Evaluation of 3D Human Intestinal Organoids as a Platform for EV-A71 Antiviral Drug Discovery.

Enteroviruses are a leading cause of upper respiratory tract, gastrointestinal, and neurological infections. Management of enterovirus-related diseases has been hindered by the lack of specific antiviral treatment. The pre-clinical and clinical development of such antivirals has been challenging, calling for novel model systems and strategies to identify suitable pre-clinical candidates. Organoids represent a new and outstanding opportunity to test antiviral agents in a more physiologically relevant system. However, dedicated studies addressing the validation and direct comparison of organoids versus commonly used cell lines are lacking. Here, we described the use of human small intestinal organoids (HIOs) as a model to study antiviral treatment against human enterovirus 71 (EV-A71) infection and compared this model to EV-A71-infected RD cells. We used reference antiviral compounds such as enviroxime, rupintrivir, and 2'-C-methylcytidine (2'CMC) to assess their effects on cell viability, virus-induced cytopathic effect, and viral RNA yield in EV-A71-infected HIOs and cell line. The results indicated a difference in the activity of the tested compounds between the two models, with HIOs being more sensitive to infection and drug treatment. In conclusion, the outcome reveals the value added by using the organoid model in virus and antiviral studies.

Infrapatellar fat pad of patients with end-stage osteoarthritis inhibits catabolic mediators in cartilage.

OBJECTIVE: Adipose tissue is known to release inflammatory cytokines and growth factors. In this exploratory study, the authors examined whether the infrapatellar fat pad (IPFP) closely located to cartilage in the knee joint can affect cartilage metabolism. In addition, the authors analysed whether the macrophage types present in IPFP could explain the effect on cartilage. METHODS: IPFP explants obtained during total knee replacement of 29 patients with osteoarthritis (OA) were used to make fat-conditioned medium (FCM). Explants of bovine cartilage were cultured with or without FCM. Nitric oxide (NO) and glycosaminoglycan release and gene expression of matrix-degrading enzymes in cartilage were analysed. To stimulate catabolic processes in the cartilage, the authors added interleukin 1ß, and the effect of six FCMs was evaluated. The presence of different types of macrophages (CD68+, CD86+ and CD206+) in OA IPFPs was compared with subcutaneous adipose tissue samples and IPFP samples from patients with an anterior cruciate ligament rupture. RESULTS: FCM alone reduced NO and glycosaminoglycan release and matrix metalloproteinase (MMP)1 gene expression by the cartilage. Moreover, when catabolic conditions were enhanced with interleukin 1ß, FCM inhibited NO production as well as MMP1 and MMP3 gene expression and increased collagen type II gene expression. Significantly more CD206+ cells were present in OA IPFP samples than in subcutaneous fat or anterior cruciate ligament IPFP samples. CONCLUSION: In contrast to the authors' expectations, medium conditioned by end-stage OA IPFP inhibited catabolic processes in cartilage. CD206+ cells present in the IPFPs used for making the FCM might have contributed to the inhibition of catabolic processes in the cartilage.

Integration of efficacy, pharmacokinetic and safety assessment of interleukin-1 receptor antagonist in a preclinical model of arthritis.

Pharmacokinetic properties and safety profile of a drug are likely influenced by the disease state of a patient. In this study, we investigated the influence of arthritic processes on pharmacokinetics and immunotoxicity of interleukin-1 receptor antagonist (Anakinra) in the rat adjuvant arthritis model. Anakinra dose-dependently suppressed joint inflammation and degradation as demonstrated by reduced clinical arthritis score, paw thickness, synovial infiltration and bone degradation. In addition, plasma levels of chemokines MCP-1 and GRO/KC were reduced. Pharmacokinetic behaviour of Anakinra was influenced by disease state of the rats as judged from a decrease in C(max) and an increase of the MRT as the disease progressed at a dose of 24 and 72 mg Anakinra/kg body weight. The pharmacokinetic parameters increased dose-dependently, but non-proportionally with increasing dose. Low level anti-Anakinra antibody formation was observed at prolonged exposure to the biologic. Safety parameters, including haematology, splenic lymphocyte subset analysis, ex vivo stimulation of spleen cells and histopathology of immune system organs were affected by the disease itself to such extent that no additional effects of Anakinra could be observed. In conclusion, we demonstrated that pharmacokinetic behaviour of Anakinra was influenced by the arthritis background of the rats resulting in decreased internal exposure.

Comprehensive LC-MS E lipidomic analysis using a shotgun approach and its application to biomarker detection and identification in osteoarthritis patients.

A fast and robust method for lipid profiling utilizing liquid chromatography coupled with mass spectrometry has been demonstrated and validated for the analysis of human plasma. This method allowed quantification and identification of lipids in human plasma using parallel alternating low energy and high energy collision spectral acquisition modes. A total of 275 [corrected] lipids were identified and quantified (as relative concentrations) in both positive and negative ion electrospray ionization mode. The method was validated with five nonendogenous lipids, and the linearity (r(2) better than 0.994) and the intraday and interday repeatability (relative standard deviation, 4-6% and 5-8%, respectively) were satisfactory. The developed lipid profiling method was successfully applied for the analysis of plasma from osteoarthritis (OA) patients. The multivariate statistical analysis by partial least-squares-discrimination analysis suggested an altered lipid metabolism associated with osteoarthritis and the release of arachidonic acid from phospholipids.

Association between weight or body mass index and hand osteoarthritis: a systematic review.

OBJECTIVE: To investigate the association between weight or body mass index (BMI) and the development of hand osteoarthritis. METHODS: Systematic review of observational studies. Medical databases were searched up to April 2008. Articles that presented data on the association between weight and hand osteoarthritis were selected. The qualities of these studies were then assessed by two independent reviewers using a 19 criteria scoring system. Using the mean scores of all studies as a cut-off value, the studies were deemed as high or low quality. Study quality and study designs were combined to determine the level of evidence using best-evidence synthesis, which consisted of five levels of evidence. RESULTS: From the 25 studies included, two had cohort, three case-control and 20 cross-sectional study designs. Fifteen studies were considered high-quality studies. Of these high-quality studies, one cohort, two case-control and seven cross-sectional studies showed a positive association between weight or BMI and hand osteoarthritis. Based on three high-quality studies with preferred study designs (one cohort and two case-control) with a positive association, the level of evidence of the association between overweight and developing hand osteoarthritis is moderate. The approximate risk ratio of this association is 1.9. CONCLUSION: Weight or BMI is associated with the development of hand osteoarthritis. The level of evidence of published studies is moderate according to best-evidence synthesis. Further high-quality cohort or case-control studies are needed to elucidate the role of weight in hand osteoarthritis.

Inhibition of glycosaminoglycan incorporation influences collagen network formation during cartilage matrix production.

To understand cartilage degenerative diseases and improve repair procedures, we investigate the influence of glycosaminoglycans (GAGs) on cartilage matrix biochemistry and functionality. Bovine articular chondrocytes were cultured in alginate beads with(out) para-nitrophenyl-beta-d-xyloside (PNPX) to inhibit GAG incorporation into newly formed proteoglycans. As expected, GAG deposition in alginate beads decreased with increasing PNPX concentration. Next to GAGs, collagen deposition and cross-linking also decreased. In the presence of PNPX, GAGs and collagen were deposited further away from the chondrocyte than in the control and increased amounts were found in the culture medium. These changes resulted in decreased functional properties of the construct. We conclude that in our culture system, intact proteoglycans play a role in deposition of collagen and thus the formation of a functional matrix. The effect of less proteoglycans on the collagen network could explain why cartilage repair is ineffective in osteoarthritis and help us with development of new therapies.

Feasibility of electrodialysis as a fast and selective sample preparation method for the profiling of low-abundant peptides in biofluids.

Considerable interest exists in endogenous peptides as potential biomarkers, since they act as signaling molecules and are formed by degradation of proteins. A crucial step in the profiling of these peptides is the sample preparation, which aims to enrich the low-abundant peptides, while removing interfering matrix compounds. In a feasibility study we examined the suitability of electrodialysis (ED) for this purpose. A custom-made device was developed from the low-binding material Kel-F. It consisted of two compartments separated by a dialysis membrane, over which a voltage was applied. One compartment served as donor (containing the sample), while the smaller acceptor compartment collected the peptides. The procedure was optimized by investigating the effect of the applied voltage, ammonium acetate buffer concentration, and ED duration using model peptides. Optimum conditions were found at 300 V (150 V/cm), 25 mM ammonium acetate buffer (pH 3.8) containing 20% v/v DMSO, and 10 min, respectively. With these optimized parameters, recoveries for the model peptides were found to be 35-85% (average 64%). Additionally, ED was successfully applied to the challenging synovial fluid biological sample (due to its high viscosity). In a synovial fluid sample from a rheumatoid arthritis patient, 27 peptides originating from 12 proteins were identified, of which a considerable fraction was not identified before with other methods. This demonstrates the usefulness and complementary nature of combining ED with nanoLC-MS for biomarker discovery. These results indicate that ED is promising as a fast and selective sample preparation method for the profiling of endogenous peptides.

Identification of regulators of the myofibroblast phenotype of primary dermal fibroblasts from early diffuse systemic sclerosis patients.

Systemic sclerosis (SSc or scleroderma) is an auto-immune disease characterized by skin fibrosis. While primary cells from patients are considered as a unique resource to better understand human disease biology, the effect of in vitro culture on these cells and their evaluation as a platform to identify disease regulators remain poorly characterized. The goal of our studies was to provide insights into the utility of SSc dermal fibroblast primary cells for therapeutic target discovery. The disease phenotypes of freshly isolated and in vitro cultured SSc dermal fibroblasts were characterized using whole transcriptome profiling, alpha smooth muscle actin (ASMA) expression and cell impedance. SSc dermal fibroblasts retained most of the molecular disease phenotype upon in vitro culture for at least four cell culture passages (approximatively 10 cell doublings). We validated an RNA interference high throughput assay that successfully identified genes affecting the myofibroblast phenotype of SSc skin fibroblasts. These genes included MKL1, RHOA and LOXL2 that were previously proposed as therapeutic anti-fibrotic target, and ITGA5, that has been less studied in fibrosis biology and may be a novel potential modifier of SSc fibroblast biology. Together our results demonstrated the value of carefully-phenotyped SSc dermal fibroblasts as a platform for SSc target and drug discovery.

Antisense Long Non-Coding RNAs Are Deregulated in Skin Tissue of Patients with Systemic Sclerosis.

Systemic sclerosis is an autoimmune disease characterized by fibrosis of skin and multiple organs of which the pathogenesis is poorly understood. We studied differentially expressed coding and non-coding genes in relation to systemic sclerosis pathogenesis with a specific focus on antisense non-coding RNAs. Skin biopsy-derived RNAs from 14 early systemic sclerosis patients and six healthy individuals were sequenced with ion-torrent and analyzed using DEseq2. Overall, 4,901 genes with a fold change >1.5 and a false discovery rate <5% were detected in patients versus controls. Upregulated genes clustered in immunologic, cell adhesion, and keratin-related processes. Interestingly, 676 deregulated non-coding genes were detected, 257 of which were classified as antisense genes. Sense genes expressed opposite of these antisense genes were also deregulated in 42% of the observed sense-antisense gene pairs. The majority of the antisense genes had a similar effect sizes in an independent North American dataset with three genes (CTBP1-AS2, OTUD6B-AS1, and AGAP2-AS1) exceeding the study-wide Bonferroni-corrected P-value (PBonf < 0.0023, Pcombined = 1.1 × 10-9, 1.4 × 10-8, 1.7 × 10-6, respectively). In this study, we highlight that together with coding genes, (antisense) long non-coding RNAs are deregulated in skin tissue of systemic sclerosis patients suggesting a novel class of genes involved in pathogenesis of systemic sclerosis.

Age-related accumulation of pentosidine in aggrecan and collagen from normal and degenerate human intervertebral discs.

During aging and degeneration, many changes occur in the structure and composition of human cartilaginous tissues, which include the accumulation of the AGE (advanced glycation end-product), pentosidine, in long-lived proteins. In the present study, we investigated the accumulation of pentosidine in constituents of the human IVD (intervertebral disc), i.e. collagen, aggrecan-derived PG (proteoglycan) (A1) and its fractions (A1D1-A1D6) in health and pathology. We found that, after maturity, pentosidine accumulates with age. Over the age range studied, a linear 6-fold increase was observed in pentosidine accumulation for A1 and collagen with respective rates of 0.12 and 0.66 nmol x (g of protein)(-1) x year(-1). Using previously reported protein turnover rate constants (k(T)) obtained from measurements of the D-isomer of aspartic residue in collagen and aggrecan of human IVD, we could calculate the pentosidine formation rate constants (k(F)) for these constituents [Sivan, Tsitron, Wachtel, Roughley, Sakkee, van der Ham, DeGroot, Roberts and Maroudas (2006) J. Biol. Chem. 281, 13009-13014; Tsitron (2006) MSc Thesis, Technion-Israel Institute of Technology, Haifa, Israel]. In spite of the comparable formation rate constants obtained for A1D1 and collagen [1.81+/-0.25 compared with 3.71+/-0.26 micromol of pentosidine x (mol of lysine)(-1) x year(-1) respectively], the higher pentosidine accumulation in collagen is consistent with its slower turnover (0.005 year(-1) compared with 0.134 year(-1) for A1D1). Pentosidine accumulation increased with decreasing buoyant density and decreasing turnover of the proteins from the most glycosaminoglycan-rich PG components (A1D1) to the least (A1D6), with respective k(F) values of 1.81+/-0.25 and 3.18+/-0.37 micromol of pentosidine.(mol of lysine)(-1) x year(-1). We concluded that protein turnover is an important determinant of pentosidine accumulation in aggrecan and collagen of human IVD, as was found for articular cartilage. Correlation of pentosidine accumulation with protein half-life in both normal and degenerate discs further supports this finding.

The type of collagen cross-link determines the reversibility of experimental skin fibrosis.

Fibrotic processes in humans are characterised by an excessive accumulation of collagen containing increased levels of hydroxyallysine-derived cross-links. The occurrence of these cross-links appears to be an important criterion in assessing the irreversibility of fibrosis. We hypothesise that increased hydroxyallysine cross-linking results in a collagenous matrix that is less susceptible to proteolytic degradation and therefore the collagen deposition is no longer reversible. In this report, we show that collagen matrices with increased hydroxyallysine cross-link levels were less susceptible to matrix metalloproteinase 1 degradation than are collagen matrices containing low hydroxyallysine levels. These data indicate that the type of collagen cross-link influences collagen catabolism. In vivo evidence for the importance of the cross-linking type in determining the reversibility of the fibrotic process was found using the bleomycin-induced skin fibrosis mouse model. The analysis of the accumulated collagen in the fibrotic skin of bleomycin-treated mice did not reveal an increase in hydroxyallysine cross-link levels. In concurrence with our hypothesis, the collagen accumulation resolved in time when the mice were no longer receiving bleomycin treatment, showing the reversibility of the fibrosis. In conclusion, our data indicate that the type of collagen cross-linking is an important factor in determining whether the outcome of the fibrotic process is reversible or not.

Corticosteroids reduce the tensile strength of isolated collagen fascicles.

BACKGROUND: Overuse tendon injuries are frequent. Corticosteroid injections are commonly used as treatment, although their direct effects on the material properties of the tendon are poorly understood. PURPOSE: To examine the influence of corticosteroids on the tensile strength of isolated collagen fascicles. STUDY DESIGN: Controlled laboratory study. METHODS: Single strands (300-500 mum) of rat-tail collagen fascicles were incubated in either high (1 mL of 40 mgmL(-1) mixed with 0.5 mL saline 9%) or low (1 mL of 40 mgmL(-1) mixed with 2 mL saline 9%) concentration of methylprednisolone acetate (Depomedrol) for 3 or 7 days, while the control segment from the same fascicle was kept in saline (N = 64). After the incubation period, the fascicles underwent displacement to failure in a mechanical test rig at 0.13 mm/s, and thereafter hydroxylysyl pyridinoline and lysyl pyridinoline cross-link content was evaluated in a high-performance liquid chromatography system. Data for each group were analyzed with a 2-way analysis of variance (time x incubation) for ultimate stress (mean +/- standard deviation). RESULTS: In the high-concentration groups, strength was reduced after 3 (16.6 +/- 4.6 MPa) and 7 (8.6 +/- 1.7 MPa) days compared to the controls (30.2 +/- 5.0 MPa and 25.6 +/- 4.6 MPa, respectively; P < .05). In the low-concentration groups, strength was reduced after 3 (12.0 +/- 3.1 MPa) and 7 days (10.9 +/- 2.5 MPa) compared to the controls (31.5 +/- 5.0 MPa and 32.4 +/- 5.6 MPa, respectively; P < .05). The amount of cross-linking was unaffected by the intervention. CONCLUSION: Data show that the tensile strength of isolated fascicles is markedly reduced after 3- and 7-day incubation in both high and low concentration of corticosteroids, although the observed effect on whole tendon remains unknown. CLINICAL RELEVANCE: Corticosteroids may weaken specific regions of the injected tendon and leave it more prone to rupture. This weakening effect is manifested in the individual collagen fascicles that constitute the tendon.

Trabecular microfracture and the influence of pyridinium and non-enzymatic glycation-mediated collagen cross-links.

The propensity of individual trabeculae to fracture (microfracture) may be important clinically since it could be indicative of bone fragility. Whether or not an overloaded trabecula fractures is determined in part by its structural ductility, a mechanical property that describes how much deformation a trabecula can sustain. The overall goal of this study was to determine the structural ductility of individual trabeculae and the degree to which it is influenced by pyridinium and non-enzymatic collagen cross-links. Vertically oriented rodlike trabeculae were taken from the thoracic vertebral bodies of 32 cadavers (16 male and 16 female, 54 - 94 years of age). A total of 221 trabeculae (4 - 9 per donor) were tested to failure in tension using a micro-tensile loading device. A subset of 76 samples was analyzed to determine the concentration of hydroxylysyl-pyridinoline (HP) and lysyl-pyridinoline (LP) cross-links as well as pentosidine, a marker of non-enzymatic glycation. Structural ductility (defined as the ultimate strain of the whole trabecula) ranged from 1.8% to 20.2% strain (8.8 +/- 3.7%, mean +/- SD) and did not depend on age (P = 0.39), sex (P = 0.57), or thickness of the sample at the point of failure (P = 0.36). Pentosidine was the only marker of collagen cross-linking measured that was found to be correlated with structural ductility (P = 0.01) and explained about 9% of the observed variance. We conclude that the ductility of individual trabeculae varies tremendously, can be substantial, and is weakly influenced by non-enzymatic glycation.

The AGE of the matrix: chemistry, consequence and cure.

Accumulation of advanced glycation endproducts (AGEs) plays a crucial part in the development of age-related diseases and diabetic complications. AGEs are formed in vivo via the so-called Maillard reaction: a reducing sugar reacts with a protein to form a labile Amadori product that is subsequently stabilized, producing an irreversible, non-enzymatic post-translational modification of the protein involved. Recently, it has become clear that, in addition to sugars, lipids play an important role in the initiation of AGE formation, and that genetic factors contribute to an individual's AGE levels. The highest AGE levels are found in tissues with slow turnover, such as tendon, skin, bone, amyloid plaques and cartilage. AGEs exert their effects by adversely affecting the mechanical properties of the matrix and by modulating tissue turnover. In cartilage, these detrimental effects result in tissue that is more prone to the development of osteoarthritis. As such, the accumulation of AGEs provides the first molecular mechanism explaining the age-related increase in the incidence of osteoarthritis. Ongoing research into anti-AGE-ing therapies, such as pyrodoxamine and thiazolium compounds, which are often developed to prevent AGE-induced diabetic complications, might also prove beneficial for the prevention of osteoarthritis.

Functional adaptation through changes in regional biochemical characteristics during maturation of equine superficial digital flexor tendons.

OBJECTIVE: To quantify and compare biochemical characteristics of the extracellular matrix (ECM) of specimens harvested from tensional and compressive regions of the superficial digital flexor tendon (SDFT) of horses in age classes that include neonates to mature horses. SAMPLE POPULATION: Tendon specimens were collected on postmortem examination from 40 juvenile horses (0, 5, 12, and 36 months old) without macroscopically visible signs of tendonitis. PROCEDURE: Central core specimens of the SDFT were obtained with a 4-mm-diameter biopsy punch from 2 loaded sites, the central part of the mid-metacarpal region and the central part of the mid-sesamoid region. Biochemical characteristics of the collagenous ECM content (ie, collagen, hydroxylysylpyridinoline crosslink, and pentosidine crosslink concentrations and percentage of degraded collagen) and noncollagenous ECM content (percentage of water and glycosaminoglycans, DNA, and hyaluronic acid concentrations) were measured. RESULTS: The biochemical composition of equine SDFT was not homogeneous at birth with respect to DNA, glycosaminoglycans, and pentosidine concentrations. For most biochemical variables, the amounts present at birth were dissimilar to those found in mature horses. Fast and substantial changes in all components of the matrix occurred in the period of growth and development after birth. CONCLUSIONS AND CLINICAL RELEVANCE: Unlike cartilage, tendon tissue is not biochemically blank (ie, homogeneous) at birth. However, a process of functional adaptation occurs during maturation that changes the composition of equine SDFT from birth to maturity. Understanding of the maturation process of the juvenile equine SDFT may be useful in developing exercise programs that minimize tendon injuries later in life that result from overuse.

Biochemical analysis of the articular cartilage and subchondral and trabecular bone of the metacarpophalangeal joint of horses with early osteoarthritis.

OBJECTIVE: To assess whether site-related changes in biochemical composition are present in the cartilage and subchondral and trabecular bone of the metacarpophalangeal joint of horses with early osteoarthritis. SAMPLE POPULATION: Right metacarpophalangeal joints from 59 mature warmblood horses. PROCEDURE: Biochemical data (cross-link, amino acid, DNA, and ash contents; denatured collagen and glycosaminoglycan [GAG] concentrations; bone mineral density; and mineral composition) were obtained from 2 differently loaded sites of phalanx I cartilage and subchondral and trabecular bone samples; data were compared with previously published values from nonosteoarthritic equine joints. RESULTS: Compared with findings in nonosteoarthritic joints, GAG concentration was lower in cartilage from osteoarthritic joints and there was a loss of site differences in cellularity and lysylpyridinoline (LP) cross-link content. In subchondral bone, LP cross-link content was decreased overall and there was a loss of site differences in osteoarthritic joints; ash content was higher in the osteoarthritic joints. Hydroxyproline content in trabecular bone from osteoarthritic joints was greater than that in nonosteoarthritic trabecular bone. In all 3 layers and at both sites, the linear increase of the pentosidine cross-link content with age had diminished or was not apparent in the horses with osteoarthritic joints. CONCLUSIONS AND CLINICAL RELEVANCE: In equine metacarpophalangeal joints with early osteoarthritis, distinct biochemical changes were detected in the cartilage and subchondral and trabecular bone. The dissimilarity in response of the different tissues and differences between the sites that are affected may be related to differences in biomechanical loading and transmission and dissipation of force.

PET-CT imaging with [(18)F]-gefitinib to measure Abcb1a/1b (P-gp) and Abcg2 (Bcrp1) mediated drug-drug interactions at the murine blood-brain barrier.

INTRODUCTION: The efflux transporters P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2) are expressed at the blood-brain barrier (BBB), and can limit the access of a wide range of drugs to the brain. In this study we developed a PET-CT imaging method for non-invasive, quantitative analysis of the effect of ABCB1 and ABCG2 on brain penetration of the anti-cancer drug gefitinib, and demonstrated the applicability of this method for identification and quantification of potential modulators of ABCB1 and ABCB2 using the dual inhibitor elacridar. METHODS: In vitro cellular accumulation studies with [(14)C]-gefitinib were conducted in LLC-PK1, MDCKII, and the corresponding ABCB1/Abcb1a and ABCG2/Abcg2 overexpressing cell lines. Subsequently, in vivo brain penetration of [(18)F]-gefitinib was quantified by PET-CT imaging studies in wild-type, Abcg2(-/-), Abcb1a/1b(-/-), and Abcb1a/1b;Abcg2(-/-) mice. RESULTS: In vitro studies showed that [(14)C]-gefitinib is a substrate of the human ABCB1 and ABCG2 transporters. After i.v. administration of [(18)F]-gefitinib (1mg/kg), PET-CT imaging showed 2.3-fold increased brain levels of [(18)F]-gefitinib in Abcb1a/1b;Abcg2(-/-) mice, compared to wild-type. Levels in single knockout animals were not different from wild-type, showing that Abcb1a/1b and Abcg2 together limit access of [(18)F]-gefitinib to the brain. Furthermore, enhanced brain accumulation of [(18)F]-gefitinib after administration of the ABCB1 and ABCG2 inhibitor elacridar (10 mg/kg) could be quantified with PET-CT imaging. CONCLUSIONS: PET-CT imaging with [(18)F]-gefitinib is a powerful tool to non-invasively assess potential ABCB1- and ABCG2-mediated drug-drug interactions (DDIs) in vivo. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: This minimally-invasive, [(18)F]-based PET-CT imaging method shows the interplay of ABCB1 and ABCG2 at the BBB in vivo. The method may be applied in the future to assess ABCB1 and ABCG2 activity at the BBB in humans, and for personalized treatment with drugs that are substrates of ABCB1 and/or ABCG2.

Matrix metalloproteinase activities and their relationship with collagen remodelling in tendon pathology.

Our aim was to correlate the activity of matrix metalloproteinases (MMPs) with denaturation and the turnover of collagen in normal and pathological human tendons. MMPs were extracted from ruptured supraspinatus tendons (n=10), macroscopically normal ("control") supraspinatus tendons (n=29) and normal short head of biceps brachii tendons (n=24). Enzyme activity was measured using fluorogenic substrates selective for MMP-1, MMP-3 and enzymes with gelatinolytic activity (MMP-2, MMP-9 and MMP-13). Collagen denaturation was determined by alpha-chymotrypsin digestion. Protein turnover was determined by measuring the percentage of D-aspartic acid (% D-Asp). Zymography was conducted to identity specific gelatinases. MMP-1 activity was higher in ruptured supraspinatus compared to control supraspinatus and normal biceps brachii tendons (70.9, 26.4 and 11.5 fmol/mg tendon, respectively; P<0.001). Gelatinolytic and MMP-3 activities were lower in normal biceps brachii and ruptured supraspinatus compared to control supraspinatus (gelatinase: 0.18, 0.23 and 0.82 RFU/s/mg tendon respectively; P<0.001; MMP-3: 9.0, 8.6 and 55 fmol/mg tendon, respectively; P<0.001). Most gelatinase activity was shown to be MMP-2 by zymography. Denatured collagen was increased in ruptured supraspinatus compared to control supraspinatus (20.4% and 9.9%, respectively; P<0.001). The % D-Asp content increased linearly with age in normal biceps brachii but not in control supraspinatus and was significantly lower in ruptured supraspinatus compared to age-matched control tendons (0.33 and 1.09% D-Asp, respectively; P<0.01). We conclude that the short head of biceps brachii tendons show little protein turnover, whereas control supraspinatus tendons show relatively high turnover mediated by the activity of MMP-2, MMP-3 and MMP-1. This activity is thought to represent a repair or maintenance function that may be associated with an underlying degenerative process caused by a history of repeated injury and/or mechanical strain. After tendon rupture, there was increased activity of MMP-1, reduced activity of MMP-2 and MMP-3, increased turnover and further deterioration in the quality of the collagen network. Tendon degeneration is shown to be an active, cell-mediated process that may result from a failure to regulate specific MMP activities in response to repeated injury or mechanical strain.

Increased formation of pyridinoline cross-links due to higher telopeptide lysyl hydroxylase levels is a general fibrotic phenomenon.

Fibrosis is characterized by an excessive accumulation of collagen which contains increased levels of pyridinoline cross-links. The occurrence of pyridinolines in the matrix is an important criterion in assessing the irreversibility of fibrosis, which suggests that collagen containing pyridinoline cross-links significantly contributes to the unwanted collagen accumulation. Pyridinoline cross-links are derived from hydroxylated lysine residues located within the collagen telopeptides (hydroxyallysine pathway). Here, we have investigated whether the increase in hydroxyallysine-derived cross-links in fibrotic conditions can be ascribed to an increased expression of one of the lysyl hydroxylases (LH1, LH2 with its splice variants LH2a and LH2b, or LH3) and/or to an increased expression of lysyl oxidase (LOX). In fibroblast cultures of hypertrophic scars, keloid and palmar fascia of Dupuytren's patients, as well as in activated hepatic stellate cells, increased levels of LH2b mRNA expression were observed. Only minor amounts of LH2a were present. In addition, no consistent increase in the mRNA expression levels of LH1, LH3 and LOX could be detected, suggesting that LH2b is responsible for the overhydroxylation of the collagen telopeptides and the concomitant formation of pyridinolines as found in the collagen matrix deposited in long-term cultures by the same fibrotic cells. This is consistent with our previous observation that LH2b is a telopeptide lysyl hydroxylase. We conclude that the increased expression of LH2b, leading to the increased formation of pyridinoline cross-links, is present in a wide variety of fibrotic disorders and thus represents a general fibrotic phenomenon.

Automated synthesis of [18F]gefitinib on a modular system.

In recent years, [(18)F]gefitinib PET has successfully been employed for a number of applications ranging from oncology to in vivo studies of drug transporter proteins. We here report a reliable, automated procedure for routine synthesis of this radiotracer on an Eckert and Ziegler modular system. The 3-step radiosynthesis followed by preparative HPLC-purification provided [(18)F]gefitinib in 17.2±3.3% (n=22) overall decay-corrected radiochemical yield with radiochemical purity >99% in a total synthesis time of about 2.5h.