This study examined how the monoblast-like human histiocytic lymphoma cell line U937 can be induced by phorbol 12-myristrate 13-acetate (PMA) to undergo differentiation. In order to study the mechanism of action of CSF-1, a CSF-1 receptor gene (c-fms) was transfected into U937 cells. Exogenous CSF-1 treatment induced an autocrine response in this CSF-1 was determined and all events were shown to be time dependent. CSF-1 stimulation also enhanced proto-oncogene c-jun and c-myc gene expression. Complementary DNA coding for Jun or Fos was introduced into U937 cells by transfection. The transfection did not generate a high level of CSF-1 gene expression which suggests that Fos and Jun alone are insufficient to induce CSF-1 synthesis.

Conditioned media prepared from umbilical cord (UC) segments or endothelial cells (EC) contain colony stimulating activity, Both UCCM and ECCM were partially purified by DEAE-Sepharose and ACA44 gel filtration chromatography. The molecular weights were estimated as 25,000 and 31,000 for UC-CSF and EC-CSF, respectively. UC-CSF was further fractionated by Con A Sepharose, IEF and HPLC on a hydrophobic phenyl column. The highly purified CSF stimulates human macrophage and granulocyte colony formation, indicating it is GM-CSF in nature. Characterization studies have revealed that both CSFs are heat stable at 60°C for 30 min. They are sensitive to digestion by protease and to periodate oxidation but are stable to treatment with sulfhydryl reagents. The synthesis of CSF in endothelial cells is inhibited by actinomycin D, cycloheximide and puromycin, indicating that protein and RNA synthesis are required for CSF production. Among the mitogens tested, only LPS exhibited stimulatory activity on the production of CSF. Metabolic modulators such as dibutyryl cAMP, isobutylmethylxanthine, PGE2 and lactoferrin inhibit CSF production, while PGF2 enhances CSF production.

Macrophages are a natural target cell for HIV-1 infection, and they contribute to the development of disease as they are important for transmission, dissemination and persistence of the virus in an infected patient. Macrophages are less well-studied than T cells and cell lines in relation to HIV-1 infection, yet macrophages are highly specialised and key aspects of the HIV-1 life cycle in these cells are already known to differ compared to other cell types. HIV-1 entry into macrophages has been suggested to occur by macropinocytosis, however the entry route in these cells has not been fully characterised. In this thesis I have tested a panel of pharmacological inhibitors of cellular proteins and uptake pathways, in order to delineate the requirements for HIV-1 entry into macrophages and to determine the nature of the entry route. My findings suggest that the following host factors are important for entry; membrane cholesterol, actin rearrangements, dynamin, sodium-hydrogen exchange, Pak1, and Rac. Other factors including clathrin, PI-3 kinase, Rho kinase and some isoforms of PKC were found to be dispensable for infection or to inhibit infection. Macrophages are a heterogeneous group of cells, and tissue macrophages from different parts of the body differ in their morphology, phenotype and function. I have used the growth factors M-CSF and GM-CSF to direct monocytes to differentiate into distinct types of macrophage. This allowed me to determine that different macrophages differ in their susceptibility to infection and in their ability to support replication. This is likely to be due to variation in HIV-1 receptor expression and the levels of key HIV-1 transcription factors, respectively. Overall this thesis contributes to existing knowledge regarding HIV-1 infection of macrophages. These findings may assist with the design of entry inhibitors, and with therapies designed to eradicate HIV-1 from infected individuals.

Recombinant human macrophage colony stimulating factor beta (rhm-CSFβ) is a glycoprotein that stimulates the proliferation, differentiation and survival of cells belonging to the monocyte-macrophage lineage. It contains nine inter-subunit and intra-subunit disulfide bonds and represents an excellent model system for studying disulfide bond formation during protein folding because the assembly of its monomeric subunits and the maturation of its biological activity depend on the progressive formation of the correct disulfide structure during in vitro folding. Knowledge obtained from these studies can be potentially useful in understanding the roles of disulfide bond formation during protein folding in general. rhm-CSF8 was modified by partial reduction of disulfide bonds, yielding CN¹⁵⁷'¹⁵⁹-modified rhm-CSFβ. The modification did not affect the biological activity, stability, or the overall conformation of the protein. However, the C-terminal regions near the modification sites were shown to exhibit faster deuterium exchange behavior as a result of the chemical modification, indicating that the C-terminal regions became more flexible. Folding kinetics of rhm-CSFβ and CN¹⁵⁷'¹⁵⁹-modified rhm-CSFβ were shown to be essentially the same, suggesting that the modification did not affect the folding kinetics of the oxidized rhm-CSFβ. The denatured and reduced rhm-CSFβ was refolded with the aid of a chemical oxidant. The data indicated that the in vitro folding rhm-CSFβ proceeded via multiple pathways involving monomeric and dimeric intermediates. Disulfide bond shuffling catalyzed by GSH/GSSG represented an important isomerization step in folding. A dimeric intermediate, D-SS8-cam2, was isolated and identified as a kinetic trap, perhaps requiring significant structural arrangement to convert to the native protein. The heterogeneous folding mixture detected by both disulfide bond quenching and H/D pulsed labeling indicate that rhm -CSFβ folding is a diffusion like process as described by the folding funnel model. / Graduation date: 2003

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.

Includes nine pages of amendments. / Bibliography: leaves 140-211. / 211, [200] leaves, [12] 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. / Aims to determine the reason for the lack of ability of many myeloid leukaemic cell populations to stimulate allogeneic lymphocytes in mixed leucocyte culture (MLC), with a view to manipulating the immunogenicity of these cells for therapeutic purposes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995

L’immunologie de la reproduction englobe les principes de l’immunologie générale et les aspects spécifiques de la reproduction et du développement. Les Colony Stimulating Factors (CSFs) sont une illustration de l'application médicale de ce domaine. Dans la famille des CSFs, le Granulocyte-Colony Stimulating Factor (G-CSF) apparaît aujourd'hui comme une thérapie innovante dans divers cas d'échec de la reproduction, bien que ses cibles et ses effets ne soient pas encore clairement établis. Dans ce travail, à travers une revue sur les CSFs dans la reproduction, une étude consacrée aux gènes cibles du G-CSF dans l'endomètre humain, et une étude consacrée aux effets de la supplémentation systémique en G-CSF sur l’implantation embryonnaire murine, nous avons essayé d'approcher certains mécanismes d'action possibles pour cette cytokine. Dans les modèles murins fertiles et pro-abortifs, la supplémentation systémique en G-CSF, ciblant spécifiquement l’endomètre préimplantatoire, modifie les taux d’implantation embryonnaire. Dans l’endomètre humain, certaines dérégulations préimplantatoires de gènes cibles du G-CSF ont également été observées chez les patients infertiles. L'influence du G-CSF sur ces gènes cibles a été également illustrée dans un modèle ex-vivo de culture endométriale. Ces cibles dont l’expression est influencée par le G-CSF sont décrites comme des molécules clés dans le processus implantatoire, intervenant sur l’adhésion embryonnaire, la migration cellulaire, le remodelage des tissus et l'angiogenèse locale. Ces données suggèrent des possibilités de diagnostic préventif et pré-conceptionnel de certains échecs de reproduction, considérés jusqu’à maintenant comme idiopathiques, et de thérapies innovantes orientées, afin d’optimiser la réceptivité du biosenseur endométrial afin de permettre une implantation embryonnaire harmonieuse et une grossesse évolutive. / Reproductive Immunology involves general immunology principles and special aspects of reproduction and development. Colony Stimulating Factors (CSFs) are an illustration of the medical application of this domain. In the CSF family, Granulocyte-Colony Stimulating Factor (G-CSF) appears today as a promising therapy in various cases of reproductive failure although its targets and effects are not clearly established. In this work, through a review on CSFs in reproduction, a study dedicated to human endometrial targets of G-CSF, and a study dedicated to systemic G-CSF supplementation effects on murine embryo implantation, we tried to approach some possible mechanisms of action of this cytokine. In the considered non-abortive and abortion-prone murine models, the timed systemic G-CSF supplementation, targeting specifically the pre implantation endometrium, influenced the embryo implantation process. Some pre conceptual human endometrial dysregulations of G-CSF target genes were also observed in infertile patients. The endometrial influence of G-CSF on these target genes was also illustrated in an ex-vivo model. These molecules under G-CSF influence are described as critically involved in embryo implantation process, by influencing embryo adhesion, cell migration, tissue remodelling and angiogenesis. These data suggest possible pre-conceptual preventive diagnosis of such reproductive failures and future orientated therapies to optimise the endometrial biosensor and the further embryo implantation and ongoing pregnancy.

Disertaciniame darbe analizuojama ankstyvo ir metastazavusio krūties vėžio prognozė priklausomai nuo skirtos chemoterapijos dozės ir molekulinių žymenų. Tyrimas susidėjo iš trijų etapų. I tyrimo etape buvo atlikta molekulinių žymenų (p53 baltymo, HER2, estrogeno ir progesterono receptorių) prognozinės vertės analizė retrospektyviojo ankstyvo krūties vėžio tyrimo metu, kartu įvertinta sumažintos chemoterapijos dozės įtaka ligos išeitims. II tyrimo etape ištirtas naujo rGKSF filgrastimo saugumas ir veiksmingumas gydant chemoterapija metastazavusį krūties vėžį. III tyrimo etape atlikta molekulinių žymenų prognozinės vertės analizė, eliminavus chemoterapijos dozės įtaką klinikinėms ligos išeitims, gydymą optimalia cheminio preparato doze užtikrinus prospekyviajame daugiacentriame naujo rGKSF saugumo ir veiksmingumo tyrime. Darbo uždaviniai 1. Nustatyti antraciklinų dozės įtaką sergančiųjų ankstyvu krūties vė¬žiu išgyvenamumui. 2. Ištirti p53 baltymo raiškos, kitų molekulinių žymenų ir gydymo ypatumų įtaką ankstyvo krūties vėžio prognozei. 3. Įvertinti febrilios neutropenijos profilaktikai skiriamo naujo rGKSF saugumą, gydant metastazavusį krūties vėžį. 4. Įvertinti febrilios neutropenijos profilaktikai skiriamo naujo rGKSF veiksmingumą, gydant metastazavusį krūties vėžį. 5. Nustatyti metastazavusiu krūties vėžiu sergančių pacienčių išgyvenamumo priklausomybę nuo atsako į skiriamą gydymą. 6. Įvertinti krūties vėžio prognozę įtakojančių molekulinių žymenų tarpusavio sąsajas. / This study investigated the clinical outcomes in early and metastatic breast cancer patients depending on chemotherapy and molecular markers. The study comprised of 3 parts: Part I - retrospective investigation of molecular markers (p53 protein, HER2 receptors, estrogene and progesterone receptors) and chemotherapy dose reduction in early stage breast cancer; Part II - prospective open-label multicenter phase IV clinical trial on safety and effectiveness of GCSF used in chemotherapy of metastatic breast cancer; Part III - investigation of prognostic value of p53 protein expression and other molecular markers in metastatic breast cancer when optimal chemotherapy dose was adjusted in prospective multicenter trial of new rGCSF on safety and effectiveness used in chemotherapy of metastatic breast cancer. Objectives: 1. To establish the influence of anthracycline dose on survival of early stage breast cancer patients. 2. To evaluate the influence of p53 protein expression and other molecular markers as well as treatment on prognosis of early stage breast cancer. 3. To establish the safety of new rGCSF prescribed for prevention of febrile neutropenia in chemotherapy of metastatic breast cancer. 4. To analyze the effectiveness of new rGCSF prescribed for prevention of febrile neutropenia in chemotherapy of metastatic breast cancer. 5. To establish the relationship between response to treatment and survival in metastatic breast cancer patients. 6. To evaluate the associations among... [to full text]

Typescript (Photocopy) / Bibliography: leaves 170-198. / xx, 198 leaves, 1 leaf of col. plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1991