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  • 1. Algenas, Cajsa
    et al.
    Agaton, Charlotta
    Fagerberg, Linn
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Bjorling, Lisa
    Bjorling, Erik
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Lundberg, Emma
    Nilsson, Peter
    Persson, Anja
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Wernerus, Henrik
    Uhlen, Mathias
    Takanen, Jenny Ottosson
    Hober, Sophia
    Antibody performance in western blot applications is context-dependent2014In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, no 3, p. 435-445Article in journal (Refereed)
    Abstract [en]

    An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well-designed conditions. Here, we have analyzed 13000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application-specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full-length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full-length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context- and application-dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross-reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data.

  • 2.
    Andersson, Ann-Catrin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Strömberg, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Bäckvall, Helena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Uhlén, Mathias
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Analysis of protein expression in cell microarrays: A tool for antibody-based proteomics2006In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 54, no 12, p. 1413-1423Article in journal (Refereed)
    Abstract [en]

    Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TIVIA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.

  • 3. Berglund, Lisa
    et al.
    Björling, Erik
    Oksvold, Per
    Fagerberg, Linn
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Szigyarto, Cristina Al-Khalili
    Persson, Anja
    Ottosson, Jenny
    Wernérus, Henrik
    Nilsson, Peter
    Lundberg, Emma
    Sivertsson, Åsa
    Navani, Sanjay
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hober, Sophia
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Uhlén, Mathias
    A genecentric Human Protein Atlas for expression profiles based on antibodies2008In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 7, no 10, p. 2019-2027Article, review/survey (Refereed)
    Abstract [en]

    An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.

  • 4.
    Brattström, Daniel
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Enh för onkologi.
    Bergqvist, Michael
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Enh för onkologi.
    Wester, Kenneth
    Department of Genetics and Pathology.
    Hesselius, Patrik
    Ren, Zhi-Ping
    Department of Genetics and Pathology.
    Scheibenpflug, Lena
    Department of Genetics and Pathology.
    Wagenius, Gunnar
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Enh för onkologi.
    Brodin, Ola
    Endothelial markers and circulating angiogenic factors and p53 may be potential markers for recurrence in surgically resected non-small cell lung cancer patients.2004In: Med Sci Monit, ISSN 1234-1010, Vol. 10, no 9, p. BR331-8Article in journal (Other scientific)
  • 5.
    Brattström, Daniel
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Enh för onkologi.
    Wester, Kenneth
    Department of Genetics and Pathology.
    Bergqvist, Michael
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Enh för onkologi.
    Hesselius, Patrik
    Malmström, Per-Uno
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Department of Surgical Sciences. BMS.
    Nordgren, Hans
    Wagenius, Gunnar
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Enh för onkologi.
    Brodin, Ola
    HER-2, EGFR, COX-2 expression status correlated to microvessel density and survival in resected non-small cell lung cancer.2004In: Acta Oncol, ISSN 0284-186X, Vol. 43, no 1, p. 80-6Article in journal (Refereed)
  • 6.
    Carlsson, Jörgen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Nordgren, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sjöström, J.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Villman, K.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Bengtsson, N. O.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Ostenstad, B.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Blomqvist, Carl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    HER2 expression in breast cancer primary tumours and corresponding metastases: Original data and literature review2004In: British Journal of Cancer, ISSN 0007-0920, E-ISSN 1532-1827, Vol. 90, no 12, p. 2344-2348Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to evaluate whether the HER2 expression in breast cancer is retained in metastases. The HER2 expression in primary tumours and the corresponding lymph node metastases were evaluated in parallel samples from 47 patients. The HercepTest was used for immunohistochemical analyses of HER2 overexpression in all cases. CISH/FISH was used for analysis of gene amplification in some cases. HER2 overexpression (HER2-scores 2+ or 3+) was found in 55% of both the primary tumours and of the lymph node metastases. There were only small changes in the HER2-scores; six from 1+ to 0 and one from 3+ to 2+ when the metastases were compared to the corresponding primary tumours. However, there were no cases with drastic changes in HER2 expression between the primary tumours and the corresponding lymph node metastases. The literature was reviewed for similar investigations, and it is concluded that breast cancer lymph node metastases generally overexpress HER2 to the same extent as the corresponding primary tumours. This also seems to be the case when distant metastases are considered. It has been noted that not all patients with HER2 overexpression respond to HER2-targeted Trastuzumab treatment. The stability in HER2 expression is encouraging for efforts to develop complementary forms of therapy, for example, therapy with radionuclide-labelled Trastuzumab.

  • 7.
    Carlsson, Jörgen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    De La Torre, Manuel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    Gardmark, Truls
    EGFR-expression in primary urinary bladder cancer and corresponding metastases and the relation to HER2-expression. On the possibility to target these receptors with radionuclides2015In: Radiology and Oncology, ISSN 1318-2099, E-ISSN 1581-3207, Vol. 49, no 1, p. 50-58Article in journal (Refereed)
    Abstract [en]

    Background. There is limited effect of tyrosine kinase inhibitors or "naked" antibodies binding EGFR or HER2 for therapy of metastasized urinary bladder canter and these methods are therefore not routinely used. Targeting radionuclides to the extracellular domain of the receptors is potentially a better possibility. Methods. EGFR- and HER2-expression was analyzed for primary tumors and corresponding metastases from 72 patients using immunohistochemistry and the internationally recommended HercepTest. Intracellular mutations were not analyzed since only the receptors were considered as targets and intracellular abnormalities should have minor effect on radiation dose. Results. EGFR was positive in 71% of the primary tumors and 69% of corresponding metastases. Local and distant metastases were EGFR-positive in 75% and 66% of the cases, respectively. The expression frequency of HER2 in related lesions was slightly higher (data from previous study). The EGFR-positive tumors expressed EGFR in metastases in 86% of the cases. The co-expression of EGFR and HER2 was 57% for tumors and 53% for metastases. Only 3% and 10% of the lesions were negative for both receptors in tumors and metastases, respectively. Thus, targeting these receptors with radionuclides might be applied for most patients. Conclusions. At least one of the EGFR- or HER2-receptors was present in most cases and co-expressed in more than half the cases. It is therefore interesting to deliver radionuclides for whole-body receptor-analysis, dosimetry and therapy. This can hopefully compensate for resistance to other therapies and more patients can hopefully be treated with curative instead of palliative intention.

  • 8.
    Ekberg, Thomas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Engström, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Nordgren, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Anniko, Matti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Expression of EGFR, HER2, HER3, and HER4 in metastatic squamous cell carcinomas of the oral cavity and base of tongue2005In: International Journal of Oncology, ISSN 1019-6439, Vol. 26, no 5, p. 1177-85Article in journal (Refereed)
    Abstract [en]

    The expressions of all four receptors in the epidermal growth factor receptor family, EGFR. HER2, HER3, and HER4 were evaluated by immunohistochemistry in 19 cases of metastatic squamous cell carcinoma of the oral cavity and base of tongue. EGFR had a similar and high expression in both primary tumours and the corresponding metastases, while the expression in normal epithelium was lower in most cases. HER2 was not expressed to the same extent as EGFR. However, when HER2 was well expressed, it was in most cases expressed to the same extent and intensity in the primary tumours, metastases, and normal epithelium. The expression of HER3 and HER4 varied and was mainly cytoplasmic in all cases studied. No overexpression of HER3 and HER4 in tumours was seen as compared to normal epithelium. In order to further investigate the distribution of HER3, two HER3 expressing cell lines originating from tongue cancer were analysed in vitro, using radiolabelled anti-HER3 antibodies directed to the extracellular domains of the receptor. The results indicated that HER3 was not present in measurable amounts in the cellular membrane. There is a need for improved diagnostics and therapy for the studied type of tumours, e.g. using radiolabelled antibodies or ligands, and EGFR seemed suitable as target since the expression was high, membrane associated and similar in the primary tumours and the corresponding metastases.

  • 9. Fagelskiold, Amanda Jabin
    et al.
    Kannisto, Kristina
    Bostrom, Anna
    Hadrovic, Banina
    Farre, Cecilia
    Eweida, Mohamed
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Islam, Md. Shahidul
    Insulin-secreting INS-1E cells express functional TRPV1 channels2012In: Islets, ISSN 1938-2014, Vol. 4, no 1, p. 56-63Article in journal (Refereed)
    Abstract [en]

    We have studied whether functional TRPV1 channels exist in the INS-1E cells, a cell type used as a model for beta-cells, and in primary beta-cells from rat and human. The effects of the TRPV1 agonists capsaicin and AM404 on the intracellular free Ca2+ concentration ([Ca2+]i) in the INS-1E cells were studied by fura-2based microfluorometry. Capsaicin increased [Ca2+] i in a concentration-dependent manner, and the [Ca2+] i increase was dependent on extracellular Ca2+. AM404 also increased [Ca2+] i in the INS-1E cells. Capsazepine, a specific antagonist of TRPV1, completely blocked the capsaicin- and AM404-induced [Ca2+] i increases. Capsaicin did not increase [Ca2+] i in the primary beta-cells from rat and human. Whole cell patch clamp configuration was used to record currents across the plasma membrane in the INS-1E cells. Capsaicin elicited inward currents that were inhibited by capsazepine. Western blot analysis detected TRPV1 proteins in the INS-1E cells and the human islets. Immunohistochemistry was used to study the expression of TRPV1, but no TRPV1 protein immunoreactivity was detected in the human islet cells and the human insulinoma cells. We conclude that the INS-1E cells, but not the primary beta-cells, express functional TRPV1 channels.

  • 10.
    Frisk, Peter
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Metallbiologisk forskning.
    Wester, Kenneth
    Yaqob, Amer
    Lindh, Ulf
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Metallbiologisk forskning.
    Selenium protection against mercury-induced apoptosis and growth inhibition in cultured K-562 cells.2003In: Biol Trace Elem Res, ISSN 0163-4984, Vol. 92, no 2, p. 105-14Article in journal (Other scientific)
  • 11.
    Gedda, Lars
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Björkelund, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Lebel, Lena
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Dubois, Louise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    Penagos, Nelly
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Malmqvist, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Evaluation of Real-Time Immunohistochemistry and Interaction Map as an Alternative Objective Assessment of HER2 Expression in Human Breast Cancer Tissue2013In: Applied immunohistochemistry & molecular morphology (Print), ISSN 1541-2016, E-ISSN 1533-4058, Vol. 21, no 6, p. 497-505Article in journal (Refereed)
    Abstract [en]

    Immunohistochemical study (IHC) is a critical tool in the clinical diagnosis of breast cancer. One common assessment is the expression level of the HER2 receptor in breast cancer tissue samples with the aim of stratifying patients for applicability of the therapeutic antibody Herceptin. In this study, we aimed to investigate whether a novel assay, real-time IHC combined with Interaction Map analysis, offers the possibility of objective assessment of HER2 expression. Interaction Map presents real-time interaction data as a collection of peaks on a surface, and it was performed on 20 patient tissue samples previously scored for HER2 expression. The result shows that the relative weight of the peaks in the maps contains novel information that could discriminate between high and low HER2 expression in an operator-independent manner (P<0.001). We conclude that the real-time IHC assay has a promising potential to complement conventional IHC and may improve the precision in the future clinical diagnostics of breast cancer.

  • 12.
    Gårdmark, Truls
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    De la Torre, Manuel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Analysis of HER2 expression in primary urinary bladder carcinoma and corresponding metastases2005In: BJU International, ISSN 1464-4096, E-ISSN 1464-410X, Vol. 95, no 7, p. 982-986Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: To evaluate the expression of HER2 receptors (previously reported to be over-expressed in malignant urothelium) in both primary tumours and metastases of transitional cell cancer, using two different staining methods and two different scoring techniques, considering the potential use of these receptors as targets for planned systemic anti-HER2 nuclide-based treatment. MATERIALS AND METHODS: HER2 expression was evaluated with two different immunohistochemical methods in 90 patients with primary urinary bladder cancer tumours and corresponding metastases. Sections were first stained with the commercially available breast cancer test kit (HercepTest, Dako, Glostrup, Denmark). Parallel sections were then stained with a modified HercepTest procedure. Two different evaluation criteria were compared; the HercepTest score that requires > or = 10% stained tumour cells (as for breast cancer) and a proposed 'Target score' that requires > 67% stained tumour cells. The latter score is assumed to be preferable for HER2-targeted radionuclide therapy. RESULTS: Using the HercepTest kit, the Target score gave lower fractions of positive primary tumours and metastases than the HercepTest score. The modified HercepTest staining procedure and Target score gave high HER2 values in 80% of primary tumours and 62% of metastases, which is considerably more than that obtained with the HercepTest staining and score. There was a significant decrease in HER2 positivity with increasing distance from the primary tumour. In nine sentinel-node metastases assessed, all but one were HER2-positive. Considering all regional metastases, 74% were positive, and of distant metastases, 47%; 72% of the patients with positive primary tumours also expressed HER2 in their metastases. CONCLUSIONS: When combining the modified HercepTest with customised evaluation criteria, more HER2-positive tumours were diagnosed. The degree of HER2 down-regulation was significantly higher in distant than in regional metastases. HER2-targeted therapy may be an alternative or complementary to other methods in the future treatment of metastatic urinary bladder carcinoma.

  • 13.
    Kampf, Caroline
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Andersson, Ann-Catrin
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Wester, Kenneth
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Björling, Erik
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Uhlen, Mathias
    Ponten, Fredrik
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Antibody-based tissue profiling as a tool for clinical proteomics2004In: Clinical Proteomics, Vol. 1, p. 285-299Article in journal (Refereed)
  • 14. Koomoa, Dana-Lynn T.
    et al.
    Go, Ramon Christopher V.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Bachmann, André S.
    Expression profile of PRAF2 in the human brain and enrichment in synaptic vesicles2008In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 436, no 2, p. 171-176Article in journal (Refereed)
    Abstract [en]

    PRA1 domain family, member 2 (PRAF2) is a novel 19-kDa protein with a prenylated Rab acceptor 1 (PRA1) motif and four transmembrane domains. Our previous studies revealed that PRAF2 is highly expressed in the brain and serves as a candidate prognostic marker in neuroblastoma (NB). PRAF2 is related to proteins PRAF1 (PRA1, prenylin, Yip3) and PRAF3 (GTRAP3-18, JWA, Arl6-IP5), both of which are enriched in the brain and implicated in cellular transport and endo/exocytic vesicle trafficking. However, the function for PRAF2 remains unknown. In this study, we analyzed the distribution and localization of PRAF2 in the mature human brain using two new antibodies specific for the protein. Analysis by immunohistochemistry revealed that in the human cerebellum, the PRAF2 protein was strongly expressed in Purkinje cells and, more moderately, in cells of the molecular and the granular layers. In the cerebral cortex, hippocampus, and lateral ventricles, PRAF2 protein was detected in neuronal cells, but not in non-neuronal cells. Intriguingly, immunoblot analysis revealed that PRAF2 is enriched in synaptic vesicles (SVs) prepared from rat brains. The expression of PRAF2 in specific regions of the brain including SVs suggest an important physiological function for this novel protein, possibly by participating in multiple aspects of SV maturation, transport, and signal transmission.

  • 15. Larsson, K.
    et al.
    Eriksson, C.
    Schwenk, J. M.
    Berglund, L.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Uhlén, M.
    Hober, S.
    Wernérus, H.
    Characterization of PrEST-based antibodies towards human Cytokeratin-172009In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 342, no 1-2, p. 20-32Article in journal (Refereed)
    Abstract [en]

    Antibody-based proteomics efforts depend on validated antibodies to ensure correct annotation of analyzed proteins. We have previously argued that a low sequence identity to other proteins is a key feature for antigens used in antibody generation. Thus, a major challenge for whole-proteome studies is how to address families of highly sequence related proteins within the context of generating specific antibodies. In this study, two non-overlapping parts of human Cytokeratin-17, a protein belonging to the intermediate filament family of highly sequence-related proteins, were selected as a model system to study the specificity and cross reactivity of antibodies generated towards such a target. These recombinantly produced Protein Epitope Signature Tags (PrESTs) were immunized in five rabbits each and the batch-to-batch variations in the obtained immune responses were studied by mapping of linear epitopes using synthetic overlapping peptides. The obtained results showed a similar but not identical immune response in the respective antibody groups with a limited number of epitopes being identified. Immunohistochemical analysis of the affinity purified monospecific antibodies on tissue micro arrays resulted in a general recognition of human cytokeratins for all analyzed binders whereas antibodies identified as binding to the most unique parts of the PrESTs showed the most Cytokeratin-17 like staining. The data presented here support the strategy to use sequence identity scores as the main criteria for antigen selection but also indicate the possibility to instead produce a single antibody recognizing a defined group of proteins when the intended targets overall sequence identity score is too high. This type of group-specific antibodies would be an important tool for antibody-based projects aiming for a complete coverage of the human proteome.

  • 16. Larsson, Karin
    et al.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Peter
    Uhlén, Mathias
    Hober, Sophia
    Wernérus, Henrik
    Multiplexed PrEST immunization for high-throughput affinity proteomics2006In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 315, no 1-2, p. 110-120Article in journal (Refereed)
    Abstract [en]

    Monospecific antibodies dfdfdfdf (msAbs) generated through antigen specific purification of polyclonal antisera are valuable tools in proteome analyses. However, proteome wide generation of msAbs would require extensive immunization programs. Therefore, it would be desirable to develop efficient immunization and purification methods to reduce the number of animals needed for such antibody-based research. Here we describe a multiplex immunization strategy for generation of msAbs towards recombinantly produced human protein fragments, denoted PrESTs. Antisera from rabbits immunized with a mixture of two, three, five and up to ten different PrESTs have been purified by a two-step immunoaffinity-based protocol and the efficiency of the purification method was analyzed using a two-color protein array concept. The obtained results showed that almost 80% of the animals immunized with antigens composed of two or three different PrESTs yielded antibodies recognizing all the included PrESTs. Furthermore, the modified two-step purification method effectively eliminated all background binding and produced pure antibody pools against individual PrESTs. This indicates that the multiplexed PrEST immunization strategy described here could become useful for high-throughput antibody-based proteomics initiatives, thus significantly reducing the number of animals needed in addition to providing a more cost-efficient method for production of msAbs.

  • 17.
    Leja, Justyna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Essaghir, Ahmed
    Essand, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular and Morphological Pathology.
    Öberg, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Tötterman, Thomas H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Lloyd, Ricardo
    Vasmatzis, George
    Demoulin, Jean-Baptiste
    Giandomenico, Valeria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Novel markers for enterochromaffin cells and gastrointestinal neuroendocrine carcinomas2009In: Modern Pathology, ISSN 0893-3952, E-ISSN 1530-0285, Vol. 22, no 2, p. 261-272Article in journal (Refereed)
    Abstract [en]

    The gene expression profile of metastasizing serotonin-producing neuroendocrine carcinomas, which arise from enterochromaffin cells in the jejunum and ileum, is still largely unknown. The aim of this study was to identify genes and proteins, which are preferentially expressed by neuroendocrine carcinoma and enterochromaffin cells and therefore potential novel biomarkers and/or therapeutic targets. Six carcinoma specimens and six normal ileal mucosas were profiled by Affymetrix microarrays. Advanced bioinformatics identified differentially and specifically expressed genes, which were validated by quantitative real-time-PCR on tumor cells extracted by laser capture microdissection and normal enterochromaffin cells extracted by immunolaser capture microdissection. We identified six novel marker genes for neuroendocrine carcinoma cells: paraneoplastic antigen Ma2 (PNMA2), testican-1 precursor (SPOCK1), serpin A10 (SERPINA10), glutamate receptor ionotropic AMPA 2 (GRIA2), G protein-coupled receptor 112 (GPR112) and olfactory receptor family 51 subfamily E member 1 (OR51E1). GRIA2 is specifically expressed by neuroendocrine carcinoma cells whereas the others are also expressed by normal enterochromaffin cells. GPR112 and OR51E1 encode proteins associated with the plasma membrane and may therefore become targets for antibody-based diagnosis and therapy. Hierarchical clustering shows high similarity between primary lesions and liver metastases. However, chemokine C-X-C motif ligand 14 (CXCL14) and NK2 transcription factor related locus 3 Drosophila (NKX2-3) are expressed to a lower level in liver metastases than in primary tumors and normal enterochromaffin cells, which implies a role in neuroendocrine carcinoma differentiation. In conclusion, this study provides a list of genes, which possess relatively specific expression to enterochromaffin and neuroendocrine carcinoma cells and genes with differential expression between primary tumors and metastases. We verified six novel marker genes that may be developed as biomarkers and/or therapeutic targets.

  • 18.
    Lindén, Mårten
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    Lind, Sara Bergström
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Mayrhofer, Corina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Segersten, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Lyutvinskiy, Yaroslav
    Zubarev, Roman
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Proteomic analysis of urinary biomarker candidates for nonmuscle invasive bladder cancer2012In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 12, no 1, p. 135-144Article in journal (Refereed)
    Abstract [en]

    Nonmuscle invasive tumors of the bladder often recur and thereby bladder cancer patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method, e.g. a urine dipstick test, for monitoring recurrence would thus be advantageous. In this study, the complementary techniques mass spectrometry (MS) and Western blotting (WB)/dot blot (DB) were used to screen the urine samples from bladder cancer patients. High resolving MS was used to analyze and quantify the urinary proteome and 29 proteins had a significantly higher abundance (p<0.05) in bladder cancer samples compared with control urine samples. The increased abundance found in urine from bladder cancer patients compared with controls was confirmed with Western blot for four selected proteins; fibrinogen β chain precursor, apolipoprotein E, α-1-antitrypsin, and leucine-rich α-2-glycoprotein 1. Dot blot analysis of an independent urine sample set pointed out fibrinogen β chain and α-1-antitrypsin as most interesting biomarkers having sensitivity and specificity values in the range of 66-85%. Exploring the Human Protein Atlas (HPA) also revealed that bladder cancer tumors are the likely source of these proteins. They have the potential of being useful in diagnosis, monitoring of recurrence and thus may improve the treatment of bladder tumors, especially nonmuscle invasive tumors.

  • 19.
    Lindén, Mårten
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    Segersten, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    Runeson, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Busch, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Bergström Lind, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    Tumour expression of bladder cancer-associated urinary proteins2013In: BJU International, ISSN 1464-4096, E-ISSN 1464-410X, Vol. 112, no 3, p. 407-415Article in journal (Refereed)
    Abstract [en]

    WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?:

    • The current basis for diagnosis and prognosis in urinary bladder cancer is based on the pathologists' assessment of a biopsy of the tumour. Urinary biomarkers are preferable as they can be non-invasively sampled. Urinary cytology is the only test with widespread use but is hampered by poor reproducibility and low sensitivity.
    • By studying the protein expression in bladder tumour tissue samples of proteins previously found in elevated levels in the urine of patients with bladder cancer, we have been able to show that these proteins originate from the tumour. The immunoreactivity of three of the investigated proteins increased with higher stage. Also a serine peptidase inhibitor was found to be predictive of progression from non-muscle-invasive to muscle-invasive tumours.

    OBJECTIVES:

    • To analyse the expression of five bladder cancer-associated urinary proteins and investigate if expression is related to the malignant phenotype of the tumour.
    • To explore the possible prognostic value of these proteins.

    PATIENTS AND METHODS:

    • Urine samples, 16 from patients with bladder cancer and 26 from controls, were used in Western Blotting experiments.
    • Tissue microarrays with bladder tissue from 344 patients diagnosed with bladder cancer between 1984 and 2005 was used in immunohistochemistry experiments.
    • The proteins apolipoprotein E (APOE), fibrinogen β chain precursor (FGB), leucine-rich α2-glycoprotein (LRG1), polymerase (RNA) I polypeptide E (POLR1E), α1-antitrypsin (SERPINA1) and topoisomerase 2A (TOP2A) were probed with antibodies validated by the Human Protein Atlas.

    RESULTS:

    • Increased expressions of APOE, FGB and POLR1E were correlated with increased tumour stage (P < 0.001).
    • Expression of SERPINA1 in Ta and T1 tumours was found to increase the risk of tumour progression (hazard ratio 2.57, 95% confidence interval 1.13-5.87; P = 0.025)

    CONCLUSIONS:

    • All proteins previously detected in urine from patients with bladder cancer were also expressed in bladder cancer tissue.
    • The expression of APOE, FGB and POLR1E increased with stage and they are potential diagnostic markers.
    • SERPINA1 was identified as a prognostic marker candidate.
  • 20.
    Loskog, Angelica
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Hedlund, Titti
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    de la Torre, Manuel
    Philipson, Lennart
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Tötterman, Thomas H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Human urinary bladder carcinomas express adenovirus attachment and internalization receptors2002In: Gene Therapy, ISSN 0969-7128, E-ISSN 1476-5462, Vol. 9, no 9, p. 547-553Article in journal (Refereed)
    Abstract [en]

    The use of adenoviral vectors as potent gene delivery systems requires expression of the Coxsackievirus/adenovirus receptor (CVADR) on the target cell surface. This receptor is important for virus attachment to the cell surface. For effective internalization of the vector into the target cell the integrins alpha(v)beta(3) and/or alpha(v)beta(5) are needed. Since there have been reports of loss of CVADR in bladder cancer cell lines, we wanted to investigate the expression of this receptor in bladder carcinoma biopsies. Surgical biopsies, as well as five human bladder cancer cell lines, were analyzed for expression of CVADR, the integrins alpha(v)beta(3) and alpha(v)beta(5) and MHC class I. Further, we studied the ability to transduce these cell lines using adenoviral vectors. Immunohistochemistry revealed that all biopsies (27/27) were positive for CVADR. Some variation in expression was evident, and superficially growing tumors stained more strongly than invasive ones. Most human tumors expressed the integrin alpha(v)beta(5) (14/24), whereas integrin alpha(v)beta(3) was less frequently seen (3/20). The established cell lines were efficiently transduced with adenoviral vectors, and transduction could be reduced with anti-CVADR antibodies. The abundance of appropriate viral receptors on tumor biopsy cells is a further argument for using adenoviral vectors in gene therapy of bladder cancer.

  • 21.
    Malmberg, Jennie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Comparative biodistribution of imaging agents for in vivo molecular profiling of disseminated prostate cancer in mice bearing prostate cancer xenografts: focus on (111)In- and (125)I-labeled anti-HER2 humanized monoclonal trastuzumab and ABY-025 Affibody2011In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 38, no 8, p. 1093-1102Article in journal (Refereed)
    Abstract [en]

    Introduction: Human epidermal growth factor receptor type 2 (HER2) overexpression supports proliferation of androgen-independent prostate cancer (PC). Radionuclide molecular imaging of HER2 expression in disseminated PC would aid in the selection of patients who are likely responders to HER2 targeting therapy. In this study, we evaluated whether ABY-025 Affibody molecule, a small (similar to 7-kDa) HER2-binding scaffold protein, produces superior tumor-to-nontumor ratios compared with those obtained through the use of radiolabeled humanized anti-HER2 antibody, trastuzumab. The influence of (111)In vs. (125)I radiolabel was evaluated for both tracers.

    Methods: ABY-025 was labeled with (111)In using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid chelator, site-specifically coupled to the C-terminus via the maleimido derivative. Trastuzumab was labeled with (111)In using a CHX-A" diethylene triamine pentaacetic acid (DTPA) chelator. An indirect radioiodination with [(125)I]-N-succinimidyl-para-iodobenzoate was used for both targeting proteins. Biodistribution of all labeled targeting proteins was evaluated in mice bearing DU-145 PC xenografts.

    Results: The use of residualizing (111)In-label facilitated better tumor uptake and better tumor-to-nontumor ratios for both targeting agents. [(111)In]-ABY-025 provided tumor uptake of 7.1 +/- 0.8% injected dose per gram of tissue (% ID/g) and tumor-to-blood ratio of 47 +/- 13 already at 6 h postinjection. The maximum tumor-to-nontumor ratios with [(111)In]-CHX-DTPA-trastuzumab were achieved at 72 h postinjection, whereas tumor uptake was 11 +/- 4% ID/g and tumor-to-blood ratio was 18 +/- 7. The biodistribution data were confirmed with gamma-camera imaging.

    Conclusions: Radiolabeled ABY-025 Affibody molecule provides higher contrast in imaging of HER2-expressing PC xenografts than radiolabeled trastuzumab. Residualizing radiometal label for ABY-025 provides better contrast in imaging of HER2-expressing PC xenografts than nonresidualizing radiohalogen.

  • 22.
    Malmstrom, PU
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Surgical Sciences.
    Ren, ZP
    Department of Genetics and Pathology.
    Sherif, A
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Surgical Sciences.
    de la Torre, M
    Department of Genetics and Pathology.
    Wester, K
    Department of Genetics and Pathology.
    Thorn, M
    Early metastatic progression of bladder carcinoma: molecular profile ofprimary tumor and sentinel lymph node.2002In: J Urol, Vol. 168, p. 2240-Article in journal (Refereed)
  • 23. Nilsson, Peter
    et al.
    Paavilainen, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Larsson, Karin
    Ödling, Jenny
    Sundberg, Mårten
    Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm.
    Andersson, Ann-Catrin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Persson, Anja
    Al-Khalili Szigyarto, Cristina
    Ottosson, Jenny
    Björling, Erik
    Hober, Sophia
    Wernérus, Henrik
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ponten, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Uhlen, Mathias
    Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling2005In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, no 17, p. 4327-4337Article in journal (Refereed)
    Abstract [en]

    A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

  • 24.
    Paavilainen, Linda
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wernérus, Henrik
    Nilsson, Peter
    Uhlén, Mathias
    Hober, Sophia
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Evaluation of monospecific antibodies: a comparison study with commercial analogs using immunohistochemistry on tissue microarrays2008In: Applied immunohistochemistry & molecular morphology (Print), ISSN 1541-2016, E-ISSN 1533-4058, Vol. 16, no 5, p. 493-502Article in journal (Refereed)
    Abstract [en]

    Generation of monospecific antibodies (msAbs) (multiepitope) through affinity purification of polyclonal antisera is a plausible strategy for high-throughput production of affinity reagents toward large sets of proteins. These antibodies are generated using readily accessible gene sequence information from publicly available databases. The resulting antibodies have the potential to be used in a variety of assays, probing differentially presented and altered proteins with high sensitivity and specificity. In the present study, 48 msAbs were compared with corresponding commercial analogs. Immunohistochemical staining properties were evaluated on tissue microarrays, representing various normal human tissues from 144 different individuals. MsAbs showed similar immunostaining patterns as compared with corresponding commercial analogs in 44 out of totally 48 (92%) antibody pairs analyzed. Although only few antibody pairs showed major discrepancies, minor dissimilarities were frequently seen. Our results suggest that msAbs are reliable and valuable tools in antibody-based proteomics, enabling analysis of protein expression patterns in cells and tissues. High-throughput strategies employing such antibodies provide a consistent approach in the exploration of the human proteome.

  • 25.
    Qu, Mingqi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurology.
    Aronica, Eleonora
    Boer, Karin
    Fällmar, David
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology.
    Kumllien, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurology.
    Nistér, Monica
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Smits, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurology.
    DLG3/SAP102 protein expression in malformations of cortical development: A study of human epileptic cortex by tissue microarray2009In: Epilepsy Research, ISSN 0920-1211, E-ISSN 1872-6844, Vol. 84, no 1, p. 33-41Article in journal (Refereed)
    Abstract [en]

    The human DLG3 gene encodes the synapse-associated protein 102 (SAP102), which is concentrated in the postsynaptic densities of excitatory synapses and involved in receptor-mediated synaptic transmission via binding to the NR2B subunit of the NMDA receptor. In this study, we investigated the expression and cellular distribution of the DLG3/SAP102 protein in human epileptic cortex. Tissue microarrays of a large number of specimens from patients operated for medically intractable epilepsy were used for immunohistochemical screening with anti-DLG3 antibody. The cellular distribution of the protein was further investigated in samples of malformations of cortical development, and the amount of DLG3 protein in the total homogenate and in the postsynaptic membrane fraction of these samples was quantified by Western blot. We found a strictly neuronal expression of DLG3/SAP102 in epileptogenic cortex as well as in non-epileptic human cortex used for control. In focal cortical dysplasia and tuberous sclerosis complex, the protein was expressed in most neurons including dysplastic neurons, but not in giant cells. Increased expression of DLG3 protein was observed in the postsynaptic membrane fraction of patients with focal cortical dysplasia. Double-labeling experiments confirmed the exclusive neuronal character of the DLG3 expressing cells and the co-localization of the DLG3 protein with the NR2B subunit. Our results suggest a putative role for DLG3/SAP102 in cortical hyperexcitability and epileptogenicity of malformations of cortical development.

  • 26.
    Rada-Iglesias, Alvaro
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wallerman, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Koch, Christoph
    Ameur, Adam
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Enroth, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Clelland, Gayle
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wilcox, Sarah
    Dovey, Oliver M
    Ellis, Peter D
    Wraight, Vicki L
    James, Keith
    Andrews, Rob
    Langford, Cordelia
    Dhami, Pawandeep
    Carter, Nigel
    Vetrie, David
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Komorowski, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Dunham, Ian
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Binding sites for metabolic disease related transcription factors inferred at base pair resolution by chromatin immunoprecipitation and genomic microarrays2005In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 14, no 22, p. 3435-3447Article in journal (Refereed)
    Abstract [en]

    We present a detailed in vivo characterization of hepatocyte transcriptional regulation in HepG2 cells, using chromatin immunoprecipitation and detection on PCR fragment-based genomic tiling path arrays covering the encyclopedia of DNA element (ENCODE) regions. Our data suggest that HNF-4α and HNF-3β, which were commonly bound to distal regulatory elements, may cooperate in the regulation of a large fraction of the liver transcriptome and that both HNF-4α and USF1 may promote H3 acetylation to many of their targets. Importantly, bioinformatic analysis of the sequences bound by each transcription factor (TF) shows an over-representation of motifs highly similar to the in vitro established consensus sequences. On the basis of these data, we have inferred tentative binding sites at base pair resolution. Some of these sites have been previously found by in vitro analysis and some were verified in vitro in this study. Our data suggests that a similar approach could be used for the in vivo characterization of all predicted/uncharacterized TF and that the analysis could be scaled to the whole genome.

  • 27.
    Ranefall, Petter
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Andersson, Ann-Catrin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Busch, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Bengtsson, Ewert
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
    Automatic quantification of immunohistochemically stained cell nuclei based on standard reference cells1998In: Analytical Cellular Pathology, ISSN 0921-8912, E-ISSN 1878-3651, Vol. 17, no 2, p. 111-23Article in journal (Refereed)
    Abstract [en]

    A fully automatic method for quantification of images of immunohistochemically stained cell nuclei by computing area proportions, is presented. Agarose embedded cultured fibroblasts were fixed, paraffin embedded and sectioned at 4 microm. They were then stained together with 4 microm sections of the test specimen obtained from bladder cancer material. A colour based classifier is automatically computed from the control cells. The method was tested on formalin fixed paraffin embedded tissue section material, stained with monoclonal antibodies against the Ki67 antigen and cyclin A protein. Ki67 staining results in a detailed nuclear texture with pronounced nucleoli and cyclin A staining is obtained in a more homogeneously distributed pattern. However, different staining patterns did not seem to influence labelling index quantification, and the sensitivity to variations in light conditions and choice of areas within the control population was low. Thus, the technique represents a robust and reproducible quantification method. In tests measuring proportions of stained area an average standard deviation of about 1.5% for the same field was achieved when classified with classifiers created from different control samples.

  • 28.
    Sandström, Karl
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Haylock, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Spiegelberg, Diana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Qvarnström, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    A novel CD44v6 targeting antibody fragment with improved tumor-to-blood ratio2012In: International Journal of Oncology, ISSN 1019-6439, Vol. 40, no 5, p. 1525-1532Article in journal (Refereed)
    Abstract [en]

    The chimeric monoclonal antibody U36 (cMAb U36) recognizes the CD44v6 antigen. Its potential as a radioimmunotargeting agent, as well as its safety, has been shown in previous studies in head and neck cancer patients. However, intact MAbs have long circulation time in the blood and tumor targeting may also be hampered due to the slow and incomplete diffusion into solid tumors. In comparison, smaller monovalent Fab' and divalent F(ab')2 fragments are expected to exhibit shorter circulating half-lives, better tumor penetration and are thus more likely to yield better imaging results. In this study, novel F(ab')2 and Fab' fragments from cMAb U36 were radiolabeled with 125I and the characteristics of the conjugates in vitro were examined. The biodistribution of the conjugates were then evaluated in nude mice bearing CD44v6-expressing xenograft tumors. Furthermore, the penetration depth and distribution in tumor tissue was assessed by autoradiography in selected tumor samples. The in vitro experiments showed that the conjugates were stable and had intact affinity to CD44v6. The biodistribution study demonstrated superior tumor-to-blood ratio for the novel cMAb U36 fragment 125I-F(ab')2 compared with both the intact MAb and the monovalent fragment form. Autoradiography also revealed better tumor penetration for 125I-F(ab')2. This study demonstrates that the use of antibody fragments may improve radioimmunotargeting and possibly improve the management of head and neck malignancies.

  • 29. Schultz, Iman J.
    et al.
    De Kok, Jacques B.
    Witjes, J. Alfred
    Babjuk, Marko
    Willems, Johannes L.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Swinkels, Dorine W.
    Tjalsma, Harold
    Simultaneous proteomic and genomic analysis of primary Ta urothelial cell carcinomas for the prediction of tumor recurrence2007In: Anticancer Research, ISSN 0250-7005, E-ISSN 1791-7530, Vol. 27, no 2, p. 1051-1058Article in journal (Refereed)
    Abstract [en]

    Background: The prediction of tumor recurrence in patients with Ta urothelial cell carcinoma is inaccurate and new prognostic markers are desirable. Materials and Methods: Surface-enhanced laser-desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) was performed on 33 primary Ta tumors (16 and 17 tumors were from patients with long and short recurrence-free periods, respectively) and data were compared to previously obtained mRNA expression profiles of 49 genes. Results: The intensities of a protein peak at m/z 33331 varied most significantly between the two patient groups (p=0.0048). This was comparable to survivin, whose mRNA expression differed most significantly (p=0.0042) of the 49 genes. ROC analysis revealed an area under the curve for protein peak 33331 and survivin of 0.78 (95% CI, 0.62-0.94) and 0.79 (95% CI, 0.63-0.94), respectively. Protein peak 33331 and survivin identified 3 (17%) and 8 (47%) patients with a recurrence-free period of at least 4 years, respectively, without generating false-negatives. Conclusion: These findings indicate that SELDI-TOF MS and real-time Q-PCR analysis on the same tissue can result in the identification of markers with comparable differential expression. Such combined analyses may yield combinations of several markers that might improve disease prognosis.

  • 30. Schultz, Iman J.
    et al.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Straatman, Huub
    Kiemeney, Lambertus A.
    Babjuk, Marko
    Mares, Jaroslav
    Willems, Johanner L.
    Swinkels, Dorine W.
    Witjes, J. Alfred
    de Kok, Jacques B.
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Prediction of recurrence in Ta urothelial cell carcinoma by real-time quantitative PCR analysis: a microarray validation study2006In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 119, no 8, p. 1915-1919Article in journal (Refereed)
    Abstract [en]

    Accurate prediction of tumor recurrence in patients with superficial urothelial cell carcinoma (UCC) might result in a significant reduction of invasive follow-up cystoscopies. A recent study identified a panel of 26 genes from a large cDNA microarray analysis of bladder tumors that discriminated between early- and late-recurring patients with superficial Ta tumors (Dyrskjot et al., Nat Genet 2003;33:90-6). We aimed to validate this panel of genes in 44 primary Ta UCCs (23 and 21 tumors from patients with short or prolonged recurrence-free periods, respectively), by real-time quantitative PCR. Statistical analysis showed marginal significant different mRNA expression levels between the 2 patient groups. To evaluate a supplementary effect of genes for the identification of patients with short or prolonged recurrence-free intervals, forward logistic regression analysis was applied. This revealed that a combination of the expression profiles of the genes HNRPK, LTB4DH and ANP32B resulted in the best performance, although the combination only marginally increased the predictive value of HNRPK alone. Comparing the receiver-operating-characteristic curves for HNRPK expression among patients with short or prolonged recurrence-free periods, revealed an area under the curve of 0.696 (95% CI, 0.537-0.855). Using the median HNRPK expression level as cut-off, a sensitivity of 69.6% and a specificity of 71.4% were obtained for the identification of patients with short or prolonged recurrence-free periods, respectively. In conclusion, we were not able to confirm the microarray gene expression pattern of the 26 genes shown by Dyrskjot et al. The discovery of accurate recurrence predictive markers, therefore, remains a challenge.

  • 31. Schultz, Iman J
    et al.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Straatman, Huub
    Kiemeney, Lambertus A
    Babjuk, Marko
    Mares, Jaroslav
    Willems, Johannes L
    Swinkels, Dorine W
    Witjes, J Alfred
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    de Kok, Jacques B
    Gene expression analysis for the prediction of recurrence in patients with primary Ta urothelial cell carcinoma2007In: European Urology, ISSN 0302-2838, E-ISSN 1873-7560, Vol. 51, no 2, p. 416-423Article in journal (Refereed)
    Abstract [en]

    Objectives

    The individual recurrence-free period after primary surgery of patients with Ta urothelial cell carcinoma (UCC) cannot be predicted accurately. This study aims at discriminating between patients with primary Ta UCC and long or short recurrence-free periods.

    Methods

    We investigated mRNA expression of 23 genes in 44 primary Ta tumours (23 and 21 tumours were from patients with long [≥4 yr] or short [≤2 yr] recurrence-free periods, respectively), using real-time quantitative polymerase chain reaction. The genes were selected from previously published studies and showed a relationship with tumour recurrence in patients with UCC.

    Results

    Differential mRNA expression between the two patient groups indicated statistical significance only for the gene survivin (p = 0.0011). Its recurrence predictive value could not be increased by a combination with any of the other genes. Comparison of the receiver operating characteristic curves for survivin expression between patients with long or short recurrence-free intervals revealed an area under the curve of 0.79 (95%CI, 0.65–0.92). Using the median expression (0.84) as cut-off level, survivin identified 71.4% (95%CI, 47.8–88.7) and 69.6% (95%CI, 47.1–86.8) of the patients with long or short recurrence-free periods, respectively.

    Conclusions

    Our study identifies survivin as the most promising candidate to distinguish between patients with primary Ta UCC and long or short recurrence-free intervals. Therefore, survivin mRNA expression analysis might help the urologist to individualise patient treatment and prevent unnecessary cystoscopies in a subgroup of these patients.

  • 32.
    Segersten, M. Ulrika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    Edlund, E. Karolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Micke, Patrick
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    de la Torre, Manuel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hamberg, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Edvinsson, Åsa E. L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Andersson, Sandra E. C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    Wester, H. Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    A novel strategy based on histological protein profiling in-silico for identifying potential biomarkers in urinary bladder cancer2009In: BJU International, ISSN 1464-4096, E-ISSN 1464-410X, Vol. 104, no 11, p. 1780-1785Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: To screen a publicly available immunohistochemistry (IHC) based web-atlas, to identify key proteins in bladder cancer that might serve as potential biomarkers. MATERIALS AND METHODS: The first version of the Human Protein Atlas (HPA 1.0), with 660 proteins, was visually examined to identify proteins with a variable staining pattern among the 12 tissue samples representing bladder cancer. None or limited previous characterization in bladder cancer, as well as a supportive Western blot, were also required. The selected proteins were then evaluated in an independent set of patient samples (106 tumour samples of differing stage and grade) represented in a tissue microarray (TMAi). The IHC expression of the identified proteins in the TMAi was scored and related to tumour stage and grade. RESULTS: The expression profiles of the 13 proteins selected from the web-atlas were confirmed in the TMAi. Expression patterns for seven proteins were significantly altered (P < 0.05) with higher stage and/or grade. Three of those (CN130, DSG3, PHF6) lack characterization in bladder cancer, whereas the remaining four proteins have previously been suggested as key proteins/potential biomarkers in cancer, some of them also in bladder cancer. CONCLUSION: New candidate proteins for urinary bladder cancer were identified through screening of the publicly available HPA 1.0. Although further evaluation is necessary, this strategy is promising in the search for new biomarkers, with potential to improve the management of patients with this disease.

  • 33.
    Söderberg, Ola
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gullberg, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ridderstråle, Karin
    Leuchowius, Karl-Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jarvius, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hydbring, Per
    Bahram, Fuad
    Larsson, Lars-Gunnar
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Direct observation of individual endogenous protein complexes in situ by proximity ligation2006In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 3, no 12, p. 995-1000Article in journal (Refereed)
    Abstract [en]

    Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-gamma (IFN-gamma) signaling and low-molecular-weight inhibitors.

  • 34. Uhlen, Mathias
    et al.
    Bjorling, Erik
    Agaton, Charlotta
    Szigyarto, Cristina Al-Khalili
    Amini, Bahram
    Andersen, Elisabet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Andersson, Ann-Catrin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Angelidou, Pia
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Asplund, Caroline
    Berglund, Lisa
    Bergström, Kristina
    Brumer, Harry
    Cerjan, Dijana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ekstrom, Marica
    Elobeid, Adila
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Eriksson, Cecilia
    Fagerberg, Linn
    Falk, Ronny
    Fall, Jenny
    Forsberg, Mattias
    Björklund, Marcus Gry
    Gumbel, Kristoffer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Halimi, Asif
    Hallin, Inga
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hamsten, Carl
    Hansson, Marianne
    Hedhammar, My
    Hercules, Görel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Larsson, Karin
    Lindskog, Mats
    Lodewyckx, Wald
    Lund, Jan
    Lundeberg, Joakim
    Magnusson, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Malm, Erik
    Nilsson, Peter
    Odling, Jenny
    Oksvold, Per
    Olsson, Ingmarie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Oster, Emma
    Ottosson, Jenny
    Paavilainen, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Persson, Anja
    Rimini, Rebecca
    Rockberg, Johan
    Runeson, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sivertsson, Asa
    Sköllermo, Anna
    Steen, Johanna
    Stenvall, Maria
    Sterky, Fredrik
    Strömberg, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sundberg, Mårten
    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden.
    Tegel, Hanna
    Tourle, Samuel
    Wahlund, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Waldén, Annelie
    Wan, Jinghong
    Wernéus, Henrik
    Westberg, Joakim
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wrethagen, Ulla
    Xu, Lan Lan
    Hober, Sophia
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    A human protein atlas for normal and cancer tissues based on antibody proteomics2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 12, p. 1920-1932Article in journal (Refereed)
    Abstract [en]

    Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, ∼400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

  • 35.
    Wei, Qichun
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Chen, Lirong
    Sheng, Liming
    Nordgren, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    EGFR, HER2 and HER3 expression in esophageal primary tumours and corresponding metastases2007In: International Journal of Oncology, ISSN 1019-6439, Vol. 31, no 3, p. 493-499Article in journal (Refereed)
    Abstract [en]

    The expression of EGFR, HER2 and HER3 receptors were analyzed in immunohistochemical preparations from primary esophageal tumours and corresponding lymph node metastases. The goal was to evaluate whether any of these receptors are suitable as targets for radionuclide based imaging and therapy. The receptor expressions were evaluated in parallel samples, primary tumour and metastasiis, from each patient (n = 51). The majority of the cases were esophageal squamous cell carcinomas, ESCC, (n = 40). The HercepTest scoring was used for the analysis of both HER2 and EGFR expression (0, 1+, 2+ or 3+). HER3 was only evaluated as negative, weak or strong staining. EGFR overexpression (2+/3+) was found in 67.5 % (27/40) of both the ESCC primary tumours and the corresponding lymph node metastases. There were only a few changes in these EGFR-scores;: two cases from 2+/3+ to 0/1+ when the primary tumours were compared to the corresponding metastases and two changes the other way around. HER2 overexpression (2+/3+) was only found only in 3three of the primary ESCC tumours and 2two of the lymph node metastases. EGFR and HER2 stainings were found mainly in the cell membranes. The HER3 staining (weak or strong) was mainly cytoplasmic and granular and was observed in about half (20/39) of the cases, for both the ESCC primary tumours and the corresponding lymph node metastases. It is was concluded that ESCC lymph node metastases generally have a strong expression of EGFR stronglyin their cell membranes and to the same extent as in the primary tumours. The stability in EGFR expression is encouraging for efforts to develop radionuclide based EGFR imaging agents. It is also possible that EGFR targeting agents (e.g. Iressa, Tarceva, Erbitux or radiolabelled antibodies) can be applied for therapy of ESCC.

  • 36.
    Wester, Kenneth
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Andersson, Ann-Catrin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ranefall, Petter
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
    Bengtsson, Evert
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Busch, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Cultured human fibroblasts in agarose gel as a multi-functional control for immunohistochemistry: Standardization Of Ki67 (MIB1) assessment in routinely processed urinary bladder carcinoma tissue2000In: Journal of Pathology, ISSN 0022-3417, E-ISSN 1096-9896, Vol. 190, no 4, p. 503-11Article in journal (Refereed)
    Abstract [en]

    Immunohistochemistry (IHC) in clinical practice is hampered by lack of standardization and by subjectivity in interpretation and quantitation. This study aimed to develop a control system for IHC in routinely fixed and histoprocessed tissues. Such a system should be easy to handle in clinical practice and should reflect variations in fixation time, section thickness, section storage conditions, and staining protocols. In addition, in image analysis quantitation of immunostained tissues, when using classifiers computed on IHC-control images, the control system should be very stable. Cultured human fibroblasts were suspended in agarose, transferred into a length of tubing and stored at 4 degrees C. Three pieces of the cellgel control were separately fixed, histoprocessed, and paraffin-embedded as external controls. One piece was prepared together with each of 18 bladder carcinoma biopsies as internal controls. Slides with sections from the biopsy and all types of cellgel controls were stored at different temperatures and then stained using three different IHC protocols. The fibroblasts were homogeneously distributed in the agarose gel. Variation in section thickness did not influence immunostaining as evaluated by the MIB1 labelling index (MIB1 LI). The external controls decreased notably in MIB1 LI with increased fixation time. This was not seen in the 18 internal controls that were each fixed with a fresh biopsy. However, section storage and immunostaining conditions influenced the MIB1 expression equally in all control types and to a similar degree to the biopsies. Furthermore, colour-based image analysis quantitation of MIB1 LI in biopsies proved stable and independent of the control type used to compute the classifier.

  • 37.
    Wester, Kenneth
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Asplund, Anna
    Bäckvall, Helena
    Micke, Patrick
    Ludwiginstitutet för Cancerforskning.
    Derveniece, Andra
    Hartmane, Ilona
    Malmström, Per-Uno
    Pontén, Fredrik
    Zinc-based fixative improves preservation of genomic DNA and proteins in histoprocessing of human tissues.2003In: Lab Invest, ISSN 0023-6837, Vol. 83, no 6, p. 889-99Article in journal (Refereed)
    Abstract [en]

    Advantageous preservation of histology and detailed cellular morphology has rendered neutral buffered formalin (NBF) the most widely used fixative in clinical pathology. Despite excellent morphology for routine diagnostics, a major drawback of NBF fixation is its detrimental effect on DNA and RNA quality. In addition to complicating analysis of genes and transcripts in complex tissues, NBF denatures proteins and thereby hampers immunohistochemical visualization of certain antigens. In the present study, we evaluated a zinc-based fixative (ZBF) regarding its effects on tissue morphology, quality of genomic DNA, and preservation of protein immunoreactivity in a broad spectrum of tissues. Four different modes of fixation were analyzed: ZBF-paraffin embedding, NBF-paraffin embedding, ZBF-fixation prior to snap-freezing, and immediate snap-freezing. Laser-assisted microdissection, allowing retrieval of a defined number of cells for PCR, was used to study DNA quality. Genomic DNA was analyzed using primers for beta2-microglobulin and the transferrin receptor. Immunohistochemistry was performed using nine antibodies. Tissue microarray blocks were used for analysis of morphology and immunoreactivity. Only slight impairment of morphologic qualities was found after ZBF-paraffin embedding, whereas ZBF prior to freezing resulted in a more crisp morphology compared with routine cryosections. A significantly higher DNA yield was observed in samples isolated from ZBF-paraffin-embedded tissues compared with NBF-paraffin-embedded tissues. Both yield and quality of DNA was comparable in frozen tissues irrespective to prior ZBF fixation. Immunoreactivity in paraffin-embedded tissue was superior in ZBF-fixated tissue compared with NBF-fixated for a majority of tested antibodies. Furthermore, for seven out of nine antibodies, antigen retrieval pretreatment proved unnecessary in ZBF-fixated tissue. Thus, despite a slight impairment of morphology, ZBF preserves protein structures well. We conclude that ZBF is superior to NBF for analysis of DNA and protein expression. Fixation of tissues in ZBF may also be an alternative strategy to freeze storage of tissue specimens, eg, in future bio-banks.

  • 38.
    Wester, Kenneth
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    Sjöström, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    de la Torre, Manuel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    HER-2: A possible target for therapy of metastatic urinary bladder carcinoma2002In: Acta Oncologica, ISSN 0284-186X, E-ISSN 1651-226X, Vol. 41, no 3, p. 282-288Article in journal (Refereed)
    Abstract [en]

    Human epidermal growth factor receptor 2, HER-2, is overexpressed in various tumours, e.g. breast- and bladder tumours. The aim of this study was to predict the potential use of HER-2 receptors as targets in systemic treatment of disseminated bladder tumours. HER-2 expression was assessed in bladder carcinoma metastases and the corresponding primary tumours, and subsequently compared with the EGFR expression. HER-2 and EGFR expression was analysed by immunohistochemistry in formalin-fixed, paraffin-embedded tissues from 21 patients with metastatic bladder carcinoma. HER-2 was overexpressed in 81% of the primary tumours and in 67% of the metastases. All HER-2-positive metastases were from HER-2-positive primary tumours. The results for EG FR were 71% of both primary and metastases-positive tumours. In 90% of the primary tumours and 86% of the metastases, at least one of the receptors was overexpressed. These results suggest that HER-2 targeted therapy can be considered as an alternative or a complement to other modalities in the treatment of metastatic urinary bladder carcinoma.

1 - 38 of 38
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