Solid tumours can undergo cycles of hypoxia, followed by reoxygenation, which can have significant implications for the success of anticancer therapies. A need therefore exists to develop methods to aid its detection and to further characterise its biological basis. We present here a novel method for decomposing systemic and tumour-specific contributions to fluctuations in tumour deoxyhaemoglobin concentration, based on magnetic resonance imaging measurements.Fluctuations in deoxyhaemoglobin concentration in two tumour xenograft models of colorectal carcinoma were decomposed into distinct contributions using independent component analysis. These components were then correlated with systemic pulse oximetry measurements to assess the influence of systemic variations in blood oxygenation in tumours, compared with those that arise within the tumour itself (tumour-specific). Immunohistochemical staining was used to assess the physiological basis of each source of fluctuation.Systemic fluctuations in blood oxygenation were found to contribute to cycling hypoxia in tumours, but tumour-specific fluctuations were also evident. Moreover, the size of the tumours was found to influence the degree of systemic, but not tumour-specific, oscillations. The degree of vessel maturation was related to the amplitude of tumour-specific, but not systemic, oscillations.Our results provide further insights into the complexity of spontaneous fluctuations in tumour oxygenation and its relationship with tumour pathophysiology. These observations could be used to develop improved drug delivery strategies. The structure of tumour vasculature is typically disorganised, with blood vessels that are tortuous, dilated, elongated and leaky, with saccular and dead-end formations , intravaTumours can also exhibit regions with temporally variable oxygenation. This can be caused by phenomena such as arteriolar vasoconstrictive motion . Consequently, blood oxygen fluctuations that are characteristic of cycling hypoxia can be estimated with measurements of temporal changes in R2* (Gradient-echo magnetic resonance imaging (GRE-MRI) sequences can be used to estimate blood oxygen variations non-invasively by mapping the blood oxygen level-dependent (BOLD) effect . BOLD ims in R2* .These fluctuations are complex and heterogeneously distributed throughout each tumour, and techniques to further evaluate and characterise them would allow the fluctuations to be better understood. To this end, we have adopted a technique that is now widely used in BOLD studies of the brain, named independent component analysis (ICA), which allows a signal to be decomposed into its constituent components. In the brain, ICA has been used to identify spatially coherent fluctuations in blood oxygenation that are associated with regions that are functionally connected  were injected subcutaneously into the flank of immunocompromised CD1 female nude mice . These two cell lines develop into xenografts with markedly different phenotypes: SW1222 tumours are well-differentiated with clearly defined glandular structures and an extensive and well-perfused vascular network, whereas LS174T tumours show moderate-to-poor differentiation and are comparatively less-well vascularised and perfused, with larger hypoxic and necrotic regions  or SW1222 (n=5) xenografts were anaesthetised using isoflurane in air and secured in a cradle using dental paste to minimise motion .During the acquisition of GEMS data, pulse oximetry recordings were obtained with a sensor clamped to the thigh of the animal , which allowed the direct measurement of systemic arterial oxyhaemoglobin saturation . This pre-processing procedure was performed for two reasons: (i) it allowed the subsequent ICA decomposition of the data to be simplified, because the great majority of the variance is concentrated on a reduced number of eigenvectors, meaning all the data can be explained in fewer components, and (ii) by doing so, we expect to filter out some of the background noise, which also facilitates the ICA decomposition . Each independent component has a (previously unknown) unique time-course and an associated spatial map, and can be studied individually. The decomposition process was modelled as:Spatial ICA was then applied to this dimensionally reduced data . A significant correlation (P<0.01) suggested that the current independent component represented systemic blood oxygen dynamics. Conversely, if the correlation was less significant (P>0.01), the time-course of the independent component, its frequency spectrum and its spatial map were studied in order to assess if it was a component modelling tumour-specific dynamics. Two selection criteria were adopted: (i) if the frequency spectrum presented a peak at a defined frequency, this was enough evidence of cycling behaviour, or (ii) if the independent component spatial maps, which were normalised and thresholded by z-value  temporal features, in order to prevent noisy independent components from being considered  were selected to represent systemically oscillating tumour regions. A similar procedure was employed to detect the most tumour-specific fluctuating areas, where only non-significant correlations (P>0.05) between the systemic O2sat and the dynamics of where Time-courses of thresholded μm-thick frozen sections of tumour tissue. Following MRI, care was taken to preserve the orientation of the tumour upon excision and during histological preparation, so that tumour sections were cut in the same plane as the MRI acquisition. Histology could not be performed on one LS174T tumour due to sample degradation following excision. We obtained distributions of vascular perfusion, vascular endothelial cells, pericytes and hypoxic regions in all tumours. Perfusion staining was achieved via intravenous injection (12.5 mg kg−1) of the blue fluorescent marker Hoechst 33342  1 min before killing the animal. Endothelial cells were stained with a rat monoclonal antibody against CD31 , revealed with Alexa Fluor-488 . Pericytes were detected by α-smooth muscle actin (α-SMA) staining with a mouse monoclonal anti-α-SMA , revealed with Alexa Fluor-546 (Life Technologies). Hypoxic regions were detected via intraperitoneal injection (60 mg kg−1) of the hypoxia marker pimonidazole  30 min before killing the animal, followed by ex vivo staining using a rabbit polyclonal anti-pimonidazole (Hypoxyprobe), revealed with Alexa Fluor-488 (Life Technologies). In addition, haematoxylin and eosin (H&E) staining was performed in immediately adjacent frozen sections to assess tumour morphology.To investigate the pathophysiology underpinning fluctuations in BOLD-ICA measurements, we performed multifluorescence labelling of 10 Fluorescence images were taken on an Axio Imager microscope , using an AxioCam digital colour camera. Images of H&E were collected on a bright-field Zeiss Axioskop2 microscope (Carl Zeiss). Both microscopes were fitted with a motorised stage and used AxioVision software (Carl Zeiss).α-SMA) was calculated to assess vascular maturation.Quantification of immunofluorescence was performed in Matlab software . Regions of interest (ROIs) were drawn around the total area of the tumour and images were thresholded to exclude background fluorescence signal. Tumour coverage of each of the four fluorescent markers was calculated as the ratio between fluorescent pixels and the total number of pixels within the ROI. In addition, the percentage of capillaries (CD31) covered with pericytes , which are thought to be caused by changes in deoxyhaemoglobin concentration, were observed in both LS174T and SW1222 tumour types. Example time-courses are shown in R2* time-courses was in the range inferior to 0.001 Hz  for both tumour types. R2* values between SW1222 and LS174T tumours were 0.12±0.02 ms−1 and 0.08±0.02 ms−1 (mean±s.d.), respectively, which were not significantly different . However, as we are interested in temporal fluctuations, the relative variations in R2* assume greater importance than the actual R2* values. Moreover, it was shown that the absolute value of R2* (or T2*) does not reliably predict pO2in vivo, although a good temporal correlation between these two quantities was observed , suggestive of spatial coherence in R2* fluctuation patterns (Decomposition of patterns . This ef2sat), whether such tumour spontaneous fluctuations included contributions from systemic variations in blood oxygenation.Alongside BOLD MRI data, pulse oximetry measurements were also acquired. An important aim of this study was to investigate, using ICA and pulse oximetry , which we considered to be specific to the tumour microenvironment (n=5) and LS174T (n=5) tumours.R2* fluctuations in each tumour line. The majority of SW1222 and LS174T tumours (4 out of 5 in each group) showed a greater occurrence of tumour-specific fluctuations than systemic fluctuations, although the difference was not statistically significant . A similar result was observed in the s.d. of R2* fluctuations .tuations , in whicAll LS174T tumours exhibited large systemic fluctuations in the tumour periphery , whereas−1 , which was better perfused (Hoechst 33342) and less hypoxic (pimonidazole) than in LS174T tumours . These oR2* fluctuations due to systemic influences was significantly correlated with tumour volume  in 4 SW1222 (n=5) and 3 LS174T (n=4) tumours  in individual tumours, whilst attempting to remove the influence of background noise inherent in the MRI acquisition process. This allowed the identification of the areas mostly affected by either mechanism in individual tumours. Low-frequency fluctuations in oxygen tension and blood flow were previously observed in both tumour and muscle by T2*-weighted gradient-echo fluctuations between tumour and contralateral muscle. They additionally looked at whether muscle and tumour time series were correlated, and concluded systemic changes did not affect the tumour microenvironment because no significant results were obtained. The technique presented here is, however, more refined, as it isolates high-variance data and separates the multitude of processes contributing to R2* fluctuations before any time series correlation. Moreover, the ICA-O2sat model provides the unique advantage of being able to identify tumour regions that are simultaneously suffering from systemic and tumour-specific effects, which would be otherwise impossible, that is, if direct correlations were used without prior ICA decomposition.In this study we have presented a novel technique, based on ICA, for characterising individual contributions to cycling hypoxia in tumours. Specifically, our approach was designed to separate systemic from tumour-specific sources of R2* fluctuations in these tumours might not correspond to cycling hypoxia, but rather to oscillations in the blood oxygenation that remains within normal oxygenation levels. In this case, ‘cycling oxygenation' might be a more accurate term than ‘cycling hypoxia'.We observed that systemically driven, high-amplitude fluctuations were preferentially located in the tumour periphery in LS174T tumours, whereas in SW1222 tumours there was no preferential location. This observation is potentially due to the less extensive vascular network evident in LS174T tumours, compared with SW1222 tumours. Fewer blood vessels can lead to impaired perfusion which, in combination with the higher microvascular density in the LS174T tumour periphery, potentially reduces the influence of systemic variations in the central area of the tumour. A similar observation was reported in an MRI study of A-07 melanoma xenografts, where blood flow fluctuations occurred preferentially in the tumour periphery, in which case the authors considered vasomotor activity in the supplying vasculature as a probable cause of fluctuations . These time scales are in the same range of the cycling hypoxia periods first reported by Chaplin and colleagues in a number of different preclinical tumour models , might not be fast enough to prevent signal interference from high-frequency oscillations. Indeed, The dominant oscillatory frequencies of RR2* changes can also reflect changes in blood volume, blood pH, haematocrit or in the fraction of oxygen extraction by the tissue (2sat model, as implemented here, did not consider any possible lag between the O2sat variations and tumour R2* fluctuations. However, the circulation time in mice is of the order of the temporal resolution of the GRE acquisition used in this study, so such effects can be assumed to be negligible. Finally, systemic influences in the tumour microenvironment can come from different sources, such as pulsatile flow and blood pressure, in addition to blood oxygenation, which might render the ICA-O2sat model an over-simplistic approach.Besides the potential issues due to the relatively low-sampling rate of acquisition, the approach used in this study suffers from additional limitations. Estimating blood oxygenation variations with gradient-echo MRI can have confounding effects, as 2sat model is a non-invasive and novel approach to directly identify systemic influences on tumour cycling hypoxia patterns. We consider it has the potential to be easily translatable to the clinic, where tumour cycling hypoxia is still poorly characterised, with only a few published studies to date (2sat or its future versions would help predict the temporal and spatial delivery of systemically applied therapies, such as chemotherapy or radioimmunotherapy, in individual patients, and consequently help tailor a more effective delivery regime. In addition, measurements of fluctuations in oxygenation could potentially provide an insight into tumour pathophysiology in a clinical context, given the relationships found with vascular characteristics in this study.Nevertheless, the ICA-O


Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood.We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate , confirmed by mass-spectrometry of ACEs tryptic digests.Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs. ACE is constitutively expressed on the surface of endothelial cells, epithelial and neuroepithelial cells and cells of the immune system  is a Zn43 is a ZIn addition to membrane-bound ACE, blood, seminal fluid and other biological fluids contain a variable amount of soluble ACE. Blood ACE likely originates from the vascular endothelium , mostly Human seminal fluid ACE likely originates from glandular epithelial cells of epididymis and prostate, that express significant amount of somatic ACE –14. HumaThere are at least two possible reasons of increased shedding: 1) Increased expression of ACE secretase in glandular epithelial cells of epididymis and prostate (but unfortunately the nature of ACE secretase is still unknown); 2) Different conformations of ACE on the surface of endothelial and epithelial cells, which, in turn, may lead to either different exposure of stalk region, where ACE secretase cleaves ACE from cell surface, or different regulation of ACE shedding from different cells by the presence of putative ACE-binding proteins/ACE effectors.in vitro and in vivo. We demonstrated recently that the pattern of mAbs binding to the conformational epitopes on the surface of ACE, “conformational fingerprint of ACE”, is an extremely sensitive marker of the local conformational changes in ACE molecule due to mutations, denaturation, inhibitor binding, presence of membrane anchor, etc. We proved the concept that the conformation of ACE is cell-and tissue-specific and stems likely from different post-translational modifications, including different glycosylation, on the example of ACE from endothelial cells and ACE from macrophages and dendritic cell of sarcoid granuloma. The ACE fingerprint, therefore, has a potential for the disclosure of the cells from which ACE originates [Species specificity of ACE is apparent; however, more subtle tissue specificity of the enzyme can influence ACE functions both iginates .We applied this approach here to detect structural  differences of ACEs originated from endothelial cells (lung ACE) and epithelial cells . We believe that structural differences in ACEs from different organs, which we found, could be a base for the development of antibodies, which will distinguish ACEs shed to the blood circulation from different organs by their origin.The work was carried out in accordance with The Code of Ethics of World Medical Association (Declaration of Helsinki) and was approved by the Institutional Review Boards of the Bakulev Center of Cardiovascular Surgery, Moscow University and the University of Illinois at Chicago. Written informed consent from the donor or the next of kin was obtained for the use of this sample in research. Human citrated plasma, seminal fluid, lung, prostate and epididymis tissue homogenates were used as sources of somatic two-domain ACE. Lung and seminal fluid ACEs were purified using lisinopril affinity chromatography exactly as in . Estimato-phtaldialdehyde. Inhibition of ACE activity by ACE inhibitors was performed by incubation of ACE (2 nM) with up to 0.1 uM enalaprilat and up to 1 uM teprotide, at pH 7.5 for one hour and then measurement of residual ACE activity with 0.2 mM ZPHL as a substrate at the same pH value. Inhibition of ACE activity with anti-catalytic anti-ACE mAbs was performed by 10 ug/ul mAbs as described in [ACE activity in blood plasma, seminal fluid, homogenates of human organs was measured using a fluorimetric assay with two ACE substrates, 2 mM Z-Phe-His-Leu (ZPHL) and 5 mM Hip-His-Leu (HHL), at pH 8.3 . Brieflyribed in .Ninety six-well plates  were coated with anti-ACE mAbs via goat anti-mouse IgG  bridge  and incuImmunochemical characterization of ACE in the presence of different effectors was performed as described above, ACE solution being mixed with a solution of effectors just before application on the plate wells. The effectors used were heat-inactivated citrated plasma, heat-inactivated seminal fluid, 3 kDa filtrate of human citrated plasma, albumin, and bilirubin. Heat-inactivated plasma and seminal fluid were prepared by heating at 65°C for 45 min and checked for the absence of ACE activity; 3 kDa filtrate was prepared by filtration of human citrated plasma on Vivaspin 500 MWCO 3000 concentrator  at 4°C and 12,000 g for 2 h.5-106, as measured by ELISA, mice were boosted and isolated splenocytes were fused to mouse myeloma cells 653-Ag8. The resulting populations grown in 96-well plates were subjected to primary screening with two pure ACEs, seminal fluid ACE and lung ACE, in plate precipitation assay in simultaneous experiments.Mice were immunized five times with pure seminal fluid ACE at 10ug per i.p. injection. The sera were tested against this immunogen in ELISA and against two ACEs, pure seminal fluid ACE and pure lung ACE, in plate precipitation assay. Upon reaching antibody titers 5x10Sialylated biantennary complex oligosaccharide was modeled with the program Insight II  and attached to the each potential site of glycosylation on the surface of the C domain (PDB 108A) of ACE. Molecular dynamics during 500 psec was applied for the estimation of the movement of the glycan and the neighboring amino acid residues within 15 Å from definite Asn residue.2, at 25°C. The process was controlled by ion-exchanged chromatography of 1 uM solutions of native and desialylated ACEs on Agilent1100 chromatograph with electrochemical cell. Chromatograms of native and desialylated ACEs were treated by the program Agilent Chemstation A.09.01 . For the calibration curve, solutions of neuraminic acids of 1, 10, and 20 uM were used.Desialylation of ACE was performed by neuraminidase from Vibrio Cholerae  in 0.1 M MES buffer, pH 6.0, containing 0.15 M KCl and 0.4 mM CaClwww.matrixscience.com) with the use of SWISS-PROT database, and then using the ExPASy online tools, FindPept (www.expasy.org/findpept) and GlycoMod (web.expasy.org/glycomod). The FindPept tool was used to find additional matching to mass spectrometry data, peptides, such as C-terminal (unspecific) tryptic peptides which could not be discriminated by the MASCOT program. Non-matched peaks from the mass spectra were further analyzed with the GlycoMod tool in order to find possible glycopeptides and N-linked glycans in the ACE. The possibility of cysteine modification with acrylamide and methionine oxidation, which are typical variable modifications in mass spectrometry of proteins, was included in the analysis as well as the possibility for one or two missed cleavages with trypsin. Since a search with the GlycoMod tool is based on a comparison of experimental masses of glycopeptides with theoretical ones, its results sometimes contain false positive hits (displaying rather exotic glycan structures). So, finally only those N-linked glycan structures were taken into consideration and further manual analysis that matched the oligosaccharide compositions contained in the UniCarbKB database.The lung ACE and seminal fluid ACE were subjected to electrophoresis in denaturing conditions. For each ACE sample, two protein bands on an electrophoretic gel were obtained, which were further subjected to mass spectrometry peptide fingerprinting as follows. A piece of ACE band from electrophoretic gel was transferred into a test tube, washed from the dye, and then the protein was digested by trypsin as is described elsewhere . PeptideWe characterized the conformation of pure lung and seminal fluid ACE, using a panel of mAbs directed against 17 different epitopes located on the N and C domains of catalytically active human ACE - “conformational fingerprint of ACE”. As appaTissue- and cell-specific post-translational modifications (PTM) are common for various proteins –36. PTMsSomatic ACE represents a characteristic N-glycosylated glycoprotein, exact glycan structures of which, as well as exact locations of actually present oligosaccharide chains on the surface of the protein globule, can vary with different protein sources. The sequence of human somatic ACE contains 17 potential sites for N-glycosylation . The strIt was shown previously that somatic ACE contains mostly complex-type oligosaccharides , 39. ModSeminal fluid ACE and lung ACE possess rather equal masses as was shown by SDS-PAGE (not shown) which indicates on the absence of major differences in the degree of glycosylation of these ACEs. However, some evidence of different modes of glycosylation of lung ACE and seminal fluid ACE was obtained by the analysis of the action of neuraminidase on both enzymes. It was found that while desialylation of seminal fluid ACE did not result in the appearance of any neuraminic acid on the ion-exchange chromatogram, desialylation of lung ACE resulted in the appearance of 5 neuraminic acid residues per molecule of the enzyme (not shown). It is known that human lung ACE is characterized by a lower pI value (5.1) compared to that of kidney ACE (5.2) which was attributed to a bigger number of neuraminic acid residues in lung ACE glycans . On the An analysis of the conformational fingerprint of lung ACE and seminal fluid ACE  allowed As for the dramatic difference in binding of mAbs 1B8 and 3F10 to seminal fluid ACE and lung ACE, it might be explained by the difference in glycosylation of Asn731, which is in the center of the epitopes for these two mAbs on the C domain of ACE  or, alteIn order to analyze the reasons for the differences in mAbs 1B8 and 3F10 binding with lung and seminal fluid ACE further we compared conformational fingerprint of ACE from lung homogenate with that from epididymis and prostate homogenates , which sThe enhanced binding of these two mAbs, as well as mAb 1B3 , to Moreover, a comparison of the conformational fingerprints of epididymal and prostate ACE with their seminal fluid ACE counterpart indicates that seminal fluid ACE could represent a mixture of ACE originated from epididymis and ACE originated from prostate. In support of this suggestion, we discovered by MALDI-TOF analysis of seminal fluid ACE digested with trypsin  a presenwww.proteinatlas.org), which demonstrates that epididymis exhibits at least 6-fold more ACE expression than prostate. However, higher content of ACE in epididymis is likely compensated by higher mass of prostate which leads to pretty equal content of ACE in both organs [Measurement of ACE activity in tissues homogenates prepared in equal conditions (tissue:buffer = 1:9) revealed that prostate exhibited much lower content of ACE (44 mU/g), than epididymis (720 mU/g), which ACE activity was comparable to that in lung—1063 mU/g with ZPHL as a substrate. These values are in accordance with lower ACE protein content in prostate than in epididymis reported earlier –12 and ch organs  and perhh organs . This coACE in prostate homogenate, however, is also characterized by a very low binding of mAb 5F1, while the binding of this mAb to ACE in epididymal homogenate and to pure seminal fluid ACE are rather high. The epitope for this mAb contains potential site of glycosylation, Asn117 . MoreoveAs a whole, the results allow us to assume that seminal fluid ACE originates likely equally from epididymis and prostate glandular epithelial cells.Somatic ACEs isolated from human lung and seminal fluid were digested with trypsin and the resulting peptides were analyzed using MALDI TOF MS. After a primary search of matching tryptic peptides with the MASCOT program, the protein sequence coverage was about 50% in both cases. After finding additional matching peptides using the FindPept tool, the sequence coverage became higher, 61% for the lung ACE and 59% for seminal fluid enzyme. Unglycosylated peptides identified by the FindPept tool in the tryptic digest of the lung ACE are listed in Analysis of the mass spectra data with the GlycoMod tool allowed revealing several peaks matching by mass potential glycopeptides in the lung ACE and seminal fluid ACE. After expert revision, we limited the number of putative glycopeptides by several ones with the most favorable structures reported in UniCarbKB. The results are presented in m/z 2468.1 to the peptides containing Asn480 or Asn666, as well as peptide with m/z 5885.4 to the peptides containing either Asn648 or Asn731. The putative N-glycosylation site Asn648 is not attributed to any known epitope for mAbs to ACE, while the N-glycosylation site Asn731 is a part of the epitopes for mAbs 1B8 and 3F10 to the C domain of ACE  and teprotide (nonapeptide), on enzymatic activity of pure lung and seminal fluid ACEs. Both ACEs were equally inhibited by ACE inhibitors with ICN domain , 27, andN domain  on ACE aN domain . InhibitHow these remarkable differences in local conformation of seminal fluid and lung ACEs may be converted into different regulation of the functions of these ACEs, including the regulation of their shedding? We demonstrated recently that the rate of recombinant human ACE shedding from the surface of CHO-ACE, HEK-ACE and endothelial cells in the presence of bovine and human serum was dramatically diminished, up to 2-times. However, the extent of the serum effect varied depending on the type of serum and cells (unpublished observation).Recently, it was found that albumin may bind to ACE –47. BearEffect of inactivated human plasma on mAbs binding to two types of ACE differed significantly , with seWe found that Low Molecular Weight (LMW) component from human plasma binds to human ACE (unpublished observation). Therefore, we tested an effect of human plasma 3kDa filtrate, containing LMW component (-s), on mAbs binding to both structurally different ACEs and showed that this filtrate had a strong (and different) effect on both types of ACE .Comparison of the effect of 3 kDa human plasma filtrate  with theWe identified recently a bilirubin as one of the components of 3 kDa plasma filtrate which is able to bind to ACE in the region of the overlapping epitopes 1G12/6A12 (unpublished observation). Therefore, we tested an effect of this novel ACE-binding partner, bilirubin, on two different, tissue specific ACE types , seen on The fact that human albumin practically abolished an effect of bilirubin on mAbs binding to both ACEs  shows thThus, significant differences in local conformation of lung ACE  and seminal fluid ACE  allow us to suggest that possible regulation of ACE shedding from epithelial cells of male reproductive tract and from endothelial cells of capillaries (blood vessels) by the constituents of blood and seminal fluid may be different due to different effect of possible regulators of ACE shedding on the structurally different ACEs in these cells.In addition, membrane-bound glycoproteins (including ACE) may often be shed into blood circulation as a by-product of the disease process. Recently an increase in blood ACE level was demonstrated for patients with epithelial ovarian carcinoma . Likely,Significant structural differences in ACE from endothelial cells (major source of blood ACE) and ACE from epithelial cells, particularly prostate ACE (demonstrated in this study) and, likely, ovary ACE, may be the base for the development of specific mAbs, which can be used to identify the tissue- or-cell source of increased ACE in blood in different diseases. In order to estimate the possibility of generating monoclonal antibodies which would discriminate between endothelial and epithelial ACEs we attempted immunization of mice with pure somatic ACE from seminal fluid which is the mixture of prostate and epididymal ACEs –16. We u


XP11.2 renal translocation carcinomas are often encountered in paediatric group of patients where they are believed to be rather indolent. They are rare but more aggressive in young adults. They are slow growing, sometimes without characteristic symptoms and their biologic behaviour is uncertain.We report two cases of this type of tumour in Slovenian young adult males with long and unusual history. Tumours were confirmed imunohistologically by positive reaction for CD10, P504S and TFE3.According to the indications in the literature prognosis of these tumours in young adults depends upon the stage. It seems that cysts, haematomas and necrosis around the kidney are often encountered in these tumours. In advanced stage with lymph nodes involvement or distant metastases, the prognosis is poor. Surgery seems to be basic mode of therapy. Bellini duct carcinoma, medullary carcinoma, Xp11.2 translocation carcinoma, mucinous tubular and spindle cell carcinoma.6Renal cell carcinoma (RCC) represents 2.9% of all carcinomas in Slovenia. The crude incidence was increasing in males from 10.6/100000 and females 6.1/100000 in the period 1993–97 to 20.4/100000 in males and 10.5/100000 in females during the period 2005–2009.et al. in his study concluded that Xp11 translation renal cell carcinoma targeted therapy achieve objective responses and prolonged progression-free survival.12Xp11.2 translocation carcinoma has been recently recognised as a distinct subtype of renal carcinoma. Xp11.2 renal cell carcinomas are defined by at lest six different translocations involving Xp11.2 chromosome, all of which result in a gene fusion involving the TFE3 (transcription factor E3) gene.We present the first two cases of Xp11.2 translocation renal cell carcinomas confirmed in Slovenia in two young males admitted to the urological department in the period of three months. The first one, aged 27, was accepted urgently due to an unbearable pain and a palpable tumour in the abdominal and lumbar region. CT scan revealed a huge solid tumour mass measuring 7.6 × 8.2 × 8.1 cm located in the lower and lateral part of right kidney with metastatic tumours of similar size in the retroperitoneal region, over and under the vena cava, between the aorta and the vena cava extending up to the liver and down to the aortal bifurcation.Four years prior the last hospitalisation, he was admitted to hospital also due to a pain in the lumbar region. At the time CT scan and ultrasound examination revealed a septal haematoma with a thick wall measuring 10 cm in diameter on the anterior side of the right kidney and an angiomyolipoma-like change on the lower pole of the same kidney measuring 3.5 × 2.5 cm. The cause of the haematoma was not clearly identified, bleeding from angiomyolipoma or trauma was suspected. Furthermore, CT scan showed a solid mass near the kidney haematoma that was not further investigated or been even overlooked. Months later, the ultrasound investigation confirmed that haematoma decreased and showed the persistent angiomyolipoma of the same size without any solid mass around the kidney. Because the patient was asymptomatic, he did not attend regular controls until lumbar and abdominal pain re-emerged after four years. Tumour biopsy verified a solid renal tumour. Due to the persistent pain and haematuria, embolization of the kidney and tumour mass was performed. Using a transabdominal surgical approach we managed to remove the kidney with the tumour and the haematoma and well delineated retroperitoneal metastases along vena cava. Tumour burden was removed radically, but eight months later local recurrence and distant lymph nodes were established. The patient refused additional surgical intervention and he preferred treatment at department for oncology. He was given sunitinib as the first line therapy, but no objective response was achieved. Treatment changed to mTOR inhibitor (everolimus), also without any objective response. Despite specific therapy the disease progressed and later only symptomatic treatment was introduced. A year after the surgery the patient died because of massive cancer involvement  and 2.The second patient, aged 31, experienced sudden respiratory distress and was admitted to the pulmonary department where pulmonary embolism was confirmed and treated. The CT scan revealed a huge cystic formation measuring 28 × 21 × 16 cm embracing the left kidney with a solid mass near compressed kidney. The formation distended to the abdominal wall. As the patient thought he has been gaining weight, has been practising slimming diets for over a year. Otherwise the patient felt no pain; his only complaint was shortness of breath. Prior the surgery cava filter was inserted into the lower vena cava. Surgical procedure was done using lumbar approach, a huge cystic cavity was isolated and 3.5 l of cloudy liquid was evacuated. A compressed kidney with adjacent tumour was removed along with the entire tumour mass. Lymph nodes were negative and tumour extension over cystic margins was not detected. Ten months after surgery the patient is still asymptomatic  and 4In the first case the kidney contained a well circumscribed solid, yellowish tumour with a central haemorrhage and necrosis, measuring 6.5 × 5.3 cm and grossly confined by the renal capsule. The uninvolved renal parenchyma was pale with dark brown spots, consistent with postembolization changes. There were five additional tumour nodules, weighing 292 g in total, all well circumscribed, some with adherent fat. On the cut surface, the tumours appeared multilobulated, soft, tan-pink, with tissue organised into papillary structures. Histologically the tumour was composed of were extensive areas of necrosis and haemorrhage surrounded by hemosiderin-laden macrophages. The neoplastic cells were diffusely immunoreactive for RCC antigen and racemase, focally for CD10 and negative for CK7, EMA, HMB45 and Melan A In the second case the tumour was cystic, the solid part of the yellowish-gray tumour with necrotic and haemorrhagic areas measured 9.4 × 7.1 cm. Microscopic examination revealed a clear cell papillary tumour with abundant hyalinised stroma. Tumour cells showed partially clear and partially eosinophilic cytoplasm and enlarged hyperchromatic nuclei. There were numerous psammoma bodies, larger calcifications and some hyaline globules. The neoplastic cells were diffusely immunoreactive for RCC antigen, racemase, CD 10 and vimentin and negative for CK7 and EMA. Angiomyolipoma was not found in the resected specimen In both cases the tumour cells showed diffuse strong nuclear immunopositivity for transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3) confirming the diagnosis of Xp11.2 translocation renal cell carcinoma We report the first two documented cases of Xp11.2 translocation/TFE3 fusion renal cell carcinomas in Slovenia in young adult males with a long and unusual history. This is a rare subtype of RCC that we were probably not sufficiently aware of since it was included in the WHO classification of RCC for the first time in 2004. Its incidence is higher in children and young adults.10Morphologically Xp11.2 translocation RCCs are quite heterogeneous. The most consistent histologic appearance is a carcinoma with mixed papillary and nested/alveolar architecture, composed of cells with clear and/or eosinophilic, granular, voluminous cytoplasm, discrete borders and the presence of extensive psammoma bodies. These features are partially consistent with clear cell RCC and partially with papillary RCC, therefore we believe, that in the past these tumour were misdiagnosed as one of this more common subtypes of RCC. The diagnosis of Xp11.2 translocation RCC is suspected based on clinical information, histology and immunochemical features, however, it is confirmed by the detection of chromosome translocation involving TFE3 gene at Xp11.2 using different methods.The biologic behaviour of Xp11.2 translocation RCC and its response to treatment is still not well defined. As described in literature, Xp11.2 translocation renal cell carcinomas are usually large tumours with focal cystic areas, haemorrhage and necrosis that present in advanced stage.19In our cases late diagnosis was established on one hand because the diagnosis of Xp11 translocation RCC may have been previously underestimated in young adults. Initial symptoms were not clear and diagnostic evaluation and imagines were underestimated too. Cysts, haematomas, necrotic tissue in or around the kidney or even extensive psammomatous calcification evident radiographically can be misinterpreted as some traumatic injury or harmless developmental abnormality in young adults. Therefore, it is necessary to diagnose this tumour entity accurately taking into consideration all available diagnostic tools to clarify unusual pains and problems in young adults.Because of the small number of Xp11.2 translocation renal cell carcinoma described in the literature, the exact biologic behaviour and impact of current treatment modalities remain to be uncertain. Increased awareness among urologists, pathologists, and oncologist is necessary in order to help identifying more cases of this phenotype in the future. Treatment is primarily surgical; in advanced-stage target agents should be a treatment of choice but with doubtful success. When dealing with younger patients we must be aware that haematomas, cysts or necrotic tissue in/around the kidneys could represent an initial stage of translocation renal cell carcinoma. In such cases we must use appropriate imaging studies to exclude reliably malignant tumour or angimyolipoma. Lesion biopsy and the use of antibodies against TFE3 in all RCC, with emphasis on young adults, may be necessary to determine the biologic nature and incidence of this tumour.


Soldiers and recreational backpackers are often required to carry heavy loads during military operations or hiking. Despite the advances in backpack design, the loads carried by soldiers still impose an extreme physiological strain (soft tissue deformation) which frequently results in discomfort, pain, and musculoskeletal injuries. Shoulder strain, which appears to be one of the limiting factors of load carriage, is thought to result from higher susceptibility to short-term injuries such as soft tissue damage and/or trapped nerves and obstruction of blood vessels. The aim of the current study was to develop a flexible model enabling to simulate real life loading scenarios  and that will help in optimising load carriage systems design and guidelines.Open-MRI scans were used for reconstructing a 3D geometrical model of an unloaded shoulder and for measuring the soft tissue deformations caused by a 25 kg backpack; subsequently, a subject-specific finite element (FE) stress-strain analyses was developed. In this model, loads were applied at the strap-shoulder contact surfaces by pulling the strap towards the shoulder until the desired load was reached. Then, the model enables to calculate and analyse the strains in the soft tissues surrounding the brachial plexus.The newly developed model successfully enabled the prediction of soft tissue deformations in the brachial plexus surrounding tissues, which are caused by different backpack loads. Increasing the loads up to 35 kg  resulted in further increase in strains of the underlying soft tissues: the maximal tensile strain in the brachial plexus for a 25 kg backpack was 12% and while carrying 35 kg the maximal tensile strain increased to 16%. It also had been found in the study that the lateral aspects of the brachial plexus are more vulnerable to a deformation-induced injury, because at these aspects the nerve plexus is less protected against the compressive loads applied during load carriage.In addition, the model allows a better understanding of the more susceptible anatomical regions in the backpack-wearer interface. Based on previous animal studies, the soft tissue strains in the area of the brachial plexus as calculated by the model are substantial and might hamper normal nerve conductivity.This method is potentially a strong working tool allowing further developments of new strap structures and materials or backpack design for alleviating the strains applied on the shoulder's soft tissues.


Introduction. Congenital nasopharyngeal teratomas are rare tumours that pose difficulties in diagnosis and surgical management. We report the first use of radiofrequency coblation in the management of such tumours. Case Report. A premature baby with a perinatal diagnosis of a large, obstructing nasooropharyngeal mass was referred to the ENT service for further investigations and management. The initial biopsy was suggestive of a neuroblastoma, but the tumour demonstrated rapid growth despite appropriate chemotherapy. In a novel use of radiofrequency coblation, the nasooropharyngeal mass was completely excised, with the final histopathology revealing a congenital nasopharyngeal teratoma. Conclusion. We report the first use of radiofrequency coblation to excise a congenital nasopharyngeal teratoma and discuss its advantages.  Teratomas are congenital germ cell tumours derived from pluripotent cells from all three germinal layers –3. NasopRadiofrequency coblation technology is a relatively new surgical tool that has been adopted for use in common otorhinolaryngology procedures including adenoidectomy, tonsillectomy, and inferior turbinoplasty . It has This case report describes the novel technique of radiofrequency coblation in the management of a congenital NPT and discusses the difficulties in diagnosis and management of such lesions.A 40-year-old female G3 P0, with a pregnancy complicated by polyhydramnios and antepartum haemorrhage, delivered a male neonate weighing 2085 grams at 33-week gestation via an emergency Caesarean section for failure to progress through labour.On birth, the neonate immediately developed respiratory distress requiring intermittent positive pressure ventilation (IPPV) and APGAR scores of 5 at 1 minute, 7 at 5 minutes, and 8 at 10 minutes. At 15 minutes of life, the neonate was admitted to NICU for continuous positive airway pressure (CPAP) ventilation and was intubated at 1 hour of life for worsening respiratory distress and carbon dioxide retention. The clinical course was consistent with hyaline membrane disease and a single dose of surfactant was given. Trial extubation occurred at day 1 of life.Immediately after extubation, the neonate developed significant apnoea with desaturations. During a traumatic reintubation, a purple mass with overlying haemorrhage on the posterior pharyngeal wall, obscuring the view of the airway, was noted.The ENT team was consulted for further investigation and management of the mass. Physical examination revealed a protruding tongue and a visible mass in the oral cavity. A laryngobronchoesophagoscopy performed on day 4 of life revealed a vascular, exophytic mass arising from the posterior pharyngeal wall, extending from the postnasal space superiorly to the level of the hypopharynx inferiorly.A MRI was performed and demonstrated a well-circumscribed, heterogeneous mass originating from the nasopharynx, measuring 33 × 22 mm in the axial plane and 32 mm craniocaudally Figures  and 2. TLaryngoscopy and core biopsy from the oropharyngeal component of the lesion were performed the following day. The pathology favoured a diagnosis of a differentiated neuroblastoma. As a treatment for neuroblastoma, the neonate received 1 course (3 days) of chemotherapy with carboplatin and etoposide, with prophylactic trimethoprim, sulfamethoxazole, and nystatin. However, chemotherapy-related complications of pancytopenia and febrile neutropenia developed and this was treated with G-CSF and vancomycin, piperacillin with tazobactam, and meropenem.At this point, a clinical examination and MRI revealed an enlargement of the mass to a size of 41 × 21 × 47 mm and a second opinion of the histopathology favoured a diagnosis of teratoma.Once the side effects of the chemotherapy were under control at 4 weeks of life, the baby underwent a perioperative tracheostomy and complete macroscopic excision of the tumour via a combined transoral and transnasal endoscopic radiofrequency coblation. A PROcise EZ View plasma wand was used to debulk the directly visualised tumour from its centre, with the overall effect of tumour collapsing in on itself.The histopathology confirmed the diagnosis of a mostly mature NPT, with small foci immature neuroepithelium, hepatocytes, and myocytes consistent with low grade immature teratoma. No evidence of a yolk sac tumour was identified.Decannulation of the tracheostomy was performed successfully 8 weeks postoperatively. At 18-month follow-up, the patient has no signs of recurrence and no other health concerns.Congenital teratomas are the most common neoplasm in a neonate, occurring in 1 in 4000 live births .The tumour in this case was a teratoma, consisting of cells from all three germ layers in varying degrees of differentiation. It exhibited mature cells, with foci of immature, poorly differentiated neuroepithelium. The latter is the likely reason for the initial biopsy being suggestive of a neuroblastoma, as the biopsy sampled an area of immature neuroepithelium, which was unrepresentative of the entire teratoma.Based on the initial histopathological analysis, the multidisciplinary consensus was to treat the tumour as a neuroblastoma even though the radiological appearance of the tumour was most consistent with the correct diagnosis of a NPT. However, when the tumour progressed despite appropriate chemotherapy, surgical resection was organised.In planning for surgery, radiofrequency coblation was selected as the preferred surgical technique given the vascularity of the lesion and relatively low blood volume of the 2.7 kg baby. Radiofrequency coblation uses bipolar waves to create a plasma field, which disassociates molecular bonds within soft tissues, providing haemostasis with minimal thermal damage and allowing for early wound healing , 8.The tumour was debulked with radiofrequency coblation from the centre of the lesion, creating an overall effect of the tumour collapsing in on itself. This method did not require extensive dissection of tissue planes and minimised intraoperative blood loss.However, there are limitations to the use of radiofrequency coblation in oncological procedures. Accurate histopathological assessment of tumour margins may be difficult as radiofrequency coblation leaves a 2-3 mm ablative margin around the tumour . This waIn summary, we report the first, novel use of radiofrequency coblation in the successful management of a congenital NPT.Nasopharyngeal teratomas are rare tumours that can cause airway obstruction.Diagnosis of nasopharyngeal teratomas may be difficult due to sampling error.Radiofrequency coblation can be used safely in excising nasopharyngeal teratomas to minimise blood loss.Determining microscopic margins is difficult when radiofrequency coblation is used.


In the intensive care unit (ICU) the need for acute renal replacement therapy (aRRT) is associated with high mortality and risk of end-stage renal disease (ESRD). We investigated long term mortality and progression to ESRD in ICU patients requiring aRRT and factors associated to these.Retrospective analysis of all adult patients admitted to the general ICU, Rigshospitalet, from 1/1-2005 to 31/12-2012, identified through the ICU database, excluding chronic dialysis patients. ESRD was defined as the need of dialysis >90 days or kidney transplant.Of 5766 patients included, 931 (16%) received aRRT, 4762 (83%) did not receive any RRT and 73 (1%) only received aRRT during a later ICU admission. The 90-day mortality was 55% for aRRT patients and 22% for those not requiring aRRT (p < 0.001). The median (IQR) age was 61 (49 - 68) for aRRT 90-day survivors and 65 (55 - 73) for aRRT non-survivors (p < 0.001). The median SAPS II score was 53 (43 - 64) for aRRT 90-day survivors and 61 (49 - 74) for aRRT non-survivors (p < 0.001). Characteristics of aRRT patients developing ESRD and those that did not, are displayed in tables The aRRT patients surviving 90 days were younger and less severely ill at ICU admission as compared to the non-survivors. The survivors developing ESRD had more frequently preexisting chronic kidney disease and higher APACHE II scores as compared to those who did not develop ESRD. aRRT patients had increased risk of ESRD up to 7 years after ICU admission, emphasizing the KDIGO recommendations to follow up AKI patients due to their increased risk for CKD.


One of the most widely employed quantitative measurements for assessing tumour response to treatment is the tumour diameter (RECIST criteria), usually determined on cross-sectional CT or MRI. This simple measurement is relatively reproducible and reduction in the maximum tumour diameter by 30% or more following therapy is taken as a sign of effective treatment. However, new targeted therapies may be effective without significantly reducing tumour size and other quantitative response criteria are being evaluated.Some of the response criteria being evaluated combine tumour diameter measurement with other quantitative assessment (e.g. CT density). These have been applied towards the evaluation of gastrointestinal stromal tumours (CHOI criteria) and renal cell carcinoma (modified or revised CHOI criteria) undergoing multi-kinase inhibitor treatment; and hepatocellular carcinoma (modified RECIST criteria) treated with novel therapeutics or chemoembolization. In these clinical settings, the applications of such criteria for tumour response assessment have shown better correlation with clinical outcome compared with conventional RECIST criteria. When immunotherapy is administered, conventional RECIST criteria may erroneously ascribe the appearance of new lesions to disease progression; whereas this phenomenon is given due consideration within the immune-related response criteria (irRC).As quantitative imaging becomes increasingly important in oncology, quantitative indices are being applied and developed in CT, MRI and PET imaging for tumour response assessment. Using perfusion CT technique, the change in CT attenuation value with time resulting from the passage of contrast through tissue can be used to calculate quantitative vascular parameters such as permeability surface area product (PS), blood flow (F) and mean transit time (MTT). Using dual energy CT, it is possible to obtain quantitative iodine contrast distribution in soft tissue.MRI is a powerful multiplex imaging technique because depending on how the scans are performed, it can yield different quantitative information. Two of the most widely employed quantitative techniques for tumour assessment are dynamic contrast enhanced MRI (DCE-MRI) and diffusion-weighted MRI (DW-MRI). The former has been used in early phase trials for the assessment of antivascular/ antiangiogenic therapies. The latter has shown potential as an early response biomarker to a range of effective treatments including chemotherapy, radiotherapy, chemoemobolization treatment and novel therapeutics. Other clinical MR techniques include intrinsic susceptible contrast imaging and magnetic resonance spectroscopy (MRS). However, these have currently limited value for assessing tumour response to treatment.PET imaging using 18FDG tracer enables the semi-quantitative standardised uptake value (SUV) to be derived. PET imaging is increasing utilized for tumour response assessment, and forms the basis of standard criteria for assessment of tumour response in lymphoma. By performing dynamic imaging, it is also possible to calculate the quantitative tracer uptake in tissues. The strength of PET imaging is the range of radiolabelled tracers that are now available clinically to probe a range of tissue properties.In early phase trials, quantitative imaging other than tumour size measurements using CT, MRI or PET can provide mechanistic information about drug action, and also therapeutic effects on specific aspects of tumour biology . By using multimodality imaging, there is also an opportunity to corroborate imaging measurements made using one technique with another. However, quantitative techniques require a process of quality control and quality assurance; as well as knowledge of their measurement reproducibility so that they can be applied with confidence in clinical practice.


A NSF grant was omitted from this article. Please view the complete funding statement below:This work was funded through a postdoctoral fellowship at the Cornell Lab of Ornithology, and a NSF grant IIS-1125098 . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Malignant pleural mesothelioma is a rare but devastating cancer of the pleural lining with no effective treatment. The tumour is often diffusely spread throughout the chest cavity, making surgical resection difficult, while systemic chemotherapy offers limited benefit. Bone marrow-derived mesenchymal stem cells (MSCs) home to and incorporate into tumour stroma, making them good candidates to deliver anticancer therapies. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic molecule that selectively induces apoptosis in cancer cells, leaving healthy cells unaffected. We hypothesised that human MSCs expressing TRAIL (MSCTRAIL) would home to an in vivo model of malignant pleural mesothelioma and reduce tumour growth. Human MSCs transduced with a lentiviral vector encoding TRAIL were shown in vitro to kill multiple malignant mesothelioma cell lines as predicted by sensitivity to recombinant TRAIL (rTRAIL). In vivo MSC homing was delineated using dual fluorescence and bioluminescent imaging, and we observed that higher levels of MSC engraftment occur after intravenous delivery compared with intrapleural delivery of MSCs. Finally, we show that intravenous delivery of MSCTRAIL results in a reduction in malignant pleural mesothelioma tumour growth in vivo via an increase in tumour cell apoptosis. Can TRAIL delivered by mesenchymal stem cells (MSCs) be an effective therapeutic option in malignant pleural mesothelioma?When delivered systemically, MSCs expressing TRAIL successfully incorporate into malignant pleural mesothelioma and induce cancer cell death.This is the first paper that uses both bioluminescent and fluorescent in vivo imaging to show MSC homing and infiltration into malignant pleural mesothelioma and delivery of TRAIL to cause a reduction in tumour burden.Malignant mesothelioma (MM) is a rare but devastating malignancy found most commonly within the pleura. It is largely caused by asbestos exposure5Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein and member of the TNF superfamily. It binds via two active transmembrane death receptors, DR4 and DR5, triggering the caspase cascade resulting in apoptosis. TRAIL is an exciting anticancer molecule as it induces cell death in cancer cells without affecting healthy cells.11Bone marrow-derived mesenchymal stem cells (MSCs) are attractive candidates as vectors for anticancer therapies for multiple reasons. In vitro migration studies have demonstrated MSC migration towards both tumour cells and their conditioned media18In this study, we show for the first time that MSCs expressing TRAIL (MSCTRAIL) induce apoptosis in MPM cells in vitro and that MSCs home to and incorporate into tumours in vivo when delivered via both intrapleural and intravenous routes. Furthermore, we demonstrate that intravenous MSCTRAIL delivery causes a significant reduction in tumour growth in an in vivo model of MPM through a mechanism involving increased intratumoural MSC retention and tumour cell apoptosis.Tissue culture reagents were purchased from Invitrogen  unless otherwise stated. Human adult bone marrow-derived MSCs were purchased from Tulane University and cultured in α-MEM with 16% fetal bovine serum (FBS), 4 mM L-glutamine with 50 U/mL penicillin and 50 µg/mL streptomycin. MSCs transduced with a Tet-inducible plasmid had FBS replaced with Tet-system approved FBS . Human MPM cell lines  and the benign mesothelial cell line Met5A were a kind gift from Professor Bruce Robinson  and were cultured in Dulbecco's Modified Eagle Medium with 10% FBS, 4 mM L-glutamine with 50 U/mL penicillin and 50 µg/mL streptomycin.TRAIL-IRES-eGFP lentivirus vector was produced as previously described.19MSCs were transduced with the TRAIL-IRES-eGFP lentivirus as previously described.6 cells/mL, then incubated with antibodies against the four TRAIL receptors or an isotype control, followed by biotinylated secondary goat antimouse IgG1 and streptavidin phycoerythrin (PE). Flow cytometry was performed to detect PE.MPM cells were harvested and resuspended at 1×10supplementary materials and methods.MSCTRAIL cells were plated in a 1:1 ratio with human MPM cells, and apoptosis and cell death were determined as described in online All animal studies were approved by the University College London Biological Services Ethical Review Committee and licensed under the UK Home Office regulations and the Evidence for the Operation of Animals (Scientific Procedures) Act 1986 . Eight-week-old female NOD/SCID mice were purchased from Harlan, kept in individually ventilated cages under specific pathogen-free conditions and had access to sterile-irradiated food and autoclaved water ad libitum.To create pleural tumours, mice were anaesthetised using 2% isofluorane, the right thoracic wall was shaved and cleaned with alcohol. A 5 mm incision was made on the right chest wall, and a right anterolateral thoracotomy was performed in the fourth intercostal space, followed by installation of luciferase-transduced MSTO-211H (MSTO-211HLuc) cells in 100 µL phosphate-buffered saline (PBS). MSCs were either delivered intrapleurally as described or intravenously using the lateral tail vein. Animals were weighed twice weekly, and bioluminescent imaging of tumour burden was performed twice weekly. Tumours were allowed to develop until mice reached 20% weight loss or showed signs of distress.4 MSTO-211HLuc were delivered intrapleurally and tumours were left to develop for 5 days. 1×106 untransduced MSCs were labelled using the fluorescent lipophilic dyes, DiI  and DiR  according to manufacturer's instructions and delivered either by intravenous or intrapleural injections 5 days post-tumour cell injection. Bioluminescence and fluorescence were determined using an in vivo imaging system  as described in online supplementary methods. At study termination, mice were killed and tumour samples were digested for flow cytometry and fixed for histochemical analysis.8×10Tumours were identified using open cavity bioluminescent imaging, removed and digested in a solution containing RPMI-1640 (Invitrogen) with 1 mg/mL collagenase (Sigma) and DNase I  for 1 h at 37°C. Red blood cells were lysed with red blood cell lysis buffer (Sigma) for 1 min, neutralised with RPMI and filtered. Flow cytometry  was performed for YFP to detect tumour cells and DiR to detect MSCs.6 MSCTRAIL or untransduced MSCs suspended in PBS or PBS alone were delivered intravenously on days 5, 9, 12, 15 and 18. For intrapleural delivery, the same protocol was followed except that animals received intrapleurally delivered MSCTRAIL, untransduced MSCs or PBS alone on days 5, 9, 12, 15 and 18. All animals received doxycycline at 2 mg/mL in sterile water containing 3% sucrose. Tumour growth was monitored via bioluminescent imaging twice weekly using IVIS and tumour burden was determined as described above. Animals were injected with 10 mg/kg 5-bromo-2′-deoxyuridine  1 h prior to sacrifice. All tumour tissue and lungs were removed and weighed prior to fixation and fixed overnight in 10% neutral buffered formalin (Roche) for histology.Pleural tumours were established as previously described. Tumours were left to develop for 5 days and for intravenous delivery 1×10Samples were processed as described in online supplementary methods. Calretinin antibody (Abcam), WT1 antibody , TRAIL, DR5 antibodies (ProSci) and Luciferase antibodies  were used as primary antibodies. BrdU-positive and TUNEL-positive cells were quantified using Volocity software.Statistical analysis was performed using GraphPad Prism V.4 (GraphPad Software). In vivo experiments with multiple groups were analysed using repeated measures ANOVA, and single-group data were assessed using Student t test. All in vitro experiments were performed in triplicate unless specified.The ability of MSCs to differentiate into fat and bone was confirmed along with their colony-forming efficiency as previously described.Immunocytochemistry confirmed both calretinin and Wilms Tumour antigen 1 (WT1) in all MPM cell lines (see online supplementary figure S1A–F), and they all possessed death receptor 5 (DR5) (see online supplementary figure S1G), the receptor responsible for the majority of TRAIL signalling. There was no correlation between the mean fluorescence intensity of DR5 staining and the sensitivity of MPM to MSCTRAIL (see online supplementary figure S1H). Met5A is shown as a normal mesothelial control.MPM tumour cell lines were co-cultured for 48 h with rTRAIL or MSCTRAIL with doxycycline to activate TRAIL production. Five out of six cell lines (83%) showed sensitivity to TRAIL. H28 cells showed a significant increase in apoptosis and death when treated with MSCTRAIL compared with either rTRAIL  or inactivated MSCTRAIL  A,D. MSTOTo confirm that cell death was not caused by treatment with doxycycline, MPM cells were incubated for 48 h with 10 µg/mL doxycycline, harvested and stained for annexin V and DAPI as before. Flow cytometry showed no significant increase in cell death or apoptosis in any of the MPM cell lines tested (see online supplementary figure S2B).MSTO-211H cells were transduced with a luciferase-YFP (MSTO-211HLuc) lentiviral vector and selected using hygromycin (200 µg/mL) to ensure a pure population A and inc4 MSTO-211HLuc cells were injected intrapleurally and tumour growth was monitored. Bioluminescence images showed increasing signal over 21 days (20To determine the kinetics of tumour growth in vivo, 8×10 21 days D,E. MSTO6 DiI-labelled MSCs injected into mice either intrapleurally or intravenously. Animals were imaged immediately after injection and 48 h later to determine the location of both MSCs and tumours. Tumours were clearly visible on bioluminescent imaging (Tumours were established as described in the methods with 1×10 imaging A–C, and  imaging E,F. No a imaging D. Immuno imaging G–J. No f6 MSCTRAIL or untransduced MSC or 100 µL PBS were delivered intravenously on days 5, 9, 12, 15 and 18. IVIS imaging demonstrated a significant reduction in tumour growth in the MSCTRAIL group compared with the PBS and untransduced MSC group (p<0.001, repeated measures ANOVA; Tumours were established as described and 1×106 MSCTRAIL or untransduced MSC or PBS. In this experiment, there was no significant reduction in tumour growth or lung weights compared with the PBS and untransduced MSC-treated groups.To determine whether a similar effect was seen with topically delivered MSCTRAIL, the experiment was repeated using intrapleural delivery of 1×10Histopathological analysis showed no significant difference in tumour cell proliferation D–F and K5 MSTO-211HLuc intrapleurally and tumours were left to establish for 10 days. 1×106 DiR-stained MSCs stained were injected either intravenously or intrapleurally and imaging was performed daily. Tumours were successfully established and fluorescent MSCs were clearly visible following both routes of delivery (To determine why MSCTRAIL is only effective when delivered intravenously, mice were given 2×10delivery A. There delivery B, which delivery C,D, althIn this study, we have shown that MSCs engineered to express TRAIL can induce death in multiple MPM cell lines in vitro and are more efficient at killing than recombinant TRAIL. We have also demonstrated that MSCs migrate to MPM tumours in vivo when delivered both intravenously and intrapleurally. However, only intravenous-delivered MSCTRAIL causes a significant reduction in tumour growth and the difference in efficacy is likely due to a higher number of MSCs engrafting within the tumours causing increased apoptosis.Using MSCs as vectors for gene therapy is becoming increasingly common as they are easy to extract from bone marrow, are highly expandable and readily transducible with viral vectors.There is some concern that exogenously delivered MSCs have an unpredictable effect on tumour biology with different in vivo models suggesting pro-tumourigenic or antitumourigenic properties,34When using MSCs as therapeutic delivery vectors, treatment efficacy is related to the level of MSC accumulation at tumour sites. Our study shows that while MSC homing occurs regardless of route of delivery, the level of MSC accumulation following intravenous delivery is significantly higher than when delivered intrapleurally. The increase in apoptosis seen in the intravenous delivery model could be a direct effect of the tumour receiving a greater number of cells and hence a higher dose of TRAIL. The first step in MSC accumulation within tumours is adhesion to vascular endothelial cells, and multiple factors are involved in this process.37In conclusion, this study shows intravenous MSCTRAIL delivery causes a reduction in tumour growth in an in vivo MPM model. MSCs home to and incorporate into tumours using both intravenous and intrapleural delivery routes but greater numbers engraft when delivered intravenously. The therapeutic effect seen with intravenous delivery could be related to greater engraftment of MSCs within the tumour and is an important finding when considering the future therapeutic role of MSCTRAIL therapy in the clinic.


The results showed that the averaged peak-to-peak disk deformations during the in vitro cyclic tests were well fitted with limited FE simulations and a quadratic response surface regression for both disk groups. The results showed that higher loading frequency increased the intradiscal pressure, decreased the total fluid loss, and slightly increased the maximum axial stress within solid matrix. Enzyme-induced degeneration decreased the intradiscal pressure and total fluid loss, and barely changed the maximum axial stress within solid matrix. The increase of intradiscal pressure and total fluid loss with loading frequency was less sensitive after the frequency elevated to 0.1 Hz for the enzyme-induced degenerated disk. Based on this study, it is found that enzyme-induced degeneration decreases energy attenuation capability of disk, but less change the strength of disk.The risk of low back pain resulted from cyclic loadings is greater than that resulted from prolonged static postures. Disk degeneration results in degradation of disk solid structures and decrease of water contents, which is caused by activation of matrix digestive enzymes. The mechanical responses resulted from internal solid–fluid interactions of degenerative disks to cyclic loadings are not well studied yet. The fluid–solid interactions in disks can be evaluated by mathematical models, especially the poroelastic finite element (FE) models. We developed a robust disk poroelastic FE model to analyze the effect of degeneration on solid–fluid interactions within disk subjected to cyclic loadings at different loading frequencies. A backward analysis combined with  Excessive cyclic loading requested by occupations in daily lives, such as repetitive lifting and vibration working environments, is recognized as a risk factor of low back pain , stab injury, and mechanical loading in animal models  and elastic modulus (E) regulate fluid outflow and disk strength, respectively. The choices of values of these two parameters affect the robustness of model predictions on fluid–solid interactions in disks during cyclic loadings. Understanding the effects of these parameters on time-dependent response of intervertebral disk can be beneficial to enhance understanding of changes in material properties during disk degeneration. The purpose of this study was to develop a robust disk poroelastic FE model to analyze the effect of degeneration on solid–fluid interactions within disk subjected to cyclic loadings at different loading frequencies.The fluid–solid interactions in disks can be evaluated by mathematical models, especially the one based on poroelastic theory  soaked gauze, sealed in plastic bag, and stored at −20°C in a refrigerator until experiment.® 3510, Bose Corporation, MN, USA).Specimens were equally assigned to intact and degeneration groups. The disk of intact group did not receive any forms of injury. Disk degeneration was simulated by injecting 0.5 ml trypsin solution (0.5%) into nucleus pulposus. Specimens were mounted in center of a home-made chamber filled with PBS solution to mimic the physiological environment of spine. A Teflon plate was attached to the actuator of the material testing machine to transfer cyclic loadings to the specimen. After preloading , a sinusoid cyclic load  was applied to the specimens with frequencies sweeping at 0.01, 0.063, 0.1, 0.63, 1, 6.3, and 10 Hz by a material testing apparatus  Figure . In thisin vitro human disk experiments for both creep  and hydraulic permeability (k), were selected as the independent variables of the FE model. To simplify the optimization procedure, the ratio of the elastic modulus for the anulus fibrosus and nucleus pulposus was assumed to be 1.67 and the ratio of hydraulic permeability were assumed to be 1 during analysis   regression  error was calculated. With 25 sets of RMS errors and input parameters (E/k), QRS model was constructed and optimized to find the best set of k/E. The results were considered to be accurate when the RMS error was <5%. If it was larger than 5%, the input ranges of independent variables were modified to spread the searching window.A full factorial design of experiment (DOE) for the FE simulation was conducted using MATLAB . Two factors, i.e., in vitro experiments and FE backward analysis, elastic modulus and hydraulic permeability of the 18 intact and enzyme-induced degenerated disks were calculated. The differences of the elastic modulus and hydraulic permeability between intact and enzyme-induced degenerated disks were evaluated using the independent t-test . Statistical differences were considered to be significant at p < 0.05. To evaluate how well the elastic modulus and hydraulic permeability can differentiate intact disks from enzyme-induced degenerated ones, a two-group linear discriminant analysis (LDA) was performed.Based on the results of the Based on the updated poroelastic FE models, intradiscal pressure, total fluid loss, and axial stress within solid matrix of intact and enzyme-induced degenerated intervertebral disk under four types of cyclic loadings were investigated. For these simulations, the boundary conditions were considered the same as the described model in Section “−16 m4/Ns . Reduction of intradiscal pressure and fluid outflow reduces energy absorption capability of disk. As well, the degraded proteoglycan also decrease the water content and damping coefficient (Hsu et al., Few limitations of this study should be addressed. First, the geometry of the FE model was axisymmetric and the posterior elements and facet joints were removed. In this study, only the axial cyclic loading was applied and displacement response was investigated. Hence, the 2D axisymmetric model was sufficient for the optimization procedure. Second, the ratio of elastic modulus and hydraulic permeability of anulus fibrosus and nucleus pulposus was assumed to be constant (Argoubi and Shirazi-Adl, in vitro experiments, and QRS regression to provide a methodology to investigate the effect of cyclic loading frequency and disk degeneration on fluid–solid interaction of disk. The novelty of this study was to analyze the effect of degeneration on solid–fluid interactions within disk subjected to cyclic loadings at different loading frequencies. The safe limits of repetitive activities and whole body vibration that avoid spinal disease would be beneficial in understanding the potentially detrimental effects of exposure to loading. During the cyclic loading, the fluid of disk is squeezed out. Reduction of fluid loss would inhibit the nutrition transport and metabolism process within the disk, hence forms a vicious circle for the disk. The results of this study may be valuable to enhance understanding of changes in time-dependent response of intact and moderate enzyme-induced degenerated disk during repetitive activities. Based on this method, it is found that enzyme-induced degeneration decreases energy attenuation capability of disk, but less change the strength of disk.In conclusion, this study combined FE simulations, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.