Uppsala, Sweden
Uppsala, Sweden

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de Leeuw N.,Radboud University Nijmegen | Dijkhuizen T.,University of Groningen | Hehir-Kwa J.Y.,Radboud University Nijmegen | Carter N.P.,Wellcome Trust Sanger Institute | And 13 more authors.
Human Mutation | Year: 2012

The range of commercially available array platforms and analysis software packages is expanding and their utility is improving, making reliable detection of copy-number variants (CNVs) relatively straightforward. Reliable interpretation of CNV data, however, is often difficult and requires expertise. With our knowledge of the human genome growing rapidly, applications for array testing continuously broadening, and the resolution of CNV detection increasing, this leads to great complexity in interpreting what can be daunting data. Correct CNV interpretation and optimal use of the genotype information provided by single-nucleotide polymorphism probes on an array depends largely on knowledge present in various resources. In addition to the availability of host laboratories' own datasets and national registries, there are several public databases and Internet resources with genotype and phenotype information that can be used for array data interpretation. With so many resources now available, it is important to know which are fit-for-purpose in a diagnostic setting. We summarize the characteristics of the most commonly used Internet databases and resources, and propose a general data interpretation strategy that can be used for comparative hybridization, comparative intensity, and genotype-based array data. © 2012 Wiley Periodicals, Inc.


Barisic I.,AIT Austrian Institute of Technology | Schoenthaler S.,AIT Austrian Institute of Technology | Ke R.,Rudbeck Laboratory | Ke R.,University of Stockholm | And 4 more authors.
Diagnostic Microbiology and Infectious Disease | Year: 2013

The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse β-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the β-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 104 DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of β-lactamase genes in dilutions of 107 cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene. © 2013 Elsevier Inc.


Laytragoon-Lewin N.,Ryhov Hospital | Laytragoon-Lewin N.,Rudbeck Laboratory | Bahram F.,Uppsala University Hospital | Rutqvist L.E.,Swedish Match AB | And 2 more authors.
Anticancer Research | Year: 2011

The adverse health effects of cigarette smoking are well established including the increased risk of various types of cancer. In this study, the direct effects of ethanol, pure nicotine, cigarette smoke extract and Swedish type smokeless tobacco (Snus) extract on normal cells were investigated. Materials and Methods: Primary normal adult human endothelial cells and fibroblasts at early passage were used. Upon exposure to pure nicotine, cigarette smoke extract, Snus extract and ethanol, these cells were assessed for DNA synthesis, gene expression profile and cellular morphology. Results: Normal human fibroblasts and endothelial cells have unique gene expression profiles. The effects of treatment with ethanol and nicotine from different sources was more prominent in endothelial cells than fibroblasts. The combination of alterated gene expressions and strongly inhibited DNA synthesis was only detected in cells exposed to smoke extract. In the presence and absence of ethanol, pure nicotine and Snus extract induced abnormalities in the cytoplasm without any significant degree of cell death. With similar doses of nicotine and ethanol, the additional components in smoke extract had a dominant effect. The smoke extract induced vast cellular abnormalities and massive cell death. Conclusion: Cigarette smoke induced massive cell death and various abnormalities at cellular and molecular levels in surviving endothelial cells and fibroblasts. The combination of genomic alterations and the chronic inflammatory microenvironment induced from massive cell death, will potentially promote tumourigenesis and various diseases in cigarette smokers.


PubMed | Uppsala University Hospital and Rudbeck Laboratory
Type: | Journal: BMC cancer | Year: 2016

Melphalan has been used in the treatment of various hematologic malignancies for almost 60 years. Today it is part of standard therapy for multiple myeloma and also as part of myeloablative regimens in association with autologous allogenic stem cell transplantation. Melflufen (melphalan flufenamide ethyl ester, previously called J1) is an optimized derivative of melphalan providing targeted delivery of active metabolites to cells expressing aminopeptidases. The activity of melflufen has compared favorably with that of melphalan in a series of in vitro and in vivo experiments performed preferentially on different solid tumor models and multiple myeloma. Melflufen is currently being evaluated in a clinical phase I/II trial in relapsed or relapsed and refractory multiple myeloma.Cytotoxicity of melflufen was assayed in lymphoma cell lines and in primary tumor cells with the Fluorometric Microculture Cytotoxicity Assay and cell cycle analyses was performed in two of the cell lines. Melflufen was also investigated in a xenograft model with subcutaneous lymphoma cells inoculated in mice.Melflufen showed activity with cytotoxic IC50-values in the submicromolar range (0.011-0.92 M) in the cell lines, corresponding to a mean of 49-fold superiority (p < 0.001) in potency vs. melphalan. In the primary cultures melflufen yielded slightly lower IC50-values (2.7 nM to 0.55 M) and an increased ratio vs. melphalan (range 13-455, average 108, p < 0.001). Treated cell lines exhibited a clear accumulation in the G2/M-phase of the cell cycle. Melflufen also showed significant activity and no, or minimal side effects in the xenografted animals.This study confirms previous reports of a targeting related potency superiority of melflufen compared to that of melphalan. Melflufen was active in cell lines and primary cultures of lymphoma cells, as well as in a xenograft model in mice and appears to be a candidate for further evaluation in the treatment of this group of malignant diseases.


Polajeva J.,Rudbeck Laboratory | Swartling F.J.,Rudbeck Laboratory | Jiang Y.,Rudbeck Laboratory | Singh U.,Rudbeck Laboratory | And 5 more authors.
BMC Cancer | Year: 2012

Background: MicroRNAs (miRNAs) and their role during tumor development have been studied in great detail during the last decade, albeit their expression pattern and regulation during normal development are however not so well established. Previous studies have shown that miRNAs are differentially expressed in solid human tumors. Platelet-derived growth factor (PDGF) signaling is known to be involved in normal development of the brain as well as in malignant primary brain tumors, gliomas, but the complete mechanism is still lacking. We decided to investigate the expression of the oncogenic miR-21 during normal mouse development and glioma, focusing on PDGF signaling as a potential regulator of miR-21.Methods: We generated mouse glioma using the RCAS/tv-a system for driving PDGF-BB expression in a cell-specific manner. Expression of miR-21 in mouse cell cultures and mouse brain were assessed using Northern blot analysis and in situ hybridization. Immunohistochemistry and Western blot analysis were used to investigate SOX2 expression. LNA-modified siRNA was used for irreversible depletion of miR-21. For inhibition of PDGF signaling Gleevec (imatinib mesylate), Rapamycin and U0126, as well as siRNA were used. Statistical significance was calculated using double-sided unpaired Student́s t-test.Results: We identified miR-21 to be highly expressed during embryonic and newborn brain development followed by a gradual decrease until undetectable at postnatal day 7 (P7), this pattern correlated with SOX2 expression. Furthermore, miR-21 and SOX2 showed up-regulation and overlapping expression pattern in RCAS/tv-a generated mouse brain tumor specimens. Upon irreversible depletion of miR-21 the expression of SOX2 was strongly diminished in both mouse primary glioma cultures and human glioma cell lines. Interestingly, in normal fibroblasts the expression of miR-21 was induced by PDGF-BB, and inhibition of PDGF signaling in mouse glioma primary cultures resulted in suppression of miR-21 suggesting that miR-21 is indeed regulated by PDGF signaling.Conclusions: Our data show that miR-21 and SOX2 are tightly regulated already during embryogenesis and define a distinct population with putative tumor cell of origin characteristics. Furthermore, we believe that miR-21 is a mediator of PDGF-driven brain tumors, which suggests miR-21 as a promising target for treatment of glioma. © 2012 Põlajeva et al.; licensee BioMed Central Ltd.


Chen D.,Rudbeck Laboratory | Juko-Pecirep I.,Rudbeck Laboratory | Hammer J.,Rudbeck Laboratory | Ivansson E.,Rudbeck Laboratory | And 8 more authors.
Journal of the National Cancer Institute | Year: 2013

Background Cervical carcinoma has a heritable genetic component, but the genetic basis of cervical cancer is still not well understood. Methods We performed a genome-wide association study of 731 422 single nucleotide polymorphisms (SNPs) in 1075 cervical cancer case subjects and 4014 control subjects and replicated it in 1140 case subjects and 1058 control subjects. The association between top SNPs and cervical cancer was estimated by odds ratios (ORs) and 95% confidence intervals (CIs) with unconditional logistic regression. All statistical tests were two-sided. Results Three independent loci in the major histocompatibility complex (MHC) region at 6p21.3 were associated with cervical cancer: the first is adjacent to the MHC class I polypeptide-related sequence A gene (MICA) (rs2516448; OR = 1.42, 95% CI = 1.31 to 1.54; P = 1.6×10-18); the second is between HLA-DRB1 and HLA-DQA1 (rs9272143; OR = 0.67, 95% CI = 0.62 to 0.72; P = 9.3×10-24); and the third is at HLA-DPB2 (rs3117027; OR=1.25, 95% CI = 1.15 to 1.35; P = 4.9×10-8). We also confirmed previously reported associations of B*0702 and DRB1*1501-DQB1*0602 with susceptibility to and DRB1*1301-DQA1*0103-DQB1*0603 with protection against cervical cancer. The three new loci are statistically independent of these specific human leukocyte antigen alleles/haplotypes. MICA encodes a membrane-bound protein that acts as a ligand for NKG2D to activate antitumor effects. The risk allele of rs2516448 is in perfect linkage disequilibrium with a frameshift mutation (A5.1) of MICA, which results in a truncated protein. Functional analysis shows that women carrying this mutation have lower levels of membrane-bound MICA. Conclusions Three novel loci in the MHC may affect susceptibility to cervical cancer in situ, including the MICA-A5.1 allele that may cause impaired immune activation and increased risk of tumor development. © 2013 The Author.


Nilsson B.,Rudbeck Laboratory | Ekdahl K.N.,Rudbeck Laboratory | Ekdahl K.N.,Uppsala University | Ekdahl K.N.,Linnaeus University | Korsgren O.,Rudbeck Laboratory
Current Opinion in Organ Transplantation | Year: 2011

Purpose of review: Transplantation of islets of Langerhans is an emerging treatment procedure for patients with severe type 1 diabetes, but despite recent progress the procedure is associated with massive tissue loss caused by an inflammatory reaction termed instant blood-mediated inflammatory reaction (IBMIR). This reaction involves activation of the complement and coagulation cascades, ultimately resulting in clot formation and infiltration of leukocytes into the islets, which leads to disruption of islet integrity and islet destruction. Recent findings: In this review we discuss basic mechanisms underlying the IBMIR and emerging strategies for therapeutic regulation of the IBMIR. These include the use of selective inhibitors of the coagulation and complement systems, different procedures to coat the surface of the islets as well as the development of composite islet-endothelial cell grafts. Summary: The IBMIR is a major cause of tissue loss in clinical islet transplantation, and most likely in other cell therapies in which cells are exposed to blood. Thus, it is an obvious target for therapeutic intervention. Due to its complexity, it is necessary to use different strategies to control the IBMIR. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.


Zhao Z.,University of Southern California | Nelson A.R.,University of Southern California | Betsholtz C.,Rudbeck Laboratory | Zlokovic B.V.,University of Southern California
Cell | Year: 2015

Structural and functional brain connectivity, synaptic activity, and information processing require highly coordinated signal transduction between different cell types within the neurovascular unit and intact blood-brain barrier (BBB) functions. Here, we examine the mechanisms regulating the formation and maintenance of the BBB and functions of BBB-associated cell types. Furthermore, we discuss the growing evidence associating BBB breakdown with the pathogenesis of inherited monogenic neurological disorders and complex multifactorial diseases, including Alzheimer's disease. Integrity of the blood-brain barrier (BBB) is essential for synaptic activity and brain connectivity. Growing evidence suggests the association between BBB breakdown and the pathogenesis of both monogenic and multifactorial neurological disorders. © 2015 Elsevier Inc.


Landegren U.,Rudbeck Laboratory
New Biotechnology | Year: 2013

After the conclusion of the second five-year period of the European Science Foundation (ESF) programme on functional genomics, it is time to take stock and evaluate its accomplishments. The programme networked leading scientists from a large number of European countries for strategy discussions about the promotion of functional genomics research, and to arrange scientific meetings and exchange programmes. In brief, I believe this programme has punched above its weight, and that it has successfully contributed to the overall organisation of molecular biosciences in Europe. With a modest annual budget the programme has created several interesting new opportunities, some of which may have yet to show their full impact. However, these mini-reviews are intended to provide a personal perspective on this functional genomics effort, and accordingly I focus on my personal experiences from the ESF programme. © 2013 Elsevier B.V.


Tolmachev V.,Rudbeck Laboratory | Malmberg J.,Uppsala University | Estrada S.,Uppsala University | Eriksson O.,Uppsala University | Orlova A.,Uppsala University
International Journal of Oncology | Year: 2014

Correct staging of prostate cancer is an unmet clinical need. Radionuclide targeting of prostate-specific membrane antigen (PSMA) with 111In- labeled capromab pendetide (ProstaScint) is a clinical option for prostate cancer staging. We propose the use of 124I-labeled capromab to decrease the retention of radioactivity in healthy organs (due to the non-residualizing properties of the radiolabel). The use of 124I as a label should increase imaging sensitivity due to the advantages of PET as an imaging modality. Capromab targets the intracellular domain of PSMA; accumulation of radioactivity in the tumor should not depend on internalization of the antigen/antibody complex. Capromab was iodinated, and its targeting properties were compared with indium labeled counterpart in LNCaP xenografts in dual isotope mode. PSMA-negative xenografts (PC3) were used as a negative control. Radioiodinated capromab bound to PSMA specifically. Biodistribution of 125I/111In-capromab showed a more rapid clearance of iodine radioactivity from liver, spleen, kidneys, bones, colon tissue, as well as tumors. Maximum tumor uptake (13±8% ID/g for iodine and 29±9% ID/g for indium) and tumor-to-non-tumor ratios for both agents were measured 5 days post-injection (pi). High tumor accumulation and low uptake of radioactivity in normal organs were confirmed using microPET/CT 5 days pi of 124I-capromab.

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