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  • 1. Acero Sanchez, Josep Ll.
    et al.
    Joda, Hamdi
    Henry, Olivier Y. F.
    Solnestam, Beata W.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sahlén, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Laddach, Nadja
    Ramakrishnan, Dheeraj
    Riley, Ian
    Schwind, Carmen
    Latta, Daniel
    O'Sullivan, Ciara K.
    Electrochemical Genetic Profiling of Single Cancer Cells2017In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 89, no 6, p. 3378-3385Article in journal (Refereed)
    Abstract [en]

    Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present asimple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.

  • 2. Forsell, M. N. E.
    et al.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology.
    Sedimbi, S. K.
    Andersson, J.
    Karlsson, M. C. I.
    Regulation of subunit-specific germinal center B cell responses to the HIV-1 envelope glycoproteins by antibody-mediated feedback2017In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 8, no JUN, article id 738Article in journal (Refereed)
    Abstract [en]

    The regulation of germinal center (GC) B cell responses to single epitopes is well investigated. How monoclonal B cells are regulated within the polyclonal B cell response to protein antigens is less so. Here, we investigate the primary GC B cell response after injection of mice with HIV-1 envelope glycoproteins. We demonstrate that single GCs are seeded by a diverse number of B cell clones shortly after a single immunization and that the presence of Env-specific antibodies can inhibit the development of early GC B cells. Importantly, the suppression was dependent on the GC B cells and the infused antibodies to target the same subunit of the injected HIV-1 envelope glycoproteins. An affinity-dependent antibody feedback has previously been shown to regulate GC B cell development. Here, we propose that this antibody-based feedback acts on GC B cells only if they target the same or overlapping epitopes. This study provides important basic information of GC B cell regulation, and for future vaccine designs with aim to elicit neutralizing antibodies against HIV-1.

  • 3. Kalderén, Christina
    et al.
    Stadler, Charlotte
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsgren, Margareta
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Elin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sydow-Bäckman, Mona
    Gelius, Stefan Svensson
    CCL2 mediates anti-fibrotic effects in human fibroblasts independently of CCR22014In: International Immunopharmacology, ISSN 1567-5769, E-ISSN 1878-1705, Vol. 20, no 1, p. 66-73Article in journal (Refereed)
    Abstract [en]

    CCL2 is known for its major role as a chemoattractant of monocytes for immunological surveillance and to site of inflammation. CCL2 acts mainly through the G-protein-coupled receptor CCR2 but has also been described to mediate its effects independently of this receptor in vitro and in vivo. Emerging pieces of evidence indicate that the CCL2/CCR2 axis is involved in fibrotic diseases, such as increased plasma levels of CCL2 and the presence of CCL2-hyperresponsive fibroblasts explanted from patients with systemic sclerosis and idiopathic pulmonary fibrosis. One of the profibrotic key mediators is the myofibroblast characterized by overexpression of alpha-smooth muscle actin and collagen I. However, the correlation between the CCL2/CCR2 axis and the activation of fibroblasts is not yet fully understood. We have screened human fibroblasts of various origins, human pulmonary fibroblasts (HPF), human fetal lung fibroblasts (HFL-1) and primary preadipocytes (SPF-1) in regard to CCL2 stimulated fibrotic responses. Surprisingly we found that CCL2 mediates anti-fibrotic effects independently of CCR2 in human fibroblasts of different origins.

  • 4.
    Kvastad, Linda
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Werne Solnestam, Beata
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Erik
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nygren, A. O.
    Laddach, N.
    Sahlén, Pelin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vickovic, Sanja
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bendigtsen, S. C.
    Aaserud, M.
    Floer, L.
    Borgen, E.
    Schwind, C.
    Himmelreich, R.
    Latta, D.
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 16519Article in journal (Refereed)
    Abstract [en]

    Single cell analysis techniques have great potential in the cancer genomics feld. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics.

  • 5. Lagae, L.
    et al.
    Liu, C. X.
    Latta, D.
    Henry, O.
    O'Sullivan, C.
    Roeser, T.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Riley, I.
    Borgen, E.
    Lab-on-chip tracing circulating tumour cells2013In: European Journal of Cancer, ISSN 0959-8049, E-ISSN 1879-0852, Vol. 49, p. S74-S74Article in journal (Other academic)
  • 6.
    Solnestam, Beata W.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Elin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nygren, A. O.
    Laddach, N.
    Sahlén, Pellin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoringManuscript (preprint) (Other academic)
  • 7. Sánchez, J. L. A.
    et al.
    Henry, O. Y. F.
    Joda, H.
    Solnestam, Beata Werne
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Erik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lladach, N.
    Ramakrishnan, D.
    Riley, I.
    O'Sullivan, C. K.
    Multiplex PCB-based electrochemical detection of cancer biomarkers using MLPA-barcode approach2016In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 82, p. 224-232Article in journal (Refereed)
    Abstract [en]

    Asymmetric multiplex ligation-dependent probe amplification (MLPA) was developed for the amplification of seven breast cancer related mRNA markers and the MLPA products were electrochemically detected via hybridization. Seven breast cancer genetic markers were amplified by means of the MLPA reaction, which allows for multiplex amplification of multiple targets with a single primer pair. Novel synthetic MLPA probes were designed to include a unique barcode sequence in each amplified gene. Capture probes complementary to each of the barcode sequences were immobilized on each electrode of a low-cost electrode microarray manufactured on standard printed circuit board (PCB) substrates. The functionalised electrodes were exposed to the single-stranded MLPA products and following hybridization, a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the amplified strand completed the genocomplex, which was electrochemically detected following substrate addition. The electrode arrays fabricated using PCB technology exhibited an excellent electrochemical performance, equivalent to planar photolithographically-fabricated gold electrodes, but at a vastly reduced cost (>50 times lower per array). The optimised system was demonstrated to be highly specific with negligible cross-reactivity allowing the simultaneous detection of the seven mRNA markers, with limits of detections as low as 25 pM. This approach provides a novel strategy for the genetic profiling of tumour cells via integrated "amplification-to-detection".

1 - 7 of 7
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