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  • 1. Ahrén-Moonga, Jennie
    et al.
    Lekander, Mats
    von Blixen, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Rönnelid, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Holmgren, Sven
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    af Klinteberg, Britt
    Levels of Tumour Necrosis Factor-Alpha and Interleukin-6 in Severely Ill Patients with Eating Disorders2011In: Neuropsychobiology, ISSN 0302-282X, E-ISSN 1423-0224, Vol. 63, no 1, p. 8-14Article in journal (Refereed)
    Abstract [en]

    Background: The underlying pathophysiology of eating disorders (ED) is dependent on complex interactions between psychological, biological and social factors. The purpose of the present study was to examine a possible increase in cytokines indicating inflammation, as measured by tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in ED patients, and to explore possible relationships between cytokines and self-reported personality traits. Methods: Female patients with severe ED (n = 26) were recruited consecutively from an inpatient clinic and were compared to age-matched healthy females (n = 12). Commercial ELISA tests developed for the measurement of serum levels of TNF-alpha and IL-6 were employed. Personality traits were measured using Karolinska Scales of Personality. Results: The patient group displayed increased levels of the cytokine TNF-alpha and a tendency towards increased IL-6 levels. Spearman's rank correlation coefficient was used to examine possible relationships between levels of cytokines and personality traits. The results showed that IL-6 levels were positively related to both somatic and psychic anxiety and to aggression scales, such as irritability and suspicion. Increased levels of TNF-alpha, in turn, were significantly correlated with high scores on the depression-related anxiety scale Inhibition of Aggression. However, increased levels of cytokines in the ED group did not seem to be mainly associated with symptoms of depression. Conclusion: We cannot rule out the possibility that comorbid conditions in the group contribute to the higher cytokine values. Further studies need to explore the possible influence of cytokines on the severity of ED and whether this might be mediated or moderated by specific personality traits.

  • 2. Amara, Umme
    et al.
    Flierl, Michael A.
    Rittirsch, Daniel
    Klos, Andreas
    Chen, Hui
    Acker, Barbara
    Brueckner, Uwe B.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Gebhard, Florian
    Lambris, John D.
    Huber-Lang, Markus
    Molecular Intercommunication between the Complement and Coagulation Systems2010In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 185, no 9, p. 5628-5636Article in journal (Refereed)
    Abstract [en]

    The complement system as well as the coagulation system has fundamental clinical implications in the context of life-threatening tissue injury and inflammation. Associations between both cascades have been proposed, but the precise molecular mechanisms remain unknown. The current study reports multiple links for various factors of the coagulation and fibrinolysis cascades with the central complement components C3 and C5 in vitro and ex vivo. Thrombin, human coagulation factors (F) XIa, Xa, and IXa, and plasmin were all found to effectively cleave C3 and C5. Mass spectrometric analyses identified the cleavage products as C3a and C5a, displaying identical molecular weights as the native anaphylatoxins C3a and C5a. Cleavage products also exhibited robust chemoattraction of human mast cells and neutrophils, respectively. Enzymatic activity for C3 cleavage by the investigated clotting and fibrinolysis factors is defined in the following order: FXa > plasmin > thrombin > FIXa > FXIa > control. Furthermore, FXa-induced cleavage of C3 was significantly suppressed in the presence of the selective FXa inhibitors fondaparinux and enoxaparin in a concentration-dependent manner. Addition of FXa to human serum or plasma activated complement ex vivo, represented by the generation of C3a, C5a, and the terminal complement complex, and decreased complement hemolytic serum activity that defines exact serum concentration that results in complement-mediated lysis of 50% of sensitized sheep erythrocytes. Furthermore, in plasma from patients with multiple injuries (n = 12), a very early appearance and correlation of coagulation (thrombin-antithrombin complexes) and the complement activation product C5a was found. The present data suggest that coagulation/fibrinolysis proteases may act as natural C3 and C5 convertases, generating biologically active anaphylatoxins, linking both cascades via multiple direct interactions in terms of a complex serine protease system. The Journal of Immunology, 2010, 185: 5628-5636.

  • 3.
    Andersson, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Sanchez, Javier
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson Ekdahl, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Elgue, Graciela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Optimal heparin surface concentration and antithrombin binding capacity as evaluated with human non-anticoagulated blood in vitro2003In: Journal of Biomedical Materials Research, ISSN 0021-9304, E-ISSN 1097-4636, Vol. 67, no 2, p. 458-466Article in journal (Refereed)
    Abstract [en]

    Contact between blood and a biomaterial surface takes place in many applications and is known to activate the coagulation and complement systems. Heparin surface coatings have been shown to reduce blood activation upon contact with artificial surfaces. To establish the optimal heparin surface concentration, blood was incubated in a tubing loop model at 37 degrees C. The tubing was coated with different surface concentrations of heparin and rotated at three different velocities. We demonstrate that the blood compatibility of a surface with regard to coagulation, complement, and platelet activation can be improved by increasing the heparin surface concentration in the 6-12 pmol antithrombin/cm2 concentration interval. The binding of factor H is not influenced by the increased heparin surface concentration, suggesting that this factor is not the primary regulator of complement on heparin surfaces. In addition, the heparin coating has no effect on the complement activation that occurs on gas surfaces in extracorporeal circuits.

  • 4.
    Ardesjö Lundgren, Brita
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Rorsman, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Portela-Gomes, Guida M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Grimelius, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ekdahl, Kristina Nilsson
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Ekwall, Olov
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Identification of complement C3 as an autoantigen in inflammatory bowel disease2010In: European Journal of Gastroenterology and Hepathology, ISSN 0954-691X, E-ISSN 1473-5687, Vol. 22, no 4, p. 429-436Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: Autoantibodies against goblet cells in the gastrointestinal mucosa have been described in patients with inflammatory bowel disease (IBD) but a corresponding autoantigen has not yet been identified. The aim of this study was to identify such an antigen. METHODS: First, 10 candidate autoantigens were discarded based on double stainings of appendiceal sections and a mucin-producing cell line (HT29-mtx). Second, an appendiceal cDNA library was immunoscreened with IBD sera. RESULTS: Three out of 48 positive clones were identified as complement C3. Using immunoprecipitation of in vitro transcribed and translated C3, seven of 17 primary sclerosing cholangitis patient sera, 15 of 65 IBD sera, and none out of 54 sera from healthy blood donors showed C3 immunoreactivity. The results were confirmed using western blot and an enzyme-linked immunosorbent assay with alternative sources of C3 protein. CONCLUSION: In conclusion, we have identified complement C3 as a potential autoantigen in IBD and primary sclerosing cholangitis.

  • 5.
    Babiker, Adil A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Hamad, Osama A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Sanchez, Javier
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Ronquist, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson Ekdahl, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Prothrombotic effect of prostasomes of metastatic cell and seminal origin2007In: The Prostate, ISSN 0270-4137, E-ISSN 1097-0045, Vol. 67, no 4, p. 378-388Article in journal (Refereed)
    Abstract [en]

    BACKGROUND. Prostasomes are secretory granules produced by the glandular epithelial cells of the prostate. Seminal prostasomes contain high amounts of Tissue Factor (TF) but no studies of TF on malignant cell prostasomes have been made. Here we compare the expression, phosphorylation, and function of TF on prostasomes of different origin. METHODS. TF was detected on prostasomes isolated from seminal fluid and human prostate cancer cell lines (PC-3, DU145, and LNCaP) using FACS and enzyme immunoassay (EIA). Incubation of prostasomes with radioactive ATP under conditions favoring protein kinase A activity led to phosphorylation of TF as detected by immunoprecipitation and SDS-PAGE. The prothrombotic effect of prostasomes was investigated in whole blood and recalcified plasma. Blocking experiments were performed using anti-TF antibodies and corn trypsin inhibitor. RESULTS. TF was expressed on all tested prostasome preparations with lowest values found for seminal ones. Prostasomal TF was the main endogenous substrate for prostasomal protein kinase A. All tested prostasome preparations greatly enhanced the rate of clot formation in a dose-dependent fashion, that is, the clotting capability of prostasomes seemed to be related to the extent of their expression of TF. In addition, the density of the clot varied between different prostasome preparations. When incubated in whole blood, prostasomes were found to associate to WBC thereby inducing them to express and release TF. CONCLUSIONS. These data show that TF is overexpressed and also subjected to phosphorylation by malignant cell prostasomes. This suggests major roles for prostasomes in thrombotic events that occur in some advanced cases of prostate cancer.

  • 6.
    Babiker, Adil A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Magnusson, Peetra U.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Ronquist, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Ekdahl, Kristina Nilsson
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Mapping pro- and antiangiogenic factors on the surface of prostasomes of normal and malignant cell origin2010In: The Prostate, ISSN 0270-4137, E-ISSN 1097-0045, Vol. 70, no 8, p. 834-847Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Angiogenesis is the formation of new blood vessels by capillary sprouting from pre-existing vessels. Tumor growth is angiogenesis-dependent and the formation of new blood vessels is associated with the increased expression of angiogenic factors. Prostasomes are secretory granules produced, stored and released by the glandular epithelial cells of the prostate. We investigated the expression of selected angiogenic and anti-angiogenic factors on the surface of prostasomes of different origins as well as the direct effect of prostasomes on angiogenesis. METHODS: VEGF, endothelin-1, endostatin, and thrombospondin-1 were determined on prostasomes from seminal fluid and human prostate cancer cell lines (DU145,PC-3,LNCaP) using different immunochemical techniques. Human dermal microvascular endothelial cells were incubated with seminal and DU145 cell-prostasomes and with radioactive thymidine. The effect of prostasomes on angiogenesis was judged by measuring the uptake of labeled thymidine. The presence of any deleterious effects of prostasomes on the endothelial cells was investigated using thymidine assay and confocal laser microscopy. RESULTS: VEGF and endothelin-1 were determined on malignant cell-prostasomes (no difference between cell lines) but not determined on seminal prostasomes. The same applies for the expression of endostatin but with much higher expression on malignant cell-prostasomes with obvious differences between them. Seminal and DU145 cell-prostasomes were found to have anti-angiogenic effect which was more expressed by DU145 cell-prostasomes. No deleterious effect of prostasomes on endothelial function was detected using either thymidine assay or microscopy. CONCLUSIONS: Prostasomes contain pro- and anti-angiogenic factors that function to counteract each other unless the impact from one side exceeds the other to bring about dysequilibrium.

  • 7.
    Babiker, Adil A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Ronquist, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Carlsson, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Nilsson Ekdahl, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Transfer of functional prostasomal CD59 of metastatic prostatic cancer cell origin protects cells against complement attck2005In: The Prostate, ISSN 0270-4137, E-ISSN 1097-0045, Vol. 62, no 2, p. 105-114Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Prostasomes are secretory granules produced, stored, and released, by the glandular epithelial cells of the prostate. They express the glycosylphosphatidylinositol (GPI)-anchored complement regulatory protein CD59, which has been shown to be transferred to spermatozoa and erythrocytes.

    METHODS: The CD59 content of prostasomes isolated from seminal fluid and malignant prostate cells (PC-3, DU145, and LNCaP) and the transfer of prostasomal CD59 to rabbit erythrocytes (RE) and to PIPLC-treated and unmanipulated cancer cells were investigated using FACS. All prostasomes were also incubated with RE and tested in a hemolytic assay.

    RESULTS: Prostasomes from cancer cells had higher expression of CD59 than those of normal cells. Prostasomal CD59 of different origin could be transferred to RE, malignant cell lines stripped of CD59 by PIPLC, or unmanipulated LNCaP cells. Malignant cell prostasomes had an increased ability to inhibit complement-mediated lysis compared to those from non-malignant cells.

    CONCLUSIONS: These results point to a novel mechanism by which prostasomes can protect prostatic malignant cells from complement attack.

  • 8.
    Babiker, Adil A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Ronquist, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson Ekdahl, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Prostasome Involvement in the Development and Growth of Prostate Cancer2010In: The Open Prostate Cancer Journal, ISSN 1876-8229, Vol. 3, p. 1-13Article, review/survey (Refereed)
    Abstract [en]

    Prostasomes are extracellularly occurring submicron, membrane-surrounded organelles produced by the epithelial cells of the prostate and present in semen after secretion. Even dedifferentiated prostate cancer cells have preserved their ability to produce and export prostasomes to the extracellular space. The precise physiological role of prostasomes is not known, although some of their properties assign them to important physiological and patho-physiological functions that could be exploited in prostate cancer growth and development. In this review, some new properties of seminal and malignant cell line (DU145, PC-3 and LNCaP) prostasomes will be discussed.

    There are typical differences in the expressions and activities of prostasomal CD59, ATPase, protein kinases and tissue factor (TF) as well as in the transfer of prostasomal CD59 to CD59-deficient erythrocytes (rabbit and human PNH erythrocytes). CD59, protein kinases and TF exhibit characteristic patterns of overexpression by malignant cell prostasomes. A high ATPase activity is recognized on seminal prostasomes with minimal activity on malignant cell prostasomes resulting in more residual ATP available for phosphorylation reactions. Several proteins are phosphorylated by prostasomal protein kinases, namely, complement component C3, fibrinogen, vitronectin and E-cadherin. Furthermore, TF is identified as the main endogenous phosphorylation substrate on prostasomes. In addition, prothrombotic effects of prostasomes are demonstrated. DU145 and PC-3 cell-derived prostasomes exert a higher clotting effect on whole blood and plasma compared to LNCaP cell-derived and seminal prostasomes.

    In conclusion, malignant cell prostasomes show an increased ability to interact with the biological system in favor of prostate cancer cell promotion and survival. The roles played by prostasomes in this context may improve the understanding of the mechanisms that help the prostate cancer cells to avoid the complement attack (CD59 transfer and phosphorylation and inactivation of C3), to promote angiogenesis (TF) and to metastasize. It may also provide a better understanding of some of the complications usually seen in some terminal prostate cancer patients like thrombotic events and tendency to develop disseminated intravascular coagulation.

  • 9.
    Bensing, Sophie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Brandt, Lena
    Tabaroj, Farnoush
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Sjöberg, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology and Transfusion Medicine.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Ekbom, Anders
    Blomqvist, Paul
    Kämpe, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Increased death risk and altered cancer incidence pattern in patients with isolated or combined autoimmune primary adrenocortical insufficiency2008In: Clinical Endocrinology, ISSN 0300-0664, E-ISSN 1365-2265, Vol. 69, no 5, p. 697-704Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: Primary adrenocortical insufficiency is mostly caused by an autoimmune destruction of the adrenal cortex. The disease may appear isolated or as a part of an autoimmune polyendocrine syndrome (APS). APS1 is a rare hereditary disorder with a broad spectrum of clinical manifestations. In APS2, primary adrenocortical insufficiency is often combined with autoimmune thyroid disease and/or type 1 diabetes. We analysed mortality and cancer incidence in primary adrenocortical insufficiency patients during 40 years. Data were compared with the general Swedish population. DESIGN AND PATIENTS: A population based cohort study including all patients with autoimmune primary adrenocortical insufficiency (3299) admitted to Swedish hospitals 1964-2004. MEASUREMENTS: Mortality risk was calculated as the standardized mortality ratio (SMR) and cancer incidence as the standardized incidence ratio (SIR). RESULTS: A more than 2-fold increased mortality risk was observed in both women (SMR 2.9, 95% CI 2.7-3.0) and men (SMR 2.5, 95% CI 2.3-2.7). Highest risks were observed in patients diagnosed in childhood. SMR was higher in APS1 patients (SMR 4.6, 95% CI 3.5-6.0) compared with patients with APS2 (SMR 2.1, 95% CI 1.9-2.4). Cancer incidence was increased (SIR 1.3, 95% CI 1.2-1.5). When tumours observed during the first year of follow-up were excluded, only the cancer risk among APS1 patients remained increased. Cause-specific cancer incidence analysis revealed significantly higher incidences of oral cancer, nonmelanoma skin cancer, and male genital system cancer among patients. Breast cancer incidence was lower than in the general population. CONCLUSIONS: Our study shows a reduced life expectancy and altered cancer incidence pattern in patients with autoimmune primary adrenocortical insufficiency.

  • 10.
    Berg, Anna-Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Frisk, Gun
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Induction of the chemokine interferon-gamma-inducible protein-10 in human pancreatic islets during enterovirus infection2006In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 49, no 11, p. 2697-2703Article in journal (Refereed)
    Abstract [en]

    Aims/hypothesis: Enterovirus infections have long been suspected to be environmental factors that may cause type 1 diabetes, but the pathways leading from infection to beta cell destruction are still unknown. We therefore examined whether enterovirus infection of human islets leads to upregulation of interferon-gamma-inducible protein (IP-10, now known as chemokine [C-X-C motif] ligand 10 [CXCL10]), a chemokine important for the induction of insulitis. Methods: Isolated human islets were infected with three different strains of Coxsackie B4 virus. IP-10 expression and secretion from the infected human islets were then measured using RT-PCR and ELISA at several time points. Results: IP-10 was clearly upregulated in and secreted from human islets during enterovirus infection. This was demonstrated with three different strains of Coxsackie B4 virus, two of which are lytic to islets and one which is non-lytic and can establish a persistent infection in human islets. Conclusions/interpretation: We propose that enterovirus-induced upregulation of IP-10 during infection of the islets in vivo is the first step towards destructive insulitis. Our findings support the idea that enterovirus infection triggers immune-mediated beta cell destruction, and for the first time suggest a possible mechanism behind enterovirus-induced diabetes.

  • 11.
    Bersztel, Adam
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
    Lorant, Tomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
    Björkland, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Johnsson, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
    Tufveson, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
    Antibody responses to xenogenic antigens: a study in the mouse-to-rat system2006In: Tissue Antigens, ISSN 0001-2815, E-ISSN 1399-0039, Vol. 68, no 6, p. 483-488Article in journal (Refereed)
    Abstract [en]

    Antibodies play a crucial role in the rejection of an organ that has been transplanted between different animal species, i.e. xenotransplantation. In previous work, we have induced a state of humoral tolerance where mouse-to-rat heart grafts continued to beat under ciclosporine A monotherapy. Initially, a combined treatment with ciclosporine A and 15-deoxyspergualin was given. This state of tolerance could not be reproduced when the vascularised heart graft was replaced with a free tissue graft or xenogeneic blood transfusions. To gain further insight into the humoral response against mouse antigens, we studied the antibody production in naive rats and rats challenged with heart transplants, heart cells, mononuclear cells (MNC) and erythrocytes from mice. Rats not challenged with any mouse cells or organs had a moderate amount of antibodies targeted against mouse MNC as well as rosette-forming cells in the spleen targeted against mouse erythrocytes. A challenge with either mouse MNC or erythrocytes lead to immunisation with antibodies of both IgM and IgG subtype directed against both MNC and erythrocytes. Antibody titres against mouse erythrocytes in animals challenged with MNC were not detectable until day 7, whereas antibody titres against mouse MNC in animals challenged with erythrocytes were detected on day 1. Immunisation with mouse erythrocytes raised the titre of rosette-forming cells in the spleen compared with naive rats (P < 0.05). Our data indicate that different xenogeneic antigens in the mouse-to-rat system are shared between heart cells, MNC and erythrocytes; however, the immunisation patterns differ regarding the time when antibodies are first detected.

  • 12. Bexborn, Fredrik
    et al.
    Andersson, Per Ola
    Chen, Hui
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Ekdahl, Kristina N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    The tick-over theory revisited: formation and regulation of the soluble alternative complement C3 convertase (C3(H2O)Bb)2008In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 45, no 8, p. 2370-2379Article in journal (Refereed)
    Abstract [en]

    The molecular interactions between the components of the C3 convertase of the alternative pathway (AP) of complement and its regulators, in both surface-bound and fluid-phase form, are still incompletely understood. The fact that the AP convertase is labile makes studies difficult to perform. According to the so called tick-over theory, hydrolyzed C3, called C3(H(2)O), forms the initial convertase in fluid phase together with factor B. In the present study, we have applied western blot analysis and ELISA together with fluorescence resonance energy transfer (FRET) to study the formation of the fluid-phase AP convertases C3(H(2)O)Bb and C3bBb and their regulation by factor H and factor I at specific time points and, with FRET, in real time. In our hands, factor B showed a higher affinity for C3(H(2)O) than for C3b, although in both cases it was readily activated to Bb. However, the convertase activity of C3bBb was approximately twice that of C3(H(2)O)Bb, as monitored by the generation of C3a. But in contrast, the C3(H(2)O)Bb convertase was more resistant to inactivation by factor H and factor I than was the C3bBb convertase. Under conditions that totally inactivated C3bBb, C3(H(2)O)Bb still retained approximately 25% of its initial activity.

  • 13. Bexborn, Fredrik
    et al.
    Engberg, Anna E.
    Sandholm, Kerstin
    Mollnes, Tom Eirik
    Hong, Jaan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson Ekdahl, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Hirudin versus heparin for use in whole blood in vitro biocompatibility models2009In: Journal of Biomedical Materials Research. Part A, ISSN 1549-3296, Vol. 89A, no 4, p. 951-959Article in journal (Refereed)
    Abstract [en]

    Heparin has traditionally been a widely used anticoagulant in blood research, but has been shown to be inappropriate for work with the complement system because of its complement-interacting properties. In this work, we have compared the effects of heparin with those of the specific thrombin inhibitor hirudin on complement and blood cells in vitro. Whole blood collected in the presence of hirudin (50 microg/mL) or heparin (1 IU/mL) was incubated in the slide chamber model. The plasma was analyzed for complement activation markers C3a and sC5b-9, and the polyvinylchloride test slides were stained for adhering cells. The integrity of the complement system was tested by incubating serum and hirudin-treated plasma in the presence of various activating agents. In contrast to heparin, the addition of hirudin generally preserved the complement reactivity, and complement activation in hirudin plasma closely resembled that in normal serum. Importantly, immunochemical staining of surface-bound cells demonstrated the inducible expression of tissue factor on bound monocytes from hirudin-treated blood, an effect that was completely abolished in heparin-treated blood. Our results indicate that hirudin as an anticoagulant produces more physiological conditions than heparin, making hirudin well-suited for in vitro studies, especially those addressing the regulation of cellular processes.

  • 14.
    Brandhorst, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Kumarasamy, Vidya
    Maatoui, Adel
    Alt, Alexandra
    Bretzel, Reinhard G.
    Brandhorst, Heide
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Porcine islet graft function is affected by pretreatment with a caspase-3 inhibitor2006In: Cell Transplantation, ISSN 0963-6897, E-ISSN 1555-3892, Vol. 15, no 4, p. 311-317Article in journal (Refereed)
    Abstract [en]

    During the isolation procedure and after transplantation islets are subjected to numerous variables associated with the induction of apoptosis. The present study investigated the effect of transient pretreatment with caspase inhibitors on function and survival of transplanted pig islets. Isolated porcine islets (3000 IEQ) were incubated overnight in 200 mu M of the caspase-3 inhibitor DEVD-CMK prior to transplantation into diabetic nude mice. Glucose-stimulated insulin release of pretreated islets was assessed during static incubation. DEVD-CMK successfully prevented the expression of capase-3 and DFF as demonstrated in heat-shocked pig islets. Nevertheless, transient pretreatment of freshly isolated pig islets with DEVD-CMK resulted in a significantly decreased final graft function of 50.0% (n = 16) compared to 85.7% (n = 14) in control islets (p < 0.05). Glucose-stimulated insulin release of porcine islets (n = 6) was not significantly effected by overnight culture with DEVD-CMK. Morphological assessment revealed that this caspase-3 inhibitor significantly increased the percentage of necrosis to a small, but nevertheless significant, extent in comparison to control islets (p < 0.05). The study demonstrates that short-time pretreatment with the caspase-3 inhibitor DEVD-CMK reduces the capacity of transplanted porcine islets to restore normoglycemia in diabetic nude mice.

  • 15.
    Brandhorst, Heide
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Asif, Sana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Andersson, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Moench, Johanna
    Friedrich, Olaf
    Raemsch-Guenther, Nicole
    Raemsch, Christian
    Steffens, Melanie
    Lambrecht, Joerg
    Schraeder, Thomas
    Kurfuerst, Manfred
    Andersson, Helene H.
    Felldin, Marie
    Foss, Aksel
    Salmela, Kaija
    Tibell, Annika
    Tufveson, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Brandhorst, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    The Effect of Truncated Collagenase Class I Isomers on Human Islet Isolation Outcome2010In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 90, no 3, p. 334-335Article in journal (Refereed)
  • 16.
    Brandhorst, Heide
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Friberg, Andrew
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Andersson, Helena H.
    Felldin, Maria
    Foss, Aksel
    Salmela, Kaija
    Lundgren, Torbjörn
    Tibell, Annika
    Tufveson, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Brandhorst, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation2009In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 87, no 3, p. 370-5Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The isolation of islets from the human pancreas critically depends on an efficient enzyme blend. Previous studies have solely focused on the presence of collagenase and neutral protease/thermolysin. Despite improved characterization of these components, the lot-related variability in efficacy still persists suggesting that additional so far disregarded enzymes are required for efficient islet cleavage. METHODS: Varying activities of a tryptic-like enzyme were identified within collagenase NB1 lots, which were selected according to a matched ratio between tryptic-like and collagenase activity (TLA-ratio). Rat and human pancreata were processed with current standard procedures. RESULTS: Increasing the TLA-ratio from 1.3% to 10% reduced pancreas dissociation time in rats by 50% without affecting islet yield, viability, or posttransplant function in diabetic nude mice. Enhancing the TLA-ratio from 1.3% to 12.6% for human pancreas processing resulted in a significant reduction of recirculation time and increased incrementally human islet yield without affecting purity, in vitro function or recovery after culture. Optimized pancreas digestion correlated with a higher percentage of islet preparations fulfilling quality criteria for clinical transplantation. CONCLUSIONS: We conclude that TLA is an effective component that should be included in moderate amounts in enzyme blends for human islet isolation to optimize the efficiency and minimize the lot-related variability.

  • 17.
    Brandhorst, Heide
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Friberg, Andrew
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Andersson, H. H.
    Felldin, M.
    Foss, A.
    Salmela, K.
    Tibell, A.
    Tufveson, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Brandhorst, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Large-Scale Comparison of Liberase HI and Collagenase NB1 Utilized for Human Islet Isolation2010In: Cell Transplantation, ISSN 0963-6897, E-ISSN 1555-3892, Vol. 19, no 1, p. 3-8Article in journal (Refereed)
    Abstract [en]

    For more than a decade Liberase HI was commonly used as the standard enzyme blend for clinical human islet isolation until enforced replacement by collagenase NB1 (NB1). This change resulted initially in a reduction in islet isolation outcome and transplant activities worldwide. This retrospective study was initiated to compare the efficiency of NB1 premium grade with Liberase in 197 human islet isolations. All pancreata were processed between January 2006 and June 2008 utilizing the same procedures for isolation and quality assessment except the administration of preselected lots of either Liberase (n = 101) or NB1 (n = 96). Utilizing Liberase significantly more digested tissue and purified islet yield was produced compared to NB1. In contrast, the use of NB1 was associated with significantly higher purity and glucose stimulation index during dynamic perifusion. The expression of proinflammatory markers was almost identical except tissue factor expression that was higher after utilization of Liberase. No difference was found in the percentage of pancreata fulfilling the criteria for clinical islet transplantation. The results suggest that Liberase is more efficient for pancreas dissociation than collagenase NB1 but seems to be more harmful to exocrine cells and islet tissue.

  • 18.
    Brandhorst, Heide
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Muehling, B
    Yamaya, H
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Henriksnäs, Johanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Carlsson, Per-Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Brandhorst, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    New class of oxygen carriers improves islet isolation from long-term stored rat pancreata2008In: Transplantation Proceedings, ISSN 0041-1345, E-ISSN 1873-2623, Vol. 40, no 2, p. 393-394Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: Pancreas shipment is frequently associated with prolonged ischemia deteriorating islet graft function. The strategy to prevent ischemic damage utilizing perfluorodecalin (PFD) for human pancreas oxygenation does not seem to improve isolation outcome. The present study investigated the efficiency of perfluorohexyloctane (F6H8), a hyperoxygen carrier characterized by low specific density (1.33 g/cm3) and lipophilic qualities, to facilitate islet isolation from long-term stored rat pancreata. MATERIALS AND METHODS: Prior to islet isolation, pancreata were intraductally flushed in situ with Kyoto solution (KS) and stored for 24 hours in KS, oxygenated PFD, or F6H8. RESULTS: Islet isolation performed after 24-hour storage in KS failed completely. The intrapancreatic pO2 in PFD- and F6H8-incubated pancreata was almost the same. In correspondence, the ATP content and viability of isolated islets were similar as well. In contrast, islet yield and in vitro function were significantly reduced after storage in PFD compared with F6H8. CONCLUSION: This study suggested that islet isolation performed after long-term pancreas preservation can be significantly improved utilizing semifluorinated alkanes as oxygen carriers.

  • 19.
    Brandhorst, Heide
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Raemsch-Guenther, N.
    Raemsch, C.
    Friedrich, O.
    Kurfuerst, M.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Brandhorst, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Degraded collagenase deteriorates islet viability2008In: Transplantation Proceedings, ISSN 0041-1345, E-ISSN 1873-2623, Vol. 40, no 2, p. 370-371Article in journal (Refereed)
    Abstract [en]

    Objective. The utilization of purified enzyme blends consisting of collagenase class I (CI) and II (CII) and neutral protease is an essential step for clinical islet isolation. Previous studies suggested that the use of enzyme lots containing degraded CI reduced islet release from human pancreata. The present study sought to assess the effect of degraded collagenase on islet function in vitro and posttransplantation. Materials and Methods. Crude collagenase was chromatographically separated into CI, CII, and a mixture of degraded CI and CII isomers. Subsequently, classes were recombined to obtain a CII/CI ratio of 0.5. Rat islets were isolated utilizing neutral protease and 20 units of recombined collagenase containing either intact (Ci) or degraded isomers (Cd). Results. Digestion time was reduced utilizing Cd (P < .001). The highest islet yield and lowest islet fragmentation were obtained with Ci (P < .01). Utilization of Cd corresponded to a reduction in viability and in vitro function (NS). Islet transplantation reversed hyperglycemia in diabetic nude mice, but revealed an absence of weight gain in recipients receiving islets isolated using Cd (P < .01). Conclusion. This study suggested that islet function posttransplantation is affected by degraded collagenase isomers. This finding has to be considered for the purification process of collagenase.

  • 20.
    Brandhorst, Heide
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Raemsch-Guenther, Nicole
    Raemsch, Christian
    Friedrich, Olaf
    Huettler, Silke
    Kurfuerst, Manfred
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Brandhorst, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    The ratio between collagenase class I and class II influences the efficient islet release from the rat pancreas2008In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 85, no 3, p. 456-61Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Previous studies indicated different roles of collagenase class I, class II and neutral protease in the enzymatic islet release from pancreatic tissue. Because no information has been available, this study was aimed to investigate the isolation efficiency of different ratios between collagenase class II and I (C-ratio) in the rat pancreas serving as model for the human pancreas without being restricted by the large variability observed in human donors. METHODS: Rat pancreata were digested using a marginal neutral protease activity and 20 PZ-U of purified collagenase classes recombined to create a C-ratio of 0.5, 1.0, or 1.5. Collagenase efficiency was evaluated in terms of isolation outcome and posttransplantation function in diabetic nude mice. RESULTS: The highest yield of freshly isolated islets was obtained using a C-ratio of 1.0. Purity and fragmentation of freshly isolated islets were not influenced by the C-ratio. After 24-hr culture performed for quality assessment, a marginal but significant reduction of viability was observed in islets isolated by means of a C-ratio of 0.5 and 1.5. Islet in vitro and posttransplantation function revealed no negative effect mediated by different C-ratios. CONCLUSIONS: The present study demonstrates that the C-ratio is of significant relevance for the outcome after enzymatic rat islet isolation. The data indicate further that purified collagenase class I or class II does not damage islet tissue even if used in excess. The present study can serve as a start for subsequent experiments in the human pancreas.

  • 21.
    Bäck, Jennie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Sanchez, Javier
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Elgue, Graciela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Ekdahl, Kristina Nilsson
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Activated human platelets induce factor XIIa-mediated contact activation2010In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 391, no 1, p. 11-17Article in journal (Refereed)
    Abstract [en]

    Earlier studies have shown that isolated platelets in buffer systems can promote activation of FXII or amplify contact activation, in the presence of a negatively charge substance or material. Still proof is lacking that FXII is activated by platelets in a more physiological environment. In this study we investigate if activated platelets can induce FXII-mediated contact activation and whether this activation affects clot formation in human blood. Human platelets were activated with a thrombin receptor-activating peptide, SFLLRN-amide, in platelet-rich plasma or in whole blood. FXIIa and FXIa in complex with preferentially antithrombin (AT) and to some extent C1-inhibitor (C1INH) were generated in response to TRAP stimulation. This contact activation was independent of surface-mediated contact activation, tissue factor pathway or thrombin. In clotting whole blood FXIIa-AT and FXIa-AT complexes were specifically formed, demonstrating that AT is a potent inhibitor of FXIIa and FXIa generated by platelet activation. Contact activation proteins were analyzed by flow cytometry and FXII, FXI, high-molecular weight kininogen, and prekallikrein were detected on activated platelets. Using chromogenic assays, enzymatic activity of platelet-associated FXIIa, FXIa, and kallikrein were demonstrated. Inhibition of FXIIa in non-anticoagulated blood also prolonged the clotting time. We conclude that platelet activation triggers FXII-mediated contact activation on the surface and in the vicinity of activated platelets. This leads specifically to generation of FXIIa-AT and FXIa-AT complexes, and contributes to clot formation. Activated platelets may thereby constitute an intravascular locus for contact activation, which may explain the recently reported importance of FXII in thrombus formation.

  • 22.
    Caballero-Corbalan, José
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Brandhorst, Heide
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Asif, Sana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Engelse, Marten
    de Koning, Eelco
    Pattou, Francois
    Kerr-Conte, Julie
    Brandhorst, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Mammalian Tissue-Free Liberase: A New GMP-Graded Enzyme Blend for Human Islet Isolation2010In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 90, no 3, p. 332-333Article in journal (Refereed)
  • 23.
    Caballero-Corbalán, José
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Friberg, Andrew S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Brandhorst, Heide
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Andersson, Helene H.
    Felldin, Maria
    Foss, Aksel
    Salmela, Kaija
    Tibell, Annika
    Tufveson, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Brandhorst, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Vitacyte Collagenase HA: A Novel Enzyme Blend for Efficient Human Islet Isolation2009In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 88, no 12, p. 1400-1402Article in journal (Refereed)
  • 24.
    Cabric, Sanja
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Eich, Torsten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Sanchez, Javier
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    A new method for incorporating functional heparin onto the surface of islets of Langerhans2008In: Tissue Engineering. Part C, Methods, ISSN 1937-3384, Vol. 14, no 2, p. 141-147Article in journal (Refereed)
    Abstract [en]

    A novel technique is described to conjugate macromolecular heparin complexes to cell surfaces. The method is based on the dual properties of avidin-expressing binding sites for both biotin and a macromolecular complex of heparin. A quartz crystal microbalance with dissipation monitoring (QCM-D) revealed sequential binding of biotin, avidin, and heparin complexes. Large particle flow cytometry confirmed functional integrity. Confocal microscopy of the heparinized islets showed evenly distributed fluorescence. An in vitro Chandler loop model demonstrated that the biocompatibility of the new method is comparable to the previous method used on artificial materials with regard to coagulation and antithrombin uptake. The technique presented allows human islets of Langerhans to successfully be covered with functional heparin as a means to reduce instant blood-mediated inflammatory reactions induced by the innate immune system.

  • 25.
    Cabric, Sanja
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Elgue, Graciela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Schmidt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Adenovirus-Mediated Expression of the Anticoagulant Hirudin in Human Islets: A Tool to Make the Islets Biocompatible to Blood2006In: Cell Transplantation, ISSN 0963-6897, E-ISSN 1555-3892, Vol. 15, no 8-9, p. 759-767Article in journal (Refereed)
    Abstract [en]

    Human islets induce an injurious clotting reaction at the time of transplantation. A potential strategy to counteract this reaction would be to allow the islets to express hirudin, a protein with direct anticoagulative activity. Human islets were transduced with an adenoviral vector encoding hirudin, an empty corresponding vector, or left untreated. Islet culture supernatants were analyzed for hirudin using an ELISA, a chromogenic substrate assay based on the thrombin-binding properties of hirudin and in a whole blood viscosimetry assay. Immunohistochemical evaluation and determination of hirudin content revealed an abundant expression of hirudin after transduction. Hirudin content in transduced islets was in the range of the insulin content levels. A delay in human whole blood clotting time could be observed after addition of supernatants taken from islet cultures expressing hirudin. However, transduced islets showed an impaired glucose-stimulated insulin release, but could readily be retrieved 6 weeks after transplantation to athymic mice. A marked expression and secretion of hirudin with functional capacity can be induced in human islets using an adenoviral vector. The impairment in glucose-stimulated insulin release in hirudin-secreting islets, compared to controls, indicates that the additional protein synthesis affects the functional capacity of the islets.

  • 26.
    Cabric, Sanja
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Sanchez, Javier
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Johansson, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Magnusson, Peetra U.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Anchoring of vascular endothelial growth factor to surface-immobilized heparin on pancreatic islets: implications for stimulating islet angiogenesis2010In: Tissue engineering. Part A, ISSN 1937-3341, Vol. 16, no 3, p. 961-970Article in journal (Refereed)
    Abstract [en]

    In pancreatic islet transplantation, early revascularization is necessary for long-term graft function. We have shown in in vitro and in vivo models that modification with surface-attached heparin protects the islets from acute attack by the innate immune system of the blood following intraportal islet transplantation. In this study, we have investigated the ability of an immobilized conjugate composed of heparin to bind the angiogenic growth factor vascular endothelial growth factor-A (VEGF-A) as a means of attracting endothelial cells (ECs) to induce angiogenesis and revascularization. We analyzed the capacity of VEGF-A to bind to immobilized heparin and how this affected the proliferation and adherence of ECs to both artificial glass surfaces and islets. Quartz crystal microbalance with dissipation monitoring and slot-blot demonstrated the binding of VEGF-A to heparin-coated surfaces upon which ECs showed protein-dependent proliferation. Also, ECs cultured on heparin-coated glass surfaces exhibited effects upon focal contacts. Heparinized islets combined with VEGF-A demonstrated unaffected insulin release. Further, covering islets with heparin also increased the adhesion of ECs to the islet surface. Immobilized heparin on the islet surface may be a useful anchor molecule for achieving complete coverage of islets with angiogenic growth factors, ultimately improving islet revascularization and engraftment in pancreatic islet transplantation.

  • 27.
    Carlsson, Björn
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Adoptive T Cell Therapy of Viral Infection and Cancer: Ex vivo Expansion of Cytomegalovirus- and Prostate Antigen-specific T Cells2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (TH) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system.

    To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65495-503 peptide, a recombinant adenovirus coding for pp65, in vitro transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8+ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4+ TH cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and TH cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells ex vivo from virtually all stem cell donors for adoptive T cell transfer.

    I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8+ T cells even when starting from undetectable frequencies (<0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8+ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer.

    List of papers
    1.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    2. Simultaneous generation of cytomegalovirus-specific CD8+ and CD4+ T lymphocytes by use of dendritic cells comodified with pp65 mRNA and pp65 protein
    Open this publication in new window or tab >>Simultaneous generation of cytomegalovirus-specific CD8+ and CD4+ T lymphocytes by use of dendritic cells comodified with pp65 mRNA and pp65 protein
    Show others...
    2005 (English)In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 192, no 11, p. 1912-20Article in journal (Refereed) Published
    Abstract [en]

    Cytomegalovirus (CMV) disease remains a severe complication in patients who have undergone transplantation. Viremia can be prevented and treated by the adoptive transfer of donor-derived CMV-directed T cells. To ensure long-term protection against CMV disease, it is important to transfer CMV antigen-specific T cells that represent both the CD8+ and the CD4+ subsets. In the present study, we used as stimulators dendritic cells (DCs) that were electroporated with in vitro-transcribed 5'-capped polyadenylated messenger RNA (mRNA) that encoded the CMV pp65 protein (i.e., pp65 mRNA). These DCs could efficiently activate CMV-directed CD8+ T cells, as assayed by tetramer staining, interferon- gamma production, and cytolytic activity. We also used DCs that were pulsed with a recombinant pp65 protein to activate CMV-directed CD4+ T cells. When DCs were comodified with pp65 mRNA and pp65 protein, large numbers of CMV-directed CD8+ and CD4+ T cells were generated simultaneously. The approach outlined in the present study can be adapted for a clinical protocol that circumvents potential virus-related biohazards and is available to all patients independently of their human leukocyte antigen haplotype.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-92710 (URN)10.1086/497700 (DOI)16267762 (PubMedID)
    Available from: 2005-03-17 Created: 2005-03-17 Last updated: 2017-12-14Bibliographically approved
    3. Generation of cytotoxic T lymphocytes specific for the prostate and breast tissue antigen TARP
    Open this publication in new window or tab >>Generation of cytotoxic T lymphocytes specific for the prostate and breast tissue antigen TARP
    2004 (English)In: The Prostate, ISSN 0270-4137, E-ISSN 1097-0045, Vol. 61, no 2, p. 161-170Article in journal (Refereed) Published
    Abstract [en]

    BACKGROUND: Expansion of cytotoxic T lymphocytes (CTL) directed against peptide epitopes from antigens that are specifically expressed by normal and neoplastic prostate epithelial cells has during the last years emerged as an interesting therapeutic approach to treat advanced prostate cancer. TCRgamma alternate reading frame protein (TARP) is a protein that in males is specifically expressed by normal prostate epithelial cells and prostate cancer cells. We have evaluated TARP for human leukocyte antigen (HLA)-A*0201-restricted peptides capable of triggering TARP-specific CTL. METHODS: Dendritic cells (DC) were pulsed either with synthetic peptides derived from the natural amino acid sequence of TARP or with cognate peptides having enhanced affinity for HLA-A*0201 due to an N-terminal anchor residue substitution. The peptide-pulsed DC were used to stimulate autologous T cells ex vivo. RESULTS: We were able to generate T cells against TARP(27-35) and TARP(4-13) and their mutated counterparts TARP(V28L)(27-35) and TARP(P5L)(4-13). The use of affinity-enhanced peptides resulted in the generation of T cells recognizing target cells displaying either wild-type or mutated peptide. We further show that TARP-specific T cells can be tetramer-sorted and subsequently expanded to large numbers by general T cell stimulation, with retained specificity and activity. Sorted and expanded T cells, obtained by stimulation with TARP(P5L)(4-13), exert moderate lysis of the TARP-expressing prostate cancer cell line, LNCaP, and breast cancer cell line, MCF-7, indicating that the TARP(4-13) epitope may be endogenously processed and presented by TARP-positive, HLA-A*0201-positive cells. CONCLUSIONS: Our findings suggest that synthetic TARP peptides, such as TARP(P5L)(4-13), may play a role in prostate and breast cancer immunotherapy.

    Keywords
    Antigens; Neoplasm/*immunology, Breast Neoplasms/*immunology, Cell Line; Tumor, Dendritic Cells/immunology, Female, Humans, Male, Nuclear Proteins/*immunology, Prostatic Neoplasms/*immunology, T-Lymphocytes; Cytotoxic/*immunology
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-92711 (URN)10.1002/pros.20091 (DOI)15305339 (PubMedID)
    Available from: 2005-03-17 Created: 2005-03-17 Last updated: 2017-12-14Bibliographically approved
    4. Identification of prostate-specific HLA-A*0201-restricted peptides and detection of prostate antigen-directed T cells in the blood of prostate cancer patients
    Open this publication in new window or tab >>Identification of prostate-specific HLA-A*0201-restricted peptides and detection of prostate antigen-directed T cells in the blood of prostate cancer patients
    Show others...
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-92712 (URN)
    Available from: 2005-03-17 Created: 2005-03-17 Last updated: 2010-01-13Bibliographically approved
  • 28.
    Carlsson, Björn
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Bengtsson, Mats
    Malmström, Per-Uno
    Tötterman, Thomas H.
    Essand, Magnus
    Identification of prostate-specific HLA-A*0201-restricted peptides and detection of prostate antigen-directed T cells in the blood of prostate cancer patientsManuscript (Other academic)
  • 29.
    Carlsson, Björn
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Cheng, Wing-Shing
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Tötterman, Thomas H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Essand, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Ex vivo stimulation of cytomegalovirus (CMV)-specific T cells using CMVpp65-modified dendritic cells as stimulators2003In: British Journal of Haematology, ISSN 0007-1048, E-ISSN 1365-2141, Vol. 121, no 3, p. 428-38Article in journal (Refereed)
    Abstract [en]

    Cytomegalovirus (CMV) infection is a dangerous complication in immunosuppressed individuals such as allogeneic stem cell transplant patients. CMV disease can be prevented by the early post-transplant transfer of donor-derived, CMV-directed, T cells. Fast and cost efficient methods to generate CMV-specific T cells are, therefore, warranted. The current study utilized peptide-pulsed and adenovirus-transduced dendritic cells (DC) to generate CMV-restricted T cells. After one stimulation with CMV pp65495-503 peptide-pulsed DC and three re-stimulations with peptide-pulsed monocytes, virtually all T cells were CD8+, expressed the relevant T cell receptor and exhibited high peptide-specific lytic activity. After only one stimulation, pp65495-503-restricted T cells could be sorted to a purity of higher than 95% and expanded up to 1000-fold in 2 weeks. This technique may prove useful for the rapid generation of large quantities of specific cytolytic T lymphocytes (CTL) for cell therapy. DC transduced with an adenoviral vector encoding the full-length pp65 protein (Adpp65) were able to simultaneously expand CTL against multiple epitopes of pp65. In addition, they activated CMV-specific CD4+ T-helper cells. This approach would stimulate multiple-epitope populations of pp65-specific T cells and could be made available to patients of any human leucocyte antigen (HLA) haplotype. DC transduced with adenoviral vectors to express full-length antigens may prove to be potent vaccines against viral pathogens and cancer.

  • 30.
    Carlsson, Björn
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Forsberg, Ole
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Bengtsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Tötterman, Thomas H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Essand, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Characterization of human prostate and breast cancer cell lines for experimental T cell-based immunotherapy2007In: The Prostate, ISSN 0270-4137, E-ISSN 1097-0045, Vol. 67, no 4, p. 389-395Article in journal (Refereed)
    Abstract [en]

    BACKGROUND. In order to develop experimental immunotherapy for prostate and breast cancer it is of outmost importance to have representative target cell lines that through human leukocyte antigen (HLA) class I molecules present relevant levels of peptides from tumor-associated antigens for cytotoxic T lymphocyte (CTL) recognition. METHODS. We sequenced the HLA-A and HLA-B loci of eight commonly used prostate and breast cancer cell lines and analyzed the surface expression of HLA-ABC, HLA-DR, CD40, CD80, CD86, and CD54 by flow cytometry. We also analyzed the cell lines for mRNA expression from 25 genes reported to be specifically or preferentially expressed by prostate cells. RESULTS. Among the analyzed cell lines we found that LNCaP, PC-346C and MCF-7 are HLA-A*0201 positive. However, the HLA-A2 expression level is low and only MCF-7 upregulates HLA-A2 in response to IFN-γ stimulation. MCF-7 also expresses high levels of CD54, which further improve its value as a CTL target cell line. On the other hand, LNCaP and PC-346C express 25 and 23 out of 25 prostate-related genes, respectively, while MCF-7 expresses 16 out of 25 genes. CONCLUSIONS. None of the analyzed prostate cancer cell lines are optimal CTL target cells. However, MCF-7 could in many cases be used as a complement to HLA-A*0201 positive prostate cancer cells. The LNCaP and PC-346C cell lines are rich sources of prostate-related antigens that may be valuable for cancer vaccine development.

  • 31.
    Carlsson, Björn
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Hou, Mingyan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Giandomenico, Valeria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Nilsson, Berith
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Tötterman, Thomas H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Essand, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Simultaneous generation of cytomegalovirus-specific CD8+ and CD4+ T lymphocytes by use of dendritic cells comodified with pp65 mRNA and pp65 protein2005In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 192, no 11, p. 1912-20Article in journal (Refereed)
    Abstract [en]

    Cytomegalovirus (CMV) disease remains a severe complication in patients who have undergone transplantation. Viremia can be prevented and treated by the adoptive transfer of donor-derived CMV-directed T cells. To ensure long-term protection against CMV disease, it is important to transfer CMV antigen-specific T cells that represent both the CD8+ and the CD4+ subsets. In the present study, we used as stimulators dendritic cells (DCs) that were electroporated with in vitro-transcribed 5'-capped polyadenylated messenger RNA (mRNA) that encoded the CMV pp65 protein (i.e., pp65 mRNA). These DCs could efficiently activate CMV-directed CD8+ T cells, as assayed by tetramer staining, interferon- gamma production, and cytolytic activity. We also used DCs that were pulsed with a recombinant pp65 protein to activate CMV-directed CD4+ T cells. When DCs were comodified with pp65 mRNA and pp65 protein, large numbers of CMV-directed CD8+ and CD4+ T cells were generated simultaneously. The approach outlined in the present study can be adapted for a clinical protocol that circumvents potential virus-related biohazards and is available to all patients independently of their human leukocyte antigen haplotype.

  • 32.
    Carlsson, Björn
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Sadeghi, Arian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Bengtsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Wagenius, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Tötterman, Thomas H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Effector T cell analysis of melanoma tumor-infiltrating lymphocyte cultures using HLA-ABC semimatched melanoma cell lines2008In: Journal of immunotherapy (1997), ISSN 1524-9557, E-ISSN 1537-4513, Vol. 31, no 7, p. 633-43Article in journal (Refereed)
    Abstract [en]

    The generation of T cells with specific reactivity against tumor-associated antigens is prerequisite for adoptive transfer therapy. Melanoma-specific lymphocyte cultures can be established from tumor-infiltrating lymphocytes (TILs) by in vitro culture with high levels of interleukin-2. In this report, we present TIL data originating from 728 attempted cultures from 33 consecutive melanoma biopsy specimens originating from 30 patients. Cultures were analyzed for the presence of interferon gamma (IFNgamma)-producing cells upon stimulation with a panel of HLA-ABC semimatched melanoma cell lines. We sought to find whether such cell lines could be used to analyze TIL reactivity. Cell lines were used as stimulators to circumvent the need for autologous primary tumor cells. Melanoma-reactive cultures were identified by flow cytometry in 25 of the 30 patients. Four hundred forty-four of 728 (60.9%) cultures contained TILs at the end of experiment. Ninety-one of 318 cultures (28.6%) contained IFNgamma-producing cells after stimulation. In HLA-A*0201 patients IFNgamma analysis was complemented with melanoma-specific tetramer staining. All but one HLA-A*0201 patient had MART-1/Melan-A27-35-directed TILs, with frequencies ranging from 0.1% to 90% of CD8 cells. In addition, tetramer analysis also identified TILs directed against gp100, Tyrosinase, and Her2Neu. Tumor material was collected via needle biopsy in 16 cases and surgery in 18 cases. Overall, surgical material generated more cultures positive for T cells. The described methods are efficient in characterizing clinically relevant melanoma-reactive TILs.

  • 33.
    Carlsson, Björn
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Tötterman, Thomas H.
    Essand, Magnus
    Generation of cytotoxic T lymphocytes specific for the prostate and breast tissue antigen TARP2004In: The Prostate, ISSN 0270-4137, E-ISSN 1097-0045, Vol. 61, no 2, p. 161-170Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Expansion of cytotoxic T lymphocytes (CTL) directed against peptide epitopes from antigens that are specifically expressed by normal and neoplastic prostate epithelial cells has during the last years emerged as an interesting therapeutic approach to treat advanced prostate cancer. TCRgamma alternate reading frame protein (TARP) is a protein that in males is specifically expressed by normal prostate epithelial cells and prostate cancer cells. We have evaluated TARP for human leukocyte antigen (HLA)-A*0201-restricted peptides capable of triggering TARP-specific CTL. METHODS: Dendritic cells (DC) were pulsed either with synthetic peptides derived from the natural amino acid sequence of TARP or with cognate peptides having enhanced affinity for HLA-A*0201 due to an N-terminal anchor residue substitution. The peptide-pulsed DC were used to stimulate autologous T cells ex vivo. RESULTS: We were able to generate T cells against TARP(27-35) and TARP(4-13) and their mutated counterparts TARP(V28L)(27-35) and TARP(P5L)(4-13). The use of affinity-enhanced peptides resulted in the generation of T cells recognizing target cells displaying either wild-type or mutated peptide. We further show that TARP-specific T cells can be tetramer-sorted and subsequently expanded to large numbers by general T cell stimulation, with retained specificity and activity. Sorted and expanded T cells, obtained by stimulation with TARP(P5L)(4-13), exert moderate lysis of the TARP-expressing prostate cancer cell line, LNCaP, and breast cancer cell line, MCF-7, indicating that the TARP(4-13) epitope may be endogenously processed and presented by TARP-positive, HLA-A*0201-positive cells. CONCLUSIONS: Our findings suggest that synthetic TARP peptides, such as TARP(P5L)(4-13), may play a role in prostate and breast cancer immunotherapy.

  • 34.
    Cheng, Wing-Shing
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    TARP Promoter-Based Prostate Cancer Gene Therapy: From Development to Application2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Prostate cancer is one leading cause of cancer-related death among men in Western countries. The standard therapies for localized prostate cancer include radical prostatectomy and radiation therapy. Such measures are relatively effective in the short term, but many patients ultimately relapse. These patients may benefit from a combination of standard therapy and oncolytic virus therapy. My work aimed to develop viruses for this purpose.

    TARP is a protein that in males is specifically expressed in prostate epithelial and cancer cells. In my thesis, I characterized the TARP promoter and showed that TARP expression is regulated at the transcriptional level by testosterone through binding of the androgen receptor in the proximal TARP promoter. I further developed TARP promoter-based regulatory sequences for prostate-specific gene expression. A sequence comprising a PSA enhancer, a PSMA enhancer and the TARP promoter was constructed and designated PPT. An adenoviral vector containing the PPT sequence shielded from transcriptional interference by an H19 insulator showed high prostate-specific transcriptional activity in human cells both in the presence and absence of testosterone. However, in experimental murine prostate cancer the PPT sequence is not active. Therefore, a two-step transcriptional amplification (TSTA) system was used together with the PPT sequence to develop an adenovirus that confers prostate-specific transgene expression also in murine cells.

    I constructed a conditionally replicating adenovirus where the E1A gene expression is controlled by an H19 insulator-shielded PPT regulatory sequence, Ad[I/PPT-E1A]. This virus exhibited absolute prostate specificity in terms of E1A expression, viral replication and cytolysis in vitro and in vivo. Importantly, our virus is active both in the presence and absence of testosterone, which may prove beneficial for patients treated by hormonal withdrawal.

    Hopefully, my work will improve existing gene therapy strategies for prostate cancer and in the long term improve the prognosis for patients with prostate cancer.

    List of papers
    1. Characterization of the Androgen-Regulated Prostate-Specific TARP Promoter
    Open this publication in new window or tab >>Characterization of the Androgen-Regulated Prostate-Specific TARP Promoter
    2003 In: Endocrinology, ISSN 0013-7227, Vol. 144, no 8, p. 3433-3440Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-92860 (URN)
    Available from: 2005-04-11 Created: 2005-04-11Bibliographically approved
    2. A novel TARP-promoter-based adenovirus against hormone-dependent and hormone-refractory prostate cancer
    Open this publication in new window or tab >>A novel TARP-promoter-based adenovirus against hormone-dependent and hormone-refractory prostate cancer
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    2004 (English)In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 10, no 2, p. 355-364Article in journal (Refereed) Published
    Abstract [en]

    TARP (T cell receptor gamma-chain alternate reading frame protein) is a protein that in males is uniquely expressed in prostate epithelial cells and prostate cancer cells. We have previously shown that the transcriptional activity of a chimeric sequence comprising the TARP promoter (TARPp) and the PSA enhancer (PSAe) is strictly controlled by testosterone and highly restricted to cells of prostate origin. Here we report that a chimeric sequence comprising TARPp and the PSMA enhancer (PSMAe) is highly active in testosterone-deprived prostate cancer cells, while a regulatory sequence comprising PSAe, PSMAe, and TARPp (PPT) has high prostate-specific activity both in the presence and in the absence of testosterone. Therefore, the PPT sequence may, in a gene therapy setting, be beneficial to prostate cancer patients that have been treated with androgen withdrawal. A recombinant adenovirus vector with the PPT sequence, shielded from interfering adenoviral sequences by the mouse H19 insulator, yields high and prostate-specific transgene expression both in cell cultures and when prostate cancer, PC-346C, tumors were grown orthotopically in nude mice. Intravenous virus administration reveals both higher activity and higher selectivity for the insulator-shielded PPT sequence than for the immediate-early CMV promoter. Therefore, we believe that an adenovirus with therapeutic gene expression controlled by an insulator-shielded PPT sequence is a promising candidate for gene therapy of prostate cancer.

    Keywords
    Adenoviridae/*genetics, Animals, Cell Line; Tumor, Enhancer Elements (Genetics)/genetics, Gene Expression Regulation; Neoplastic, Gene Therapy/*methods, Genes; Reporter/genetics, Genetic Vectors/genetics, Humans, Insulator Elements/genetics, Luciferases/analysis/genetics, Male, Mice, Neoplasms; Hormone-Dependent/genetics/*metabolism/therapy, Nuclear Proteins/*genetics, Promoter Regions (Genetics)/*genetics, Prostatic Neoplasms/genetics/*metabolism/therapy, Research Support; Non-U.S. Gov't, Testosterone/metabolism
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-92861 (URN)10.1016/j.ymthe.2004.05.022 (DOI)15294182 (PubMedID)
    Available from: 2005-04-11 Created: 2005-04-11 Last updated: 2017-12-14Bibliographically approved
    3. An oncolytic conditionally replicating adenovirus for hormone-dependent and hormone-independent prostate cancer
    Open this publication in new window or tab >>An oncolytic conditionally replicating adenovirus for hormone-dependent and hormone-independent prostate cancer
    Show others...
    2006 (English)In: Cancer Gene Therapy, ISSN 0929-1903, E-ISSN 1476-5500, Vol. 13, no 1, p. 13-20Article in journal (Refereed) Published
    Abstract [en]

    The use of conditionally replicating adenoviruses offers an attractive complementary treatment strategy for localized prostate cancer. We have produced a replicating adenovirus, Ad[I/PPT-E1A], where E1A gene expression is controlled by a recombinant regulatory sequence designated PPT. The PPT sequence comprises a PSA enhancer, a PSMA enhancer and a T-cell receptor gamma-chain alternate reading frame protein promoter, and it is shielded from transcriptional interference from adenoviral backbone sequences by an H19 insulator. Ad[I/PPT-E1A] yields prostate-specific E1A protein expression, viral replication and cytolysis in vitro. Furthermore, Ad[I/PPT-E1A] considerably regresses the growth of subcutaneous LNCaP prostate cancer tumors in nude mice. Importantly, the viral replication and cytolytic effect of Ad[I/PPT-E1A] are independent of the testosterone levels in the prostate cancer cells. This may be beneficial in a clinical setting since many prostate cancer patients are treated with androgen withdrawal. In conclusion, Ad[I/PPT-E1A] may prove to be useful in the treatment of localized prostate cancer.

    Keywords
    Adenoviridae/*metabolism, Adenovirus E1A Proteins/genetics/metabolism, Animals, Gene Expression Regulation; Neoplastic, Genetic Vectors/metabolism/therapeutic use, Humans, Male, Mice, Mice; Inbred C57BL, Mice; Nude, Neoplasms; Hormone-Dependent/genetics/*metabolism, Prostatic Neoplasms/*metabolism, Testosterone/metabolism, Time Factors, Transfection, Virus Replication
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-92862 (URN)10.1038/sj.cgt.7700881. (DOI)16052227 (PubMedID)
    Available from: 2005-04-11 Created: 2005-04-11 Last updated: 2017-12-14Bibliographically approved
    4. Two-step transcriptional amplification (TSTA) of the PPT regulatory sequence in murine and human prostate cancer cell lines
    Open this publication in new window or tab >>Two-step transcriptional amplification (TSTA) of the PPT regulatory sequence in murine and human prostate cancer cell lines
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-92863 (URN)