyrosine kinases, receptor serine tyrosine kinases, G protein-coupled receptors and elements of their downstream signalling systems have been identified in larval trematodes. A majority of this work has focused on the blood flukes, Schistosoma spp. and therefore represents a narrow sampling of the diverse digenean helminth taxon. Despite this fact and given the substantial evidence supporting the existence of such signalling systems, the question then becomes, how are these systems employed by larval trematodes to aid them in interpreting signals received from their immediate environment to initiate appropriate responses in cells and tissues comprising the developing parasite stages? High-throughput, genome-wide analysis tools now allow us to begin to functionally characterize genes differentially expressed throughout the development of trematode larvae. Investigation of the systems used by these parasites to receive and transduce external signals may facilitate the creation of technologies for achieving control of intramolluscan schistosome infections and also continue to yield valuable insights into the basic mechanisms regulating motility and behaviour in this important group of helminths.

Safety of plasmin in the setting of concomitant aspirin and heparin administration in an animal model of bleeding.

Plasmin is a direct thrombolytic which has been shown to have a strikingly favorable benefit to risk profile in comparison with plasminogen activators, notably tissue plasminogen activator (t-PA). As heparin is known to increase the risk of hemorrhage when co-administered with a plasminogen activator, we asked whether adjunct antithrombotic agents such as aspirin and heparin would affect the safety of plasmin. Three groups of rabbits were administered plasmin at a dose (4 mg kg-1) designed to induce significant decreases in antiplasmin, fibrinogen and factor (F)VIII, to about 25, 40 and 40%, respectively, of baseline values, but not cause prolongation of the ear puncture bleeding time. In a blinded and randomized trial, the results show that an intravenous aspirin bolus plus heparin administered as a bolus followed by a maintenance continuous infusion did not significantly prolong the bleeding time during plasmin infusion. These data indicate that in the rabbit, concomitant use of aspirin plus heparin does not affect the safety of a therapeutic dose of plasmin.

Sonographic estimate of birth weight: relative accuracy of sonographers versus maternal-fetal medicine specialists.

OBJECTIVE: To compare the relative accuracy of predicting birth weight among registered diagnostic medical sonographers versus maternal-fetal medicine specialists. STUDY DESIGN: Over 7 months all patients who delivered within 2 weeks and had sonographic measurements of femur length and head and abdominal circumferences by sonographers and physicians were included in the analysis. The exclusion criteria were multiple gestation and anomalous fetuses. Receiver operating-characteristic curves (ROC) were constructed to determine the ability to detect intrauterine growth restriction (IUGR; birth weight < 2,500 g) and macrosomia (birth weight > or = 4,000 g) among term (gestational age > or = 37 weeks) parturients. A level of p < 0.05 was considered significant. RESULTS: Among 365 patients recruited, the mean gestational age was 37.3 +/- 2.4 weeks with a mean birth weight of 3,083 +/- 72.5 g. Among term patients the prevalence of IUGR was 7.5% (18/238) and of macrosomia 12% (29/238). A significantly higher percentage of predictions were within 10% of the birth weight when obtained by sonographers (70%) than physicians (54%; p < 0.0001). Registered sonographers were significantly more likely to detect IUGR than the specialists (area under the ROC curves 0.97 +/- 0.02 vs. 0.92 +/- 0.02, respectively; p = 0.02). Both groups had similar accuracy in detecting macrosomic fetuses (area under the ROC curves 0.92 +/- 0.02 for sonographers and 0.90 +/- 0.02 for physicians; p = 0.40). CONCLUSIONS: Prediction of birth weight is significantly more accurate when sonographers rather than maternal-fetal medicine specialists perform the ultrasonographic examination.