Influence of ultraviolet-B irradiation on engraftment, graft-versus-host disease and graft-versus-leukemia effect in a rat model for allogeneic bone marrow transplantation

Ultraviolet-B (UVB) irradiation is known to inhibit lymphocyte activity and consequently to reduce the incidence of graft-versus-host disease (GVHD) in experimental models for allogeneic bone marrow transplantation (BMT). GVHD is frequently associated with morbidity and mortality, but also with the beneficial graft-versus-leukemia (GVL) effect, demonstrated by a reduction in the incidence of leukemia relapse. In this study, we investigated whether UVB treatment of allogeneic T cells could prevent GVHD while sparing the beneficial GVL effect following allogeneic BMT in the Brown Norway myelocytic leukemia (BNML) rat model analogous to human acute myelocytic leukemia (AML). The dose of UVB required to abolish lethal GVHD in the rat allogeneic BMT model (WAG/Rij donors into BN recipients) was 4000 J/m2. However, this UVB dose simultaneously abrogated all GVL activity mediated by the T cells in the graft, while the radio-protective capacity of rat BM cells was strongly reduced. The number of allogeneic BM cells required to protect lethally irradiated BN rats was increased 50 to 100-fold. It is concluded that UVB acts as a non-selective form of T cell inactivation, and that UVB pretreatment of an allogeneic marrow graft is unlikely to be useful clinically as a preventive measure for GVHD, since other means of reduction of the number of functional T cells are less damaging to bone marrow stem cells.

Ultraviolet irradiation for the prevention of graft-versus-host disease and graft rejection in bone marrow transplantation

This review describes an approach to the prevention of graft-versus-host disease (GVHD) and graft rejection following allogeneic BMT that differs from conventional methods. Ultraviolet (UV) irradiation inhibits the proliferative responses of lymphoid cells to mitogens and alloantigens by inactivation of T lymphocytes and dendritic cells, and in animal models this can prevent both GVHD and graft rejection. It is important that the marrow repopulating capacity of haemopoietic stem cells is not damaged by the irradiation process. We have found that polymorphic microsatellite markers are a sensitive way of assessing the impact of UV irradiation on chimerism after BMT in rodents.

In-Situ, Simultaneous Irradiation and Monitoring of a Photocatalysed Organic Oxidation Reaction in a TiO2-coated NMR Tube

The semiconductor photocatalysed (SPC) oxidation of toluene is performed inside an NMR spectrometer and the reaction monitored simultaneously in-situ, using a fibre optic probe/diffuser to provide the UV light to activate the titania photocatalyst coating on the inside of the NMR tube. Such a system has great potential for the simple rapid screening of a wide range of SPC mediated organic reactions.

The dual EGFR/HER2 inhibitor lapatinib synergistically enhances the antitumor activity of the histone deacetylase inhibitor panobinostat in colorectal cancer models

As key molecules that drive progression and chemoresistance in gastrointestinal cancers, epidermal growth factor receptor (EGFR) and HER2 have become efficacious drug targets in this setting. Lapatinib is an EGFR/HER2 kinase inhibitor suppressing signaling through the RAS/RAF/MEK (MAP/ERK kinase)/MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase)/AKT pathways. Histone deacetylase inhibitors (HDACi) are a novel class of agents that induce cell cycle arrest and apoptosis following the acetylation of histone and nonhistone proteins modulating gene expression and disrupting HSP90 function inducing the degradation of EGFR-pathway client proteins. This study sought to evaluate the therapeutic potential of combining lapatinib with the HDACi panobinostat in colorectal cancer (CRC) cell lines with varying EGFR/HER2 expression and KRAS/BRAF/PIK3CA mutations. Lapatinib and panobinostat exerted concentration-dependent antiproliferative effects in vitro (panobinostat range 7.2-30 nmol/L; lapatinib range 7.6-25.8 μmol/L). Combined lapatinib and panobinostat treatment interacted synergistically to inhibit the proliferation and colony formation in all CRC cell lines tested. Combination treatment resulted in rapid induction of apoptosis that coincided with increased DNA double-strand breaks, caspase-8 activation, and PARP cleavage. This was paralleled by decreased signaling through both the PI3K and MAPK pathways and increased downregulation of transcriptional targets including NF-κB1, IRAK1, and CCND1. Panobinostat treatment induced downregulation of EGFR, HER2, and HER3 mRNA and protein through transcriptional and posttranslational mechanisms. In the LoVo KRAS mutant CRC xenograft model, the combination showed greater antitumor activity than either agent alone, with no apparent increase in toxicity. Our results offer preclinical rationale warranting further clinical investigation combining HDACi with EGFR and HER2-targeted therapies for CRC treatment.

The dual EGFR/HER-2 tyrosine kinase inhibitor lapatinib sensitizes colon and gastric cancer cells to the irinotecan active metabolite SN-38

Members of the human epidermal receptor (HER) family are frequently associated with aggressive disease and poor prognosis in multiple malignancies. Lapatinib is a dual tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) and HER-2. This study evaluated the therapeutic potential of lapatinib, alone and in combination with SN-38, the active metabolite of irinotecan (CPT-11), in colon and gastric cancer cell lines. Concentration-dependent antiproliferative effects of both lapatinib and SN-38 were observed in all colon and gastric cancer cell lines tested but varied significantly between individual cell lines (lapatinib range 0.08-11.7 muM; SN-38 range 3.6-256 nM). Lapatinib potently inhibited the growth of a HER-2 overexpressing gastric cancer cell line and demonstrated moderate activity in gastric and colon cancer cells with detectable HER-2 expression. The combination of lapatinib and SN-38 interacted synergistically to inhibit cell proliferation in all colon and gastric cancer cell lines tested. Cotreatment with lapatinib and SN-38 also resulted in enhanced cell cycle arrest and the induction of apoptosis with subsequent cellular pharmacokinetic analysis demonstrating that lapatinib promoted the increased intracellular accumulation and retention of SN-38 when compared to SN-38 treatment alone. Finally, the combination of lapatinib and CPT-11 demonstrated synergistic antitumor efficacy in the LoVo colon cancer mouse xenograft model with no apparent increase in toxicity compared to CPT-11 monotherapy. These results provide compelling preclinical rationale indicating lapatinib to be a potentially efficacious chemotherapeutic combination partner for irinotecan in the treatment of gastrointestinal carcinomas.

The application of a novel fluidised photo reactor under UV-Visible and natural solar irradiation in the photocatalytic generation of hydrogen

With advancements in the development of visible light responsive catalysts for H2 production frequently being reported, photocatalytic water splitting has become an attractive method as a potential ‘solar fuel generator’. The development of novel photo reactors which can enhance the potential of such catalyst, however, is rarely reported. This is particularly important as many reactor configurations are mass transport limited, which in term limits the efficiency of more effective photocatalysts in larger scale applications. This paper describes the performance of a novel fluidised photo reactor for the production of H2 over two catalysts under UV-Visible light and natural solar illumination. Catalysts Pt-C3N4 and NaTaO3.La were dispersed in the reactor and the rate of H2 was determined by GC-TCD analysis of the gas headspace. The unit was an annular reactor constructed from stainless steel 316 and quartz glass with a propeller located in the base to control fluidisation of powder catalysts. Reactor properties such as propeller rotational speed were found to enhance the photo activity of the system through the elimination of mass transport limitations and increasing light penetration. The optimum conditions for H2 evolution were found to be a propeller rotational speed of 1035 rpm and 144 W of UV-Visible irradiation, which produced a rate of 89 µmol h-1 g-1 over Pt-C3N4. Solar irradiation was provided by the George Ellery Hale Solar Telescope, located at the California Institute of Technology.

Assessment of DNA double-strand breaks induced by intravascular iodinated contrast media following in vitro irradiation and in vivo, during paediatric cardiac catheterization

Paediatric cardiac catheterizations may result in the administration of substantial amounts of iodinated contrast media and ionizing radiation. The aim of this work was to investigate the effect of iodinated contrast media in combination with in vitro and in vivo X-ray radiation on lymphocyte DNA. Six concentrations of iodine (15, 17.5, 30, 35, 45, and 52.5 mg of iodine per mL blood) represented volumes of iodinated contrast media used in the clinical setting. Blood obtained from healthy volunteers was mixed with iodinated contrast media and exposed to radiation doses commonly used in paediatric cardiac catheterizations (0 mGy, 70 mGy, 140 mGy, 250 mGy and 450 mGy). Control samples contained no iodine. For in vivo experimentation, pre and post blood samples were collected from children undergoing cardiac catheterization, receiving iodine concentrations of up to 51 mg of iodine per mL blood and radiation doses of up to 400 mGy. Fluorescence microscopy was performed to assess γH2AX-foci induction, which corresponded to the number of DNA double-strand breaks. The presence of iodine in vitro resulted in significant increases of DNA double-strand breaks beyond that induced by radiation for ≥17.5 mg/mL iodine to blood. The in vivo effects of contrast media on children undergoing cardiac catheterization resulted in a 19% increase in DNA double-strand breaks in children receiving an average concentration of 19 mg/mL iodine to blood. A larger investigation is required to provide further information of the potential benefit of lowering the amount of iodinated contrast media received during X-ray radiation investigations. 

Generation of overdense and high-energy electron-positron-pair plasmas by irradiation of a thin foil with two ultraintense lasers

A scheme for enhanced quantum electrodynamics (QED) production of electron-positron-pair plasmas is proposed that uses two ultraintense lasers irradiating a thin solid foil from opposite sides. In the scheme, under a proper matching condition, in addition to the skin-depth emission of gamma-ray photons and Breit-Wheeler creation of pairs on each side of the foil, a large number of high-energy electrons and photons from one side can propagate through it and interact with the laser on the other side, leading to much enhanced gamma-ray emission and pair production. More importantly, the created pairs can be collected later and confined to the center by opposite laser radiation pressures when the foil becomes transparent, resulting in the formation of unprecedentedly overdense and high-energy pair plasmas. Two-dimensional QED particle-in-cell simulations show that electron-positron-pair plasmas with overcritical density 10(22) cm(-3) and a high energy of 100s of MeV are obtained with 10 PW lasers at intensities 10(23) W/cm(2), which are of key significance for laboratory astrophysics studies.

Dynamics and structure of self-generated magnetics fields on solids following high contrast, high intensity laser irradiation

The dynamics of self-generated magnetic B-fields produced following the interaction of a high contrast, high intensity (I > 10¹⁹W cm^-2) laser beam with thin (3 μm thick) solid (Al or Au) targets is investigated experimentally and numerically. Two main sources drive the growth of B-fields on the target surfaces. B-fields are first driven by laser-generated hot electron currents that relax over ∼10-20 ps. Over longer timescales, the hydrodynamic expansion of the bulk of the target into vacuum also generates B-field induced by non-collinear gradients of density and temperature. The laser irradiation of the target front side strongly localizes the energy deposition at the target front, in contrast to the target rear side, which is heated by fast electrons over a much larger area. This induces an asymmetry in the hydrodynamic expansion between the front and rear target surfaces, and consequently the associated B-fields are found strongly asymmetric. The sole long-lasting (>30 ps) B-fields are the ones growing on the target front surface, where they remain of extremely high strength (∼8-10 MG). These B-fields have been recently put by us in practical use for focusing laser-accelerated protons [B. Albertazzi et al., Rev. Sci. Instrum. 86, 043502 (2015)]; here we analyze in detail their dynamics and structure.

Development of a potent and selective cell penetrant Legumain inhibitor

This Letter describes the continued SAR exploration of small molecule Legumain inhibitors with the aim of developing a potent and selective in vitro tool compound. Work continued in this Letter explores the use of alternative P2-P3 linker units and the P3 group SAR which led to the identification of 10t, a potent, selective and cellularly active Legumain inhibitor. We also demonstrate that 10t has activity in both cancer cell viability and colony formation assays.

A novel inhibitor of channel activating proteases: Putting the CAP on ENaC

Background: Excessive activation of epithelial sodium channels (ENaC) contributes to CF lung pathophysiology due to the resultant dehydration of the airway surface liquid (ASL) and impaired mucociliary clearance. Regulated proteolysis of the endogenous α and γ subunits of ENaC by apical membrane-bound Channel Activating Proteases (CAPs) is a fundamental regulatory mechanism for channel activity. In the CF lung a stark imbalance between the levels of CAPs and their natural inhibitors drives the activation of normally inactive ENaC. On this basis inhibition of CAPs-ENaC signalling represents a potential therapeutic intervention. To this end we have developed a novel cell impermeable active-site directed compound (QUB-TL1) designed to inactivate key trypsin-like CAPs highly relevant in this regard. Objectives & Methods: Utilize differentiated non-CF and CF human airway epithelial cells to assess the impact of QUB-TL1 on a range of parameters including surface CAP activities, ENaC subunit processing/channel activity, ASL height and mucociliary clearance. Results: Treatment of airway epithelial cells with QUB-TL1 results in the significant downregulation of key endogenous CAP activities found to be excessively active at the surface of CF cultures. QUB-TL1-mediated CAP inhibition subsequently causes the internalisation of a pool of processed (active) ENaCγ prominent at the apical surface of CF cultures which correlates with a decline in channel activity. This downregulation of ENaC activity results in an increase in ASL height and improved mucociliary clearance in CF cells. We further find QUB-TL1 uniquely inhibits the ENaC activating enzyme furin, which is in contrast to the alternate trypsin-like CAP inhibitors camostat mesylate and aprotinin. QUB-TL1-mediated furin inhibition correlates with a reduction in neutrophil elastase-induced ENaC activation. Moreover we find QUB-TL1 treatment protects CF cultures from Pseudomonas aeruginosa exotoxin A-induced cytotoxicity. Pseudomonas aeruginosa exotoxin A is a major toxic product activated by furin and positively associated with mortality. Conclusion: The novel inhibitor (QUB-TL1) dampens CAPs-ENaC signalling which improves hydration status mucociliary clearance in CF airway epithelial cell cultures. Moreover this compound provides additional benefit by preventing Pseudomonas aeruginosa exotoxin A-induced cytotoxicity.

MErCuRIC1: A Phase I study of MEK1/2 inhibitor PD-0325901 with cMET inhibitor crizotinib in RASMT and RASWT (with aberrant c-MET) metastatic colorectal cancer (mCRC) patients.

Background: RAS is mutated (RASMT) in ~55% of mCRC, and phase III studies have shown that patients harbouring RAS mutations do not benefit from anti-EGFR MoAbs. In addition, ~50% of RAS Wild Type (RASWT) will not benefit from the addition of an EGFR MoAb to standard chemotherapy. Hence, novel treatment strategies are urgently needed for RASMT and > 50% of RASWT mCRC patients. c-MET is overexpressed in ~50-60%, amplified in ~2-3% and mutated in ~3-5% of mCRC. Recent preclinical studies have shown that c-MET is an important mediator of resistance to MEK inhibitors (i) in RASMT mCRC, and that combined MEKi/METi resulted in synergistic reduction in tumour growth in RASMT xenograft models (1). A number of recent studies have highlighted the role of c-MET in mediating primary/secondary resistance to anti-EGFR MoAbs in mCRC, suggesting that patient with RASWT tumours with aberrant c-MET (RASWT/c-MET+) may benefit from anti-c-MET targeted therapies (2). These preclinical data supported the further clinical evaluation of combined MEKi/METi treatment in RASMT and RASWT CRC patients with aberrant c-MET signalling (overexpression, amplification or mutation; RASWT/c-MET+). Methods: MErCuRIC1 is a phase I combination study of METi crizotinib with MEKi PD-0325901. The dose escalation phase, utilizing a rolling six design, recruits 12-24 patients with advanced solid tumours and aims to assess safety/toxicity of combination, recommended phase II (RPII) dose, pharmacokinetics (PK) and pharmacodynamics (PD) (pERK1/2 in PBMC and tumour; soluble c-MET). In the dose expansion phase an additional 30-42 RASMT and RASWT/c-MET mCRC patients with biopsiable disease will be treated at the RPII dose to further evaluate safety, PK, PD and treatment response. In the dose expansion phase additional biopsy and blood samples will be obtained to define mechanisms of response/resistance to crizotinib/PD-0325901 therapy. Enrolment into the dose escalation phase began in December 2014 with cohort 1 still ongoing. EudraCT registry number: 2014-000463-40. (1) Van Schaeybroeck S et al. Cell Reports 2014;7(6):1940-55; (2) Bardelli A et al. Cancer Discov 2013;3(6):658-73. Clinical trial information: 2014-000463-40.

Design, synthesis and validation of an inhibitor of CGRP degradation

Introduction: In addition to their afferent role in detection and signalling noxious stimuli, neuropeptide-containing sensory nerves may initiate and maintain chronic inflammation in diseases such as periodontitis by an efferent process known as neurogenic inflammation. Neuropeptides are susceptible to cleavage by peptidases, and therefore, the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies in our laboratory showed that enzyme components of gingival crevicular fluid (GCF) from periodontitis sites selectively inactivated the neuropeptide calcitonin gene-related peptide (CGRP), known to have a role in inhibiting osteoclastic bone resorption. Objectives: The aim of this study was to design and synthesise a specific inhibitor to prevent the degradation of CGRP by components of GCF. Methods: A hydroxamate-based inhibitor with a biotinylated tag was designed to ensure selectivity for CGRP and ease of use for future purification strategies. The biotinylated peptide hydroxamate contained the P1-P4 amino acid sequence of the potential CGRP cleavage site and was synthesised by solid-phase methods using standard Fmoc chemistry. Inhibition of CGRP metabolism by GCF was determined by MALDI-mass spectrometry (MALDI-MS) using pooled GCF samples from periodontitis patients as a crude source of the CGRP-degrading enzyme. Results: MALDI-MS analysis of CGRP degradation showed almost complete inhibition in the presence of the biotinylated inhibitor. Our results showed that the rate-limiting step in the cleavage of CGRP is endopeptidase cleavage, followed by carboxypeptidase attack. Conclusion: This study demonstrates that the enzyme component of GCF responsible for the degradation of CGRP can be inhibited by a biotinylated hydroxamate modelled on a potential endopeptidase cleavage site. The biotin tag on the inhibitor will facilitate our future purification of the CGRP-cleavage enzyme using a streptavidin-agarose column.