G-CSF besitzt direkte Effekte auf die Aktivierung bakterizider Eigenschaften neutrophiler Granulozyten und verbessert das Überleben bakteriell infizierter Tiere. Daher wurde in der hier vorliegenden Arbeit der Effekt einer prophylaktischen oder therapeutischen Gabe von G-CSF bei experimenteller Pneumonie durch Klebsiella pneumoniae in Mäusen untersucht. Unerwarteterweise verschlechterte aber eine prophylaktische G-CSF-Gabe das Überleben und führte dosisabhängig zu einer Steigerung der bakteriellen Dissemination von der Lunge in Leber und Milz. Im Gegensatz dazu konnte ein spezifisch gegen K2-Kapselpolysaccharid (K2-KPS) von K. pneumoniae gerichteter monoklonaler Antikörper signifikant die Vermehrung der Bakterien in Lunge, Leber und Milz reduzieren. Die Blockierung von TNF?? durch Pentoxifyllin hingegen verzögerte die Letalität nach Induktion der Pneumonie, verhinderte sie jedoch nicht. In vitro konnte hier nachgewiesen werden, daß G-CSF spezifisch an K. pneumoniae bindet und daß diese Bindung an mehrere Proteine mit einem Molekulargewicht von 41, 25 und 21 kDa erfolgt. Die Bindung von G-CSF an K. pneumoniae führte zu einer signifikant erhöhten Produktion des wichtigsten Virulenzfaktors, K2-KPS. Dies verminderte in vitro signifikant eine Phagozytose der Bakterien durch neutrophile Granulozyten. Damit gelang es zum ersten Mal, die Bindung von G-CSF an ein gram-negatives Bakterium, K. pneumoniae, nachzuweisen und zu zeigen, daß diese Bindung in vitro zu einer erhöhten Produktion des wichtigsten Virulenzfaktors und in vivo zur Verschlechterung einer experimentellen Pneumonie durch erhöhte bakterielle Disseminierung bei prophylaktischer Gabe von G-CSF vor Infektion führt. Die weitere Untersuchung dieser Phänomene hinsichtlich einer möglichen Bindung von G-CSF auch an andere Bakterien könnte zu einer differenzierten supportiven Therapie bakterieller Infektionen mit G-CSF in nicht neutropenischen Patienten führen. / Besides its well-established effects on granulocytopoiesis, granulocyte colony-stimulating factor (G-CSF) has been shown to have direct effects on the recruitment and bactericidal ability of neutrophils, resulting in improved survival of experimentally infected animals. The effect of G-CSF on the course of experimental pneumonia induced by Klebsiella pneumoniae was studied. Using a highly reproducible murine model, the paradoxical finding that mortality from infection was significantly increased when animals received G-CSF before induction of pneumonia could be demonstrated. Administration of G-CSF promoted replication of bacteria in the liver and spleen, thus indicating an impairment rather than an enhancement of antibacterial mechanisms. By contrast, a monoclonal antibody against Klebsiella K2 capsule significantly reduced bacterial multiplication in the lung, liver, and spleen, and abrogated the increased mortality caused by G-CSF. Blocking of TNF-? with pentoxifylline, however, could not prevent increased mortality caused by G-CSF. In vitro studies showed a direct effect of G-CSF on K pneumoniae resulting in inreased capsular polysaccharide (CPS) production. When bacteria were coincubated with therapeutically achievable concentrations of G-CSF, phagocytic uptake and killing by neutrophils was impaired. Western blot analysis showed three binding sites of G-CSF to K pneumoniae. Thus, in this model, the direct effect of G-CSF on a bacterial virulence factor, CPS production, outweighed any beneficial effect of G-CSF on recruitment and stimulation of leukocytes. Further investigations of possible binding of G-CSF to other bacteria might influence a differentiated supportive therapy of bacterial infections in non-neutropenic patients with this growth factor.

Les cellules Natural Killer (NK) sont capables de lyser les cellules tumorales sans la nécessité de reconnaitre un antigène tumoral spécifique. Cette propriété leur confère un avantage par rapport aux lymphocytes T et les rend intéressantes à utiliser en tant que cellules effectrices pour l’immunothérapie adoptive. A ce jour, le potentiel thérapeutique des cellules NK n’a pas été complétement exploré notamment dans le contexte du traitement de la rechute post-allogreffe de cellules souches hématopoïétiques. Actuellement, les patients en rechute post-greffe sont traités avec des injections de lymphocytes du donneur (DLI) parfois issues de petites fractions du greffon de cellules souches hématopoïétiques congelées. Les cellules souches périphériques étant fréquemment utilisées comme source de cellules souches et parfois utilisées comme DLI, nous avons souhaité évaluer l’impact du G-CSF sur le phénotype et les fonctions des cellules NK présentes dans ces fractions. Dans cet objectif, nous avons comparé différentes sources de cellules NK isolées à partir de sang de donneurs sains, de sang mobilisé de donneurs sains ou de patients et observé l’évolution des différentes sous-populations de cellules NK issues de ces prélèvements au décours d’une expansion en présence d’IL-15. Nos résultats ont montré que l’administration de G-CSF diminuait la proportion de cellules NK CD56brightCD16+ au profit d’une population CD16-, diminuait la prolifération des cellules NK lors de l’expansion en culture, et modifiait les propriétés fonctionnelles des cellules NK. / The ability of natural killer (NK) cells to kill tumor cells without the need to recognize a tumor-specific antigen provides advantages over T cells and makes them appealing for a use as effectors for adoptive immunotherapy. However, the full therapeutic potential of NK cell-based immunotherapy has not been fully investigated in the context of leukemic relapse after hematopoietic stem cell transplantation. Today, patients relapsing after hematopoietic stem cell transplantation are often treated with donor lymphocyte infusion (DLI) based on small cell fractions frozen at the time of the stem cell transplantation. Since peripheral blood stem cells are increasingly used as stem cell source and as source of cells for DLI, we aimed to evaluate the impact of G-SCF mobilization on NK cell phenotype and functions. Therefore, we compared the expansion capacity, the phenotype and the function of NK cells from blood for healthy donors, from allogeneic HSCT healthy donors or from autologous HSCT from patients. We also determine the impact of G-CSF on NK cell subset repartition before and after expansion in presence of IL-15. Our results showed that G-CSF administration to patients decreases CD56brightCD16+ NK cell population, proliferation and function. Overcoming this impairment in lymphoid capacity may be important to facilitate post-transplant immunotherapy.

published_or_final_version / Medicine / Master / Master of Philosophy

Human colony-stimulating factor-1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line. The four-step procedure included chromatography on DEAE Sepharose, Con A Sepharose and HPLC on phenyl column and reverse-phase C-3 column. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS—PAGE) as a single diffuse band with a molecular weight (Mr) of 42,000-50,000 and was further confirmed by a single amino-terminal amino acid residue of glutamate. Under reducing conditions, purified CSF-1 appeared on SDS-PAGE as a single protein band with a Mr of 21,000-25,000 and concurrently lost its biological activity, indicating that human CSF-1 consists of two similar subunits and that the intact quaternary structure is essential for biological activity. When treated with neuraminidase and endo-8~D~N—acetylglucosaminidase D, the Mr of CSF-1 was reduced to 36,000-40,000 and to a Mr of 18,000-20,000 in the presence of mercaptoethanol.

Recent studies suggest that synthesis of the Colony-stimulating factor (CSF) is a well regulated process. However, the molecular mechanisms of the signal transduction of the various inducers of CSF such as monokines and lymphokines are not well understood. Using Interleukin 1 (IL-1) stimulation of CSF-1 in the MIA PaCa-2 cell line as a model system, the involvement of G-protein has been studied. The IL-1 induction of CSF-1 synthesis can be inhibited by both Pertussis toxin and Cholera toxin, which are known to modify the Gᵢ and Gₛ proteins respectively, thus activating adenylate cyclase to release more cAMP. The toxin inactivation can be prevented by inhibitors of the ADP-ribosylation such as, benzamide and MBAMG. Addition of dibutyryl-cAMP inhibits the IL-1 induced CSF production. Both Theophylline and Forskolin which increase cAMP by inhibiting phosphodiesterase and stimulating adenylate cyclase respectively, also inhibit CSF-1 production. Results from these studies have shown that cAMP level inversely regulates the biosynthesis of CSF-1. Preincubation of MIA PaCa-2 cells with IL-1 and 5'- guanylylimidodiphosphate (GppNHp) prevents the inhibitory effect of pertussis toxin on CSF-1 production. These data are consistent with the hypothesis that IL-1 binds to its receptor and couples to Gᵢ∝ resulting in the inhibition of adenylate cyclase and reducing cAMP level. Lowering of the' cAMP level leads to the activation of CSF-1 gene expression. The activity of another inducer of CSF-1 production in this system, 12-0-tetradecanoylphorbol-13-acetate (TPA), can be abolished by 1- (5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), which is a specific inhibitor of protein kinase C. However, H-7 failed to inhibit IL-1 stimulated CSF-1 production. Other known activators of protein kinase C namely, Ca²⁺ and L-α-l-oleoyl-2-acetoyl-sn- 3-glycerol (OAG), also increase CSF production. On the other hand, Indomethacin which is known to inhibit prostaglandin E (PGE), stimulates CSF-1 production in MIA PaCa-2 cells. These data suggest that different mechanisms for stimulation of CSF-1 synthesis exist in MIA PaCa-2 cells depending on the inducer. The IL-1 stimulated pathway which does not require PKC activity and appears to be associated with adenylyl cyclase regulation whereas phorbol ester induced pathway involves protein kinase C in the signaling process as expected.

Copies of author's previously published articles inserted. / Includes bibliographical references. / vi, 135, [109] leaves, [23] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Studies the structure-function properties of the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF) in order to generate molecules with novel biological properties. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995

Addendum pasted on front end papers. / Includes bibliographies. / 109, [99] leaves, [5] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes the investigation as to whether the mouse granulocyte-macrophage colony-stimulating factor and interleukin-3 genes are regulated in a similar manner as those of the human, focussing on regulation through an enhancer. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1996

Copies of author's previous publications inserted. / Bibliography: leaves 153-182. / xv, 183, [10] leaves, [27] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / From experimental data presented, combined with molecular modelling, proposes a hexameric model of active IL-3, IL-5 and GM-CSF receptor complexes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1998?

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.