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Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

Envelope proteins of the human immunodefiency virus (HIV) use the cell surface CD-4 molecule of target cells to initiate infection which eventually lead to the acquired immunodeficiency syndrome (AIDS). HIV-1 strains form three groups, namely the M, N and O, with the former group further divided into at least ten equidistant subtypes or clades (i.e. A through J) classified on the basis of sequence homologies in the envelope gene. Recombinant envelope proteins expressed in transfected Chinese hamster ovary (CHO) cells were isolated and purified here (~ 0.01 mg yield). An economical but efficient purification procedure using affinity chromatography and freeze-drying was developed. The results obtained through SDS-PAGE, western blotting, specific ELISA (using Galanthus nivalis a lectin with affinity for ENV glycoproteins) and partial sequencing confirmed the purity (~ 85 - 90 %) and identity of the proteins. Since these proteins were derived in a clade A (Uganda) and B (USA) environment we anticipated limited crossreactivity with immune responses induced in a subtype C (RSA) environment. This was assessed using ELISA (titers of 1000) and western blot analysis. The ability to induce apoptosis was used to demonstrate functionality of the purified protein (Results showed that in-vitro induction of apoptosis (65 %) using the continuous cell line PM1 was achieved). / Dr. Debra Meyer

This dissertation mainly deals with some biochemical aspects regarding the efficacy of novel platinum anticancer compounds, as part of a broader study in which both chemistry and biochemistry are involved. Various novel diamine and N-S donor chelate compounds of platinum II and IV have been developed in which factors such as stereochemistry, ligand exchange rate and biocompatibility were considered as additional parameters. In the first order testing, each of these compounds was tested with reference to their “killing” potential by comparing their rate of killing, over a period of 48 hours with those of cisplatin and oxaliplatin. Some 80 compounds were tested in this way. Although only a few could be regarded as equal to or even better than cisplatin and oxaliplatin, the testing of these compounds on cancer cells provided useful knowledge for the further development of novel compounds. Four of the better compounds, namely Y9, Y14, Y16 and Lt16.2 were selected for further studies to obtain more detailed knowledge of their anticancer action, including some flow cytometric studies. In addition to the above, cisplatin resistant cells were produced for each of the three different cell lines tested, namely, HeLa, HT29 and MCF7 cancer cell lines, by intermittent and incremental exposure to cisplatin (all the cell lines tested became resistant to cisplatin). Each of the selected compounds were exposed to the cells in the same manner, in order to attempt the induction of resistance against these compounds in the three cell lines tested (i.e. whether these cells will become resistant to the various compounds). Each of these selected platinum containing compounds were subsequently tested against the “cisplatin resistant” cell lines in order to determine their efficacy against such cells. One such compound could be singled out, since cervical cancer cells (HeLa cells) do not become resistant to it. This behaviour is similar to that of oxaliplatin against cervical cancer and colon cancer (HT29) cells (oxaliplatin is the number one treatment for colon cancer at present). This compound also proved to be more active against cisplatin resistant cell lines. It was found that all the compounds induced apoptosis in the cell lines tested as well as inhibit the DNA cycle at one or more phase. Finally, an effort was made to evaluate the different compounds by comparing them with respect to their properties relating to anticancer action.

Myasthenia Gravis (MG) is an autoimmune disorder of neuromuscular transmission. Clinically, the disease is manifested by abnormal muscle fatigue with recovery on resting. Circulating nicotinic acetylcholine receptor antibodies (nAchR Ab) are highly characteristic of myasthenia gravis. These antibodies have been shown to be directly pathogenic at the muscle endplate and are responsible for impaired neuromuscular transmission through several mechanisms. While it is clear that the immune system does not function normally in MG, the mechanisms by which the response to nAchR is initiated and perpetuated remain unknown. Moreover, it is not clear whether immunoregulatory defects actually precede development of MG or are secondary features of the disease. The overall goal of the present investigation has been to more clearly define the nature of the immune regulatory defects existing in MG, both at the cellular level and in terms of possible relationship to disease progression. To begin these studies it was necessary to develop an assay that could be used to measure nAchR Ab secreted by lymphocytes in culture. Thus, we modified the original nAchR Ab immunoassay described by Lindstrom (1976) for this purpose. Additionally, in order to gain access to an appropriate patient base for our study, we established a further modification with improved sensitivity for detection of serum nAchR Ab. This important diagnostic test had not been available in this country. Therefore, our assay was made available in Canada for clinical purposes. Through the study of in vitro nAchR Ab and polyclonal IgG secretion by peripheral blood mononuclear cells (PBMNC), we were able to identify two previously unrecognized subgroups of seropositive, generalized MG patients. PBMNC from patients with long disease duration had low capacity for in vitro Ab production (Nonsecretors). Among patients of short disease duration, PBMNC produced nAchR Ab and also secreted higher than normal levels of polyclonal IgG (Secretors). The data suggested that there were nonspecific abnormalities affecting the immune response in myasthenia gravis. Moreover, regulation of B lymphocyte mediated immune function appeared to be related to disease progression. It was hypothesized that circulating auto-antibody may contribute to deregulation of the immune response at certain stages of disease through direct interactions with leukocyte determinants. Separation/reconstitution experiments with CD4+ enriched, T-helper/inducer lymphocytes and B enriched (E- cells) lymphocytes suggested that the control of antibody production in myasthenia gravis was operative at the T-helper/inducer level. Preliminary studies with serum pretreated, CD4+ enriched, T-helper/inducer lymphocytes suggested that serum of Secretor MG patients indeed contained a factor(s) which interfered with the function of a CD4+ lymphocyte subset. We further hypothesized that nAchR Ab would have the potential to behave as anti-lymphocyte Ab if nAchR were expressed on lymphocytes. Accordingly, direct binding studies, using the nicotinic antagonist, alpha-bungarotoxin, were carried out to look for such receptors on PBMNC. Specific, saturable binding of alpha-bungarotoxin to the rhabdomyosarcoma cell line, TE671, was confirmed and characterized. However, in parallel studies, alpha-bungarotoxin binding to PBMNC of healthy individuals or MG patients was not detected. These results suggested that nicotinic acetylcholine receptors, of the type expressed by muscle endplate, do not occur on human peripheral blood mononuclear cells. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Mitogen driven differentiation of mononuclear cells is a useful model of antibody synthesis and secretion in humans. We have studied Pokeweed mitogen (PWM) induced immunoglobulin secretion in vitro in both healthy individuals and multiple sclerosis patients. Within the healthy population we have identified individuals who consistently secrete low levels of IgG in response to PWM and others who secrete very high levels. The underlying mechanisms involved in low response are not well understood. We have observed that the peripheral blood mononuclear cells (PBMC) obtained from low responders differ from those obtained from high responders in each of the following: Their T-helper cell subset contains a higher ratio of T suppressor-inducer cells over T helper-inducer cells; their PBMC contain a higher level of in vivo radiation-sensitive suppression; their PBMC generate a lower autologous mixed lymphocyte response; and their B lymphocytes secrete lower amounts of IgG when mixed with heterologous high responder T helper cells. These results suggest the response involves the interactions between T helper cell subsets, T suppressor cells and B lymphocytes and that the level of response is the sum of the contribution of each subset. PWM induced immunoglobulin secretion was measured in multiple sclerosis patients during different phases of clinical disease activity. Relapsing-remitting multiple sclerosis patients in early relapse secreted less immunoglobulin than patients with prolonged relapse, suggesting that immune function varies with clinical disease activity. Testing the level of PWM induced immunoglobulin secretion in relapsing-remitting multiple sclerosis patients during the clinically stable phase suggested that those patients who secreted high levels of IgG in response to PWM were more likely to suffer a clinical relapse within 6 months than those patients who secreted a low amount. Chronic progressive multiple sclerosis patients secreted higher amounts of immunoglobulin in this assay than healthy control individuals. This group of multiple sclerosis patients also had; (i) reduced Concanavalin A (Con A) suppressor cell activity measured both by the ability to suppress a/ Con A induced proliferation and b/ PWM induced IgG secretion in heterologous cell cultures and; (ii) reduced percentages of T cells expressing T suppressor and T suppressor-inducer markers. The treatment of chronic progressive multiple sclerosis patients in vivo with lymphoblastoid interferon resulted in a dramatic reduction in level of PWM induced immunoglobulin secretion without alteration in Concanavalin A induced suppression or in the percentages of T cells expressing subset specific markers. The PWM induced IgG secretion assay is a valuable technique for investigating the regulation of humoral immunity in both health and disease. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

The progressive growth of tumors in human cancer patients and experimental animals has frequently been associated with a generalized depression of immunological responsiveness. Suppressor cells have been implicated as mediators of tumor-associated immunosuppression, but the identities of the cells causing suppression and the mechanisms by which they act have been unclear. The object of this thesis was thus to determine: if suppressor cells capable of non specifically suppressing immune responses were present in anergic mice bearing methylcholanthrene-induced sarcomas; the cell types responsible for suppression; and the mechanisms by which suppression occurs. The spleens of mice with large tumors were found to contain two distinct populations of non specific suppressor cells. One population inhibited the proliferative responses of normal lymphocytes to the T cell mitogen, Concanavalin A (Con A) and the B cell mitogen, lipopolysaccharide (LPS). These cells also inhibited the generation of antibody forming cells by normal lymphoid cells stimulated in vitro with the T cell dependent antigen, sheep red blood cells (SRBC) and the T cell independent antigen, dinitrophenylated-lipopolysaccharide (DNP-LPS). These suppressor cells appeared to be from the macrophage/monocyte line since they adhered to plastic and nylon wool, were removed by carbonyl iron and magnet, and were inactivated by carragheenan treatment, but were not removed by anti-Thy-1 or anti-mouse immunoglobulin sera plus complement. They were among the less dense spleen cells since they were retained in the light fraction after centrifugation on hypaque-ficoll of specific gravity 1.08 and did not appear to require cell division in order to suppress- since mitomycin C treatment did not inactivate them. Cell-cell contact appeared to be essential for suppression. The second population of suppressor cells, which pelleted to the bottom of a hypaque-ficoll gradient, inhibited only the generation of plaque forming cells to the T cell dependent antigen, SRBC. These cells appeared to be T cells since they were non adherent to plastic or nylon wool, were not removed by carbonyl iron and magnet, but were removed by anti-Thy-1 serum plus complement. Cell division was necessary since suppressive activity was totally removed by mitomycin C treatment. Suppression by this cell type appeared to be mediated by a soluble factor with a molecular weight of about 3,500 to 12,000. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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