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Since 2011, the Faculty of Social Sciences (FSW) and the Institute of Environmental Sciences (CML) of Leiden University, in cooperation with Isabela State University and the Mabuwaya Foundation in the Philippines organize an annual month–long international, interdisciplinary course on sustainable development, water issues and water management in the Cagayan River basin in Northeast Luzon in the Philippines. Thirty students participate in this course, 15 through Leiden University and 15 through Isabela State University. The students are enrolled in different studies, encompassing social, natural and applied sciences. The annual report contains background information, all student reports and the online blog that the students maintained during the course.

Premature termination codon (PTC) mutations in the ATP-Binding Cassette, Sub-Family A, Member 7 gene (ABCA7) have recently been identified as intermediate-to-high penetrant risk factor for late-onset Alzheimer’s disease (LOAD). High variability, however, is observed in downstream ABCA7 mRNA and protein expression, disease penetrance, and onset age, indicative of unknown modifying factors. Here, we investigated the prevalence and disease penetrance of ABCA7 PTC mutations in a large early onset AD (EOAD)—control cohort, and examined the effect on transcript level with comprehensive third-generation long-read sequencing. We characterized the ABCA7 coding sequence with next-generation sequencing in 928 EOAD patients and 980 matched control individuals. With MetaSKAT rare variant association analysis, we observed a fivefold enrichment (p = 0.0004) of PTC mutations in EOAD patients (3%) versus controls (0.6%). Ten novel PTC mutations were only observed in patients, and PTC mutation carriers in general had an increased familial AD load. In addition, we observed nominal risk reducing trends for three common coding variants. Seven PTC mutations were further analyzed using targeted long-read cDNA sequencing on an Oxford Nanopore MinION platform. PTC-containing transcripts for each investigated PTC mutation were observed at varying proportion (5–41% of the total read count), implying incomplete nonsense-mediated mRNA decay (NMD). Furthermore, we distinguished and phased several previously unknown alternative splicing events (up to 30% of transcripts). In conjunction with PTC mutations, several of these novel ABCA7 isoforms have the potential to rescue deleterious PTC effects. In conclusion, ABCA7 PTC mutations play a substantial role in EOAD, warranting genetic screening of ABCA7 in genetically unexplained patients. Long-read cDNA sequencing revealed both varying degrees of NMD and transcript-modifying events, which may influence ABCA7 dosage, disease severity, and may create opportunities for therapeutic interventions in AD. © 2017, The Author(s).

Introduction. Matrix metalloproteinase 9 (MMP-9) is an endopeptidase involved in various cellular processes, such as tumour development and metastatic spread. In biological samples, MMP-9 can occur as pro-MMP-9 and active MMP-9, or these factors complexed with the inhibitor TIMP-1. An assay, which can measure active and total MMP-9 in biological samples, has been used on the urine from bladder cancer patients and demonstrated a significant correlation between MMP-9 and clinical parameters. The prognostic value of these measurements has never been investigated. Using this assay we have investigated the prognostic influence of total and active MMP-9 in urine from bladder cancer patients. Material and methods. Fresh voided urines from 188 consecutive patients diagnosed with bladder cancer were collected and frozen at diagnosis. After 15 years follow-up 13 patients were still alive, and 175 patients had died. MMP-9 was measured with an immunocapture activity assay. Results. Median MMP-9total was 173.7 units/10 g creatinine (range 034 792), and median MMP-9active was 14 units/10g creatinine (range, 0294 757). The two factors were correlated (Spearmańs rho 0.74, p

Background and Purpose - We studied matrix metalloproteinases (MMP) 2, 8, and 9 and extracellular matrix metalloproteinase inducer (EMMPRIN) levels in relation to carotid atherosclerotic plaque characteristics. Methods - Carotid atherosclerotic plaques (n=150) were stained and analyzed for the presence of collagen, smooth muscle cell (SMC), and macrophages. Adjacent segments were used to isolate total protein to assess MMP-2 and MMP-9 activities and gelatin breakdown, MMP-8 activity, and EMMPRIN levels. Results - Macrophage-rich lesions revealed higher MMP-8 and MMP-9 activities, whereas SMC-rich lesions showed higher MMP-2 activity. The levels of less glycosylated EMMPRIN-45kD were higher in SMC-rich lesions and lower in macrophage-rich plaques. EMMPRIN-45kD was associated with MMP-2 levels, whereas EMMPRIN-58kD was related to MMP-9 levels. Conclusions - MMP-2, MMP-8, and MMP-9 activities differed among carotid plaque phenotypes. Different EMMPRIN glycosylation forms are associated with either MMP-2 or MMP-9 activity, which suggests that EMMPRIN glycosylation may play a role in MMP regulation and plaque destabilization. © 2005 American Heart Association, Inc. Chemicals / CAS: collagen, 9007-34-5; gelatin, 9000-70-8; gelatinase A, 146480-35-5; gelatinase B, 146480-36-6; Antigens, CD147, 136894-56-9; Collagen, 9007-34-5; Matrix Metalloproteinase 2, EC 3.4.24.24; Matrix Metalloproteinase 8, EC 3.4.24.34; Matrix Metalloproteinase 9, EC 3.4.24.35

Objective. To investigate the effects of doxycycline on disease activity and joint destruction in patients with rheumatoid arthritis (RA). Methods. A 36 week double blind, placebo controlled crossover trial was conducted. Patients (n = 66) received 50 mg doxycycline or placebo twice a day during 12, 24, or 36 weeks. Patient assessments were performed before the treatment was administered, at 6, 12, 24 and 36 weeks of treatment, and finally at 4 weeks after cessation of treatment. Patient assessments, swollen and tender joint counts, duration of morning stiffness, erythrocyte sedimentation rate, and Modified Disease Activity Score were used as measures of disease activity. Effects on joint destruction were assessed by urinary excretion of the pyridinolines hydroxylysylpyridinoline and lysylpyridinoline and by scoring radiographic damage of hands and feet before and after treatment. Results. The changes of clinical or laboratory disease activity measures, pyridinoline excretion, or progression of radiographic joint damage during doxycycline or placebo treatment did not differ significantly. Conclusion. The results indicate that 50 mg doxycycline twice a day provided no therapeutic benefit for patients with RA.

Gelatinase-B has been shown to be produced by a variety of different cells, fibroblasts in particular. It has also been shown to be elevated in the sera of patients with specific disease states, for example hepatocellular carcinoma. A novel technology is described for determining the activity of matrix metalloproteinases (MMPs) using a colorimetric assay. The assay is based on a modified pro-urokinase constructed by protein engineering. In the modified prourokinase the activation sequence, normally recognised by plasmin (Pro-Arg-Phe-Lys # Ile-Ile-Gly-Gly), was replaced by a sequence that is specifically recognised by MMPs (Arg-Pro-Leu-Gly # Ile-Ile-Gly-Gly). A chromogenic peptide substrate for urokinase is then used to measure the active urokinase, generated through MMP activation of the modified urokinase. The assay uses a MMP-9 specific monoclonal antibody, coated 96 well microtitre plate to confer MMP-9 specificity. MMP-9 is captured from biological fluids or cell culture media through overnight incubation of the samples at 4°C, in the microtitre plate wells. Immobilised MMPs are then activated using APMA for a 2hour incubation period at 37°C. Differential activation with APMA, enables the analysis of the activity of both the active and latent forms of MMP-9. Detection incubation with modified urokinase and the substrate S-2444 continues at 37°C for 2 to 6 hours. The normal assay range is between 32 and 2ng/ml for a 2 hour incubation. A more sensitive assay range between 8 and 0.5ng/ml, enabled a detection sensitivity of 0.5ng/ml for a 6 hour detection incubation period. Cell culture and serum/plasma samples were used to validate the activity assay. U937 cell cultures, stimulated under different conditions, were assayed in the activity assay and in a variety of MMP-9 specific ELISAs. The APB RPN 2614 ELISA used, specifically detects pro MMP-9. In addition other antibodies were used to construct ELISAs for the measurement of total MMP-9 and pro and active MP-9/TIMP- 1 complexes. Further validation work was performed using serum and a variety of plasma types, for linearity and recovery experiments. It was observed that greater than a 1:16 dilution of these samples was required to obtain a high recovery. Normal latent MMP-9 levels were found to be detectable at this dilution. MMP-9 activity in normal biological samples was detected using the novel activity assay. The activity assay results were shown to correlate with results obtained from the MMP-9 specific ELIS As. A separate study determined the levels of Gelatinase B (MMP-9) activity in saliva samples from patients with Sjögren s syndrome. An assay based on radiolabelled gelatinated collagen showed that gelatinase activity was slightly, although not significantly, increased in Sjögren s syndrome. The novel immunocapture activity assay however, specifically measuring MMP-9, showed that MMP-9 levels were increased in Sjögren s patients compared with healthy controls: MMP-9 (already active): patients 8.9 + 2.5 U/mg, controls 1.0+0.5 U/mg (p=0.002); latent plus active MMP-9: patients 53.1 ±9.8 U/mg, controls 16.5 + 2.6 U/mg (p=0.010). © Warcourt Brace & Co. Ltd 1999.

Atherosclerosis is a multifactorial disease that is influenced by both genetic and environmental factors. Recent epidemiological studies have shown that the combination of elevated VLDL/LDL concentrations and elevated fibrinogen levels results in a strong increase of the risk for cardiovascular disease as compared to the individual risk factors. In humans, controlled studies on the relative contribution of the different factors are hampered by the heterogeneity in both environmental and genetic factors. To circumvent these limitations the APOES-Leiden transgenic mice model was developed. This model provides the opportunity to induce, modulate and measure atherosclerosis in a quantitative and standardized manner. The causal relation between increased VLDL/LDL levels and atherosclerosis has been well established, whereas for fibrinogen such a causative relationship is still uncertain. Because fibrinogen is an acutephase protein, we studied the possibility that plasma fibrinogen is a marker of the disease and becomes elevated as a consequence of inflammatory reactions that occur during the development of atherosclerotic plaques. The APOESLeiden mice were put on high cholesterol diet and at time intervals ranging from 4 to 30 weeks the plasma fibrinogen concentration was measured by a clotting rate assay and an ELISA. The progression of atherosclerosis in these mice was analyzed by histochemical methods. The fibrinogen levels in the plasma of APOES-Leiden mice on a high cholesterol diet did not change with time and were similar to the levels in APOES-Leiden mice which were on a regular mouse diet. The atherosclerosis measured in the APOES-Leiden mice on the high cholesterol diet ranged from no atherosclerosis to the presence of foam cells and the development of fatty streaks. The APOES-Leiden mice on regular diet showed no signs of atherosclerosis after 30 weeks. These results indicate that the fibrinogen concentration in the APOES-Leiden mice is not elevated secondarily to an early stage of atherosclerotic plaque formation and that the clotting properties of the fibrinogen molecules remain unchanged. Whether more advanced stages of plaque formation induce an increase in plasma fibrinogen needs to be determined. Also the role of fibrinogen as a causal factor should be assessed. © Pearson Professional Ltd 1996.

Degradation of the extracellular matrix and other tissue barriers by proteases like plasminogen activators (PAs) is a prerequisite for neoplastic growth and metastasis. Recently, we reported that highly metastatic behavior of human melanoma cells in nude mice correlates with urokinase-type PA (u-PA) expression and activity and with PA inhibitor type 1 and 2 (PAI-1, PAI-2) expression. Here we report on the occurrence of components of the PA system in the various stages of human melanoma tumor progression in situ. We studied the protein distribution on freshly frozen lesions of common nevocellular nevi (n = 25), dysplastic (= atypical) nevi (n = 16), early primary melanomas (n = 8), advanced primary melanomas (n = 11), and melanoma metastases (n = 17). Tissue-type PA was present in endothelial cells in all lesions, whereas in metastases it could be detected in tumor cells in a minority of the lesions. u-PA, its receptor, PAI-1, and PAI-2 could not be detected in benign and in early stages but appeared frequently in advanced primary melanoma and melanoma metastasis lesions. u-PA was detected in stromal cells and in tumor cells at the invasive front, the u-PA receptor and PAI-2 in tumor cells, and PAI-1 in the extracellular matrix surrounding tumor cells. Localization of the corresponding messenger RNAs and enzyme activities revealed a similar distribution. We conclude that plasminogen activation is a late event in melanoma tumor progression.

Matrix metalloproteinases (MMPs) are involved in tumor growth and metastasis, promoting the migration and invasion of cells. In this study, the amount of MMP-2 and MMP-9 activity was measured in urine from superficial bladder carcinoma patients (pTa, pT1) to evaluate their possible diagnostic value. The active and total amount of MMP-2 and MMP-9, respectively, in urine from tumor patients were compared with the levels in urine from age- and gender-matched healthy volunteers. Both MMP-2 and MMP-9 activity levels were significantly enhanced in urine from patients with high invasive cancers (pT2, PT3), whereas in urine from healthy controls no or very low MMP activities were found. More importantly, a substantial number of urine samples from patients with superficial tumors contained elevated MMP-2 and MMP-9 activities, suggesting that enhanced urinary MMP activity levels, indeed, might be indicative for early-stage bladder cancer. Overall, urinary MMP-2 and MMP-9 activity levels were significantly correlated to each other, with some individual exceptions. A comparison between urinary MMP-9 activity and a recently proposed urinary marker for bladder cancer, NMP-22, showed slightly lower numbers of patients with elevated levels for MMP-9. But because MMP-9 and NMP-22 levels were not correlated, enhanced urinary MMP activity might be useful as a marker for superficial bladder carcinoma like, or especially in combination with, other markers.