Clinical trial designs for rare diseases: Studies developed and discussed by the International Rare Cancers Initiative

BACKGROUND: The past three decades have seen rapid improvements in the diagnosis and treatment of most cancers and the most important contributor has been research. Progress in rare cancers has been slower, not least because of the challenges of undertaking research.

SETTINGS: The International Rare Cancers Initiative (IRCI) is a partnership which aims to stimulate and facilitate the development of international clinical trials for patients with rare cancers. It is focused on interventional--usually randomized--clinical trials with the clear goal of improving outcomes for patients. The key challenges are organisational and methodological. A multi-disciplinary workshop to review the methods used in ICRI portfolio trials was held in Amsterdam in September 2013. Other as-yet unrealised methods were also discussed.

RESULTS: The IRCI trials are each presented to exemplify possible approaches to designing credible trials in rare cancers. Researchers may consider these for use in future trials and understand the choices made for each design.

INTERPRETATION: Trials can be designed using a wide array of possibilities. There is no 'one size fits all' solution. In order to make progress in the rare diseases, decisions to change practice will have to be based on less direct evidence from clinical trials than in more common diseases.

Discussion and debate: Reflections on Pleistocene Island Occupation

Editorial Note: Within a 48-hour period during January 2014, the JMA co-editors received two papers—those of Curtis Runnels and Thomas P. Leppard printed above—that, quite fortuitously, each addressed the topic of Mediterranean island colonization by archaic hominins, albeit from radically different perspectives. Neither author was aware of the other’s paper, nor has either article subsequently been revised to take account of the other. Realizing the widespread current interest in this subject and the possibility for productive debate prompted by such variant approaches, we commissioned three sets of comments and invited Runnels and Leppard to respond. We are pleased to publish this discussion around questions of great importance for our understanding of the earliest insular prehistory of the Mediterranean, and with significant implications reaching well beyond it.

Journal of Mediterranean Archaeology 27.2 (2014) 255-278

Climatic signals in the life histories of insects:the distribution and abundance of heather psyllids (Strophingia spp.) in the UK

1. The population density and age structure of two species of heather psyllid Strophingia ericae and Strophingia cinereae, feeding on Calluna vulgaris and Erica cinerea, respectively, were sampled using standardized methods at locations throughout Britain. Locations were chosen to represent the full latitudinal and altitudinal range of the host plants.

2. The paper explains how spatial variation in thermal environment, insect life-history characteristics and physiology, and plant distribution, interact to provide the mechanisms that determine the range and abundance of Strophingia spp.

3. Strophingia ericae and S. cinereae, despite the similarity in the spatial distribution patterns of their host plants within Britain, display strongly contrasting geographical ranges and corresponding life-history strategies. Strophingia ericae is found on its host plant throughout Britain but S. cinereae is restricted to low elevation sites south of the Mersey-Humber line and occupies only part of the latitudinal and altitudinal range of its host plant. There is no evidence to suggest that S. ericae has reached its potential altitudinal or latitudinal limit in the UK, even though its host plant appears to reach its altitudinal limit.

4. There was little difference in the ability of the two Strophingia spp. to survive shortterm exposure to temperatures as low as - 15 degrees C and low winter temperatures probably do not limit distribution in S. cinereae.

5. Population density of S. ericae was not related to altitude but showed a weak correlation with latitude. The spread of larval instars present at a site, measured as an index of instar homogeneity, was significantly correlated with a range of temperature related variables, of which May mean temperature and length of growing season above 3 degrees C (calculated using the Lennon and Turner climatic model) were the most significant. Factor analysis did not improve the level of correlation significantly above those obtained for single climatic variables. The data confirmed that S. ericae has a I year life cycle at the lowest elevations and a 2 year life cycle at the higher elevations. However, there was no evidence, as previously suggested, for an abrupt change from a one to a 2 year life cycle in S. ericae with increasing altitudes or latitudes.

6. By contrast with S. ericae, S. cinereae had an obligatory 1 year life cycle, its population decreased with altitude and the index of instar homogeneity showed little correlation with single temperature variables. Moreover, it occupied only part of the range of its host plant and its spatial distribution in the UK could be predicted with 96% accuracy using selected variables in discriminant analysis.

7. The life histories of the congeneric heather psyllids reflect adaptations that allow them to exploit host plants with different distributions in climatic and thereby geographical space. Strophingia ericae has the flexible life history that enables it to exploit C. vulgaris throughout its European boreal temperate range. Strophingia cinereae has a less flexible life history and is adapted for living on an oceanic temperate host. While the geographic ranges of the two Strophingia spp. overlap within the UK, the psyllids appear to respond differently to variation in their thermal environment.

Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months' streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy.

Oscar Wilde at Home

Oscar, Constance, Speranza (and Bosie) are “at home” in the exquisitely beautiful Florence Court House this Mayday weekend. House guests will include Jack, Algy, Gwendolen and Cecily from the Importance of Being Earnest, (not to mention the redoubtable Lady Bracknell), Lord and Lady Windermere, the impetuous Lord Darlington, and the precocious Dorian Gray. Visitors will be conducted throughout the house where a range of short scenes from Wilde’s most popular works will be performed live by actors in each of the principal rooms. Is it life or is it art? Come and decide for yourself as we welcome you to the witty, complex and contradictory world of Oscar Wilde. Conceived and directed by David Grant.

Dramatis Personae
Oscar Wilde (and Lord Henry Wotton) – Donal Morgan
Constance Wilde (Lady Wotton, and Cecily Cardew) – Julie Lamberton
Lady Speranza Wilde (Lady Bracknell and Mrs Erlynne) – Antoinette Morelli
Lord Alfred Douglas (and Dorian Gray) – Sydney Bull
Lord Darlington and Basil Hallward – Richard Croxford
Lady Windermere and Gwendolen Fairfax – Stephanie Dale
Jack Worthing – Patrick McBrearty
Algernon Moncrieff and Lord Windermere – Stefan Dunbar
Lane and Parker – Curtis Reed and tbc

Production Team
Costume Design – Enda Kenny
Sound Design – Sydney Bull
Sound Operator – Seth Taylor
Stage Manager – Bronagh McFeely
Company Manager – Eamon Quinn
Director – David Grant

Evidence supporting a role for N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine accumulation in Muller glia dysfunction and death in diabetic retinopathy

Purpose: Recent evidence suggests that neuroglial dysfunction and degeneration contributes to the etiology and progression of diabetic retinopathy. Advanced lipoxidation end products (ALEs) have been implicated in the pathology of various diseases, including diabetes and several neurodegenerative disorders. The purpose of the present study was to investigate the possible link between the accumulation of ALEs and neuroretinal changes in diabetic retinopathy.

Methods: Retinal sections obtained from diabetic rats and age-matched controls were processed for immunohistochemistry using antibodies against several well defined ALEs. In vitro experiments were also performed using a human Muller (Moorfields/Institute of Ophthalmology-Muller 1 [ MIO-M1]) glia cell line. Western blot analysis was used to measure the accumulation of the acrolein-derived ALE adduct N epsilon-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) in Muller cells preincubated with FDP-lysine-modified human serum albumin (FDP-lysine-HSA). Responses of Muller cells to FDP-lysine accumulation were investigated by analyzing changes in the protein expression of heme oxygenase-1 (HO-1), glial fibrillary acidic protein (GFAP), and the inwardly rectifying potassium channel Kir4.1. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) were determined by reverse transcriptase PCR (RT-PCR). Apoptotic cell death was evaluated by fluorescence-activated cell sorting (FACS) analysis after staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide.

Results: No significant differences in the levels of malondialdehyde-, 4-hydroxy-2-nonenal-, and 4-hydroxyhexenal-derived ALEs were evident between control and diabetic retinas after 4 months of diabetes. By contrast, FDP-lysine immunoreactivity was markedly increased in the Muller glia of diabetic rats. Time-course studies revealed that FDP-lysine initially accumulated within Muller glial end feet after only a few months of diabetes and thereafter spread distally throughout their inner radial processes. Exposure of human Muller glia to FDP-lysine-HSA led to a concentration-dependent accumulation of FDP-lysine-modified proteins across a broad molecular mass range. FDP-lysine accumulation was associated with the induction of HO-1, no change in GFAP, a decrease in protein levels of the potassium channel subunit Kir4.1, and upregulation of transcripts for VEGF, IL-6, and TNF-alpha. Incubation of Muller glia with FDP-lysine-HSA also caused apoptosis at high concentrations.

Conclusions: Collectively, these data strongly suggest that FDP-lysine accumulation could be a major factor contributing to the Muller glial abnormalities occurring in the early stages of diabetic retinopathy.

CaV3.1 T-Type Ca2+ Channels Contribute to Myogenic Signalling in Rat Retinal Arterioles

Purpose: Although L-type Ca2+ channels are known to play a key role in the myogenic reactivity of retinal arterial vessels, the involvement of other types of voltage-gated Ca2+ channels in this process remains unknown. In the present study we have investigated the contribution of T-type Ca2+ channels to myogenic signalling in arterioles of the rat retinal microcirculation.

Methods: Confocal immunolabelling of wholemount preparations was used to investigate the localisation of CaV3.1-3 channels in retinal arteriolar smooth muscle cells. T-type currents and the contribution of T-type channels to myogenic signalling were assessed by whole-cell patch-clamp recording and pressure myography of isolated retinal arteriole segments.

Results: Strong immunolabelling for CaV3.1 was observed on the plasma membrane of retinal arteriolar smooth muscle cells. In contrast, no expression of CaV3.2 or CaV3.3 could be detected in retinal arterioles, although these channels were present on glial cell end feet surrounding the vessels and retinal ganglion cells, respectively. TTA-A2 sensitive T-type currents were recorded in retinal arteriolar myocytes with biophysical properties distinct from those of the L-type currents present in these cells. Inhibition of T-type channels using TTA-A2 or ML-218 dilated isolated, myogenically active, retinal arterioles.

Conclusions: CaV3.1 T-type Ca2+ channels are functionally expressed on arteriolar smooth muscle cells of retinal arterioles and play an important role in myogenic signalling in these vessels. The work has important implications concerning our understanding of the mechanisms controlling blood flow autoregulation in the retina and its disruption during ocular disease.

Interacting Supernovae and Supernova Impostors: SN 2009ip, is this the End?

We report the results of a three-year-long dedicated monitoring campaign of a restless luminous blue variable (LBV) in NGC 7259. The object, named SN 2009ip, was observed photometrically and spectroscopically in the optical and near-infrared domains. We monitored a number of erupting episodes in the past few years, and increased the density of our observations during eruptive episodes. In this paper, we present the full historical data set from 2009 to 2012 with multi-wavelength dense coverage of the two high-luminosity events between 2012 August and September. We construct bolometric light curves and measure the total luminosities of these eruptive or explosive events. We label them the 2012a event (lasting ~50 days) with a peak of 3 × 1041 erg s-1, and the 2012b event (14 day rise time, still ongoing) with a peak of 8 × 1042 erg s-1. The latter event reached an absolute R-band magnitude of about -18, comparable to that of a core-collapse supernova (SN). Our historical monitoring has detected high-velocity spectral features (~13,000 km s-1) in 2011 September, one year before the current SN-like event. This implies that the detection of such high-velocity outflows cannot, conclusively, point to a core-collapse SN origin. We suggest that the initial peak in the 2012a event was unlikely to be due to a faint core-collapse SN. We propose that the high intrinsic luminosity of the latest peak, the variability history of SN 2009ip, and the detection of broad spectral lines indicative of high-velocity ejecta are consistent with a pulsational pair-instability event, and that the star may have survived the last outburst. The question of the survival of the LBV progenitor star and its future fate remain open issues, only to be answered with future monitoring of this historically unique explosion.

The progress in understanding and treatment of diabetic retinopathy

In most countries, diabetic retinopathy is the most frequently occurring complication of diabetes mellitus and remains a leading cause of vision loss globally. Its etiology and pathology have been extensively studied for half a century, yet there are disappointingly few therapeutic options. Although some new treatments have been introduced for diabetic macular edema (DME) (e.g. intravitreal vascular endothelial growth factor inhibitors ('anti-VEGFs') and new steroids), up to 50% of patients fail to respond. Furthermore, for people with proliferative diabetic retinopathy (PDR), laser photocoagulation remains a mainstay therapy, even though it is inherently a destructive procedure. This review summarizes the clinical features of diabetic retinopathy and its risk factors. It describes details of retinal pathology and the cell culture approaches and animal models that are used to mimic its key components, advance understanding of its pathogenesis, and enable identification of new therapeutic targets. We emphasise that although there have been significant advances, there is still a pressing need for a better understanding basic mechanisms to enable development of reliable and robust means to identify patients at highest risk, and to intervene effectively before vision loss occurs.

TNF alpha-Induced p38MAPK Activation Regulates TRPA1 and TRPV4 Activity in Odontoblast-Like Cells

The transient receptor potential (TRP) channels are unique cellular sensors that are widely expressed in many neuronal and nonneuronal cells. Among the TRP family members, TRPA1 and TRPV4 are emerging as candidate mechanosensitive channels that play a pivotal role in inflammatory pain and mechanical hyperalgesia. Odontoblasts are nonneuronal cells that possess many of the features of mechanosensitive cells and mediate important defense and sensory functions. However, the effect of inflammation on the activity of the odontoblast's mechanosensitive channels remains unknown. By using immunohistochemistry and calcium microfluorimetry, we showed that odontoblast-like cells express TRPA1 and TRPV4 and that these channels were activated by hypotonicity-induced membrane stretch. Short treatment of odontoblast-like cells with tumor necrosis factor (TNF)-α enhanced TRPA1 and TRPV4 responses to their chemical agonists and membrane stretch. This enhanced channel activity was accompanied by phospho-p38 mitogen-activated protein kinase (MAPK) expression. Treatment of cells with the p38 inhibitor SB202190 reduced TNF-α effects, suggesting modulation of channel activity via p38 MAPK. In addition, TNF-α treatment also resulted in an up-regulation of TRPA1 expression but down-regulation of TRPV4. Unlike TRPV4, enhanced TRPA1 expression was also evident in dental pulp of carious compared with noncarious teeth. SB202190 treatment significantly reduced TNF-α-induced TRPA1 expression, suggesting a role for p38 MAPK signaling in modulating both the transcriptional and non-transcriptional regulation of TRP channels in odontoblasts.

Scene From a Different Angle: Attempts to Internationalize Northern Ireland’s Conflict Experience

Reviews the books, Lessons From the Northern Ireland Peace Process edited by Timothy J. White (2013) and Human Rights as War by Other Means by Jennifer Curtis (2014). Edited by a U.S.-based academic with an enduring interest in Ireland, the first book draws together an interdisciplinary group of academics from across North America and the U.K. (though notably not Northern Ireland itself) to cover such topics as third party intervention, nationalism, grassroots change, and community development. The second text to be reviewed may be seen as a thorough analysis of this particular point: what is the role played by human rights in Northern Ireland’s peace process?

TNF-α Sensitizes Mechanoreceptors in Human Odontoblasts

Background: Mechanotransduction in the dental pulp is mediated by mechano-sensitive trigeminal afferents but accumulating evidence suggests odontoblasts also contribute to mechano-sensory functions of the pulp as evidenced by expression of TRP channels, calcium-activated potassium channels and TREK-1 potassium channels. Activation of these mechano-sensitive channels is considered critical for the mechanotransduction of fluid movement within dentinal tubules into electrical signals transmitted by the pulpal afferents to elicit tooth sensitivity and pain. Since tooth pain and sensitivity are potentiated by inflammation we hypothesise that the inflammatory cytokine TNF-α sensitizes odontoblast responses to mechanical stimuli. Objective: To investigate the effect of TNF-α on the response of odontblast-like cells to mechanical stimuli. Method: Odontoblast-like cells were derived from dental pulp cells of immature third molars as previously described (El-karim et al 20112011 Pain, 152, 2211-2223). Odontoblast response to mechanical stimuli (application of hypotonic solution) was determined using ratiometric calcium imaging. Cells were treated with TNF-α for either 24hrs or short application for 10 mins prior to calcium imaging. Result: Odontoblast-like cells responded to hypotonic solution (230 mOSM) by increase in cytoplasmic Ca2+ concentration [Ca+2]i that was reduced to near base line in the presence of the TRPV4 antagonist RN-1734. Incubation of odontoblast -like cells with TNFα for 24 hrs resulted in a significant increase in cytoplasmic Ca2+ concentration in response to hypotonic stimuli compared to untreated cells. Similar results were obtained when cells were treated with TNF-α for 10 mins prior to imaging. Conclusion: Both short and long term treatment of odontoblasts-like cells with TNF-α resulted in enhanced responses to mechanical stimuli mediated via TRPV4 channel suggesting a role for this channel in inflammatory dental pain.

TRP Channels and calcium signalling in dental pulp stem cells

Introduction: Ca2+ ion is an important intracellular messenger essential for the regulation of various cellular functions including proliferation, differentiation and apoptosis. Transient Receptor Potential (TRP) channels are calcium permeable cationic channels that play important role in regulation of free intracellular calcium ([Ca2+]i) in response to thermal, physical and chemical stimuli. Ca2+ signalling in human dental pulp stem cells (hDPSCs) and the ion channels regulating Ca2+ are largely not known. Objectives: Investigate changes in [Ca2+]i and determine the ion channels that regulate calcium signalling in hDPSCs. Methods: DPSCs were derived from immature third molars and cells less than passage 6 were used in all the experiments. Changes in [Ca2+]i were studied with Fura2 calcium imaging. RNA was extracted from DPSCs and a panel of TRP channel gene expression was determined by qPCR employing custom designed FAM TRP specific primers and probes (Roche, UK) and the Light Cycler 480 Probes Master (Roche). Results: hDPSCs express gene transcripts for all TRP families including TRPV1, V2, V4, TRPA1, TRPC3, TRPC5, TRPC6, TRPM3, TRPM7 and TRPP2. Stimulation of cells with appropriate TRP channel agonist induced increase in [Ca2+]i and similar responses were obtained when cell were mechanically stimulated by membrane stretch with application of hypotonic solution. Conclusion: TRP channels mediate calcium signalling in hDPSCs that merit further investigation.

Nerve Growth Factor Sensitisation of TRPA1 on Sensory Neurones

Background: Sensory neurones from the trigeminal nerve innervate the oro-facial region and teeth. Transient receptor potential channels (TRPs) expressed by these neurones are responsible for relaying sensory information such as changes in ambient temperature, mechanical sensations and pain. Study of TRP channel expression and regulation in human sensory neurones therefore merits investigation to improve our understanding of allodynia and hyperalgesia. Objective: The objective of this study was to differentiate human dental pulp stem cells (hDPSCs) towards a neuronal phenotype (peripheral neuronal equivalents; PNEs) and employ this model to study TRP channel sensitisation. Method: hDPSCs were enriched by preferential adhesion to fibronectin, plated on coverslips (thickness 0) coated with poly-l-ornithine and laminin and then differentiated for 7 days in neurobasal A medium with additional supplementation. A whole cell patch clamp technique was used to investigate whether TRP channels on PNE membranes were modulated in the presence of nerve growth factor (NGF). PNEs were treated with NGF for 20 minutes immediately before experimentation and then stimulated for TRPA1 activity using cinnamaldehyde. Peak currents were read at 80 mV and -80 mV and compared to peak currents recorded in untreated PNEs. Data were analysed and plotted using Clampfit9 software (Molecular Devices, Sunnyvale, California, USA). Result: Results showed for the first time that pre-treatment of PNEs by NGF produced significantly larger inward and outward currents demonstrating that TRPA1 channels on PNE membranes were capable of becoming sensitised following treatment with NGF. Conclusion: Sensitisation of TRPA1 by NGF provides evidence of a mechanism for rapid neuronal sensitisation that is independent of TRPA1 gene expression