In the present study, we characterized the ability of a novel oncolytic rhabdovirus - Maraba MG1 to boost Natural Killer (NK) cell activity. In tandem, we addressed the ability of this enhanced NK cell functionality to reduce the incidence of post-cancer surgery micrometastases. Due to the potential safety barriers associated with the use of a live virus immediately prior to surgery in cancer patients, we generated a single cycle replication virus (MG1-Gless) and UV-inactivated MG1 to stimulate NK cell function and reduce post-operative metastases. Our in vivo data demonstrate that significant NK cell activation and a similar level of reduction in postoperative tumor metastases was achieved with live MG1, MG1-Gless and UV-inactivated MG1, concluding that viral replication is important, but not necessary for NK cell activation. Mechanistically, we observed that dendritic cells (DCs) are necessary intermediates for MG1-induced NK cell activation. Finally, we characterized and compared a panel of UV-inactivated MG1 (2mins to 2hrs) to better understand the requirements for NK cell activation. Our results suggest that intact viral particle and cellular recognition and association are essential for NK cell mediated anti-tumor responses. These findings provide the preclinical rationale to develop safe and viable virotherapy-based interventional protocols that might reduce the risk of metastatic disease after cancer surgery.

No description available.

Type 2 (Th2) immune responses are required for immune defence against helminths, but can also have pathogenic effects in allergic conditions. This thesis examined two factors which may influence Th2 immunity at a cellular and molecular level: cross-talk between Natural Killer (NK) cells and dendritic cells (DCs) and the cell surface organisation of DCs. Cross-talk between NK cells and DCs is well-established to impact Th1 responses against tumours and infection; however the influence of this interaction during Th2 inflammation is unknown. To investigate this, human monocyte-derived DCs were stimulated in vitro with different pathogen-associated molecules; LPS or Poly(I:C) which polarise a Th1 response, or soluble egg antigen (SEA) from the helminth worm Schistosoma mansoni, a potent Th2-inducing antigen. These cells were then combined with autologous NK cells. Confocal microscopy showed polarisation of the NK cell microtubule organising centre (MTOC) and accumulation of LFA-1 at contacts between NK cells and immature or Th2-polarising DCs, but not Th1-polarising DCs, indicative of the assembly of an activating immune synapse. NK cells did not lyse DCs treated with LPS or Poly(I:C), but degranulated to and lysed both immature DCs and Th2 polarising DCs. Antibody blockade of NK cell activating receptors NKp30 and DNAM-1 prevented this lysis. Furthermore, depletion of NK cells in mice which were then transferred with Th2 polarising DCs led to an enhanced Th2 recall response. Thus, these data indicate a previously unrecognised role of NK cell cytotoxicity in restricting the pool of DCs involved in Th2 immune responses. Secondly, this thesis investigated the nanoscale organisation of MHC-II on the surface of Th1 and Th2 polarising DCs using ground state depletion super-resolution microscopy. MHC-II was relatively homogenously distributed across the membrane with no significant changes in clustering between immature, Th1 and Th2 polarising DCs. In contrast, imaging CD74, which can mediate internalisation of MHC-II, revealed increased expression and a more homogenous distribution of this receptor on the surface of Th2-polarising DCs compared to Th1-polarising DCs. These data suggest that changes in the clustering of CD74 could modulate MHC-II surface expression during Th2 responses. Overall, the results in this thesis indicate that both molecular and cellular level modulation of DC function contribute to the development of Th2 responses.

The purpose of this study was to investigate natural killer cell activity and the possible role of beta- endorphin in a natural model of autoimmune orchitis, the dark mink. An assay was developed to study natural killer cell activity in the mink and base-line levels of activity in this specie were determined. Natural killer cell activity was assessed in fertile mutation mink, primary infertile dark mink and secondary infertile Utah dark mink with autoimmune orchitis. The study included three sampling times: November, March and April. Natural killer cell activity was significantly lower in mink studied in April than it was in. mink studied in March or November, and there was a significant correlation of activity to fertility. The addition of beta-endorphin to natural killer cell cultures had no effect on activity. A preliminary study was done to determine concentration levels of beta-endorphin in mink plasma, pituitary, hypothalamus and testes. Measurable amounts were found in all tissue types studied. Although the number of samples was too limited to draw significant conclusions, there appears to be a trend toward lower beta-endorphin concentration, in pituitary tissue and plasma samples, of mink with autoimmune orchitis. Beta-endorphin levels did not appear to correlate with natural killer cell activity in the mink. Two assays, the natural killer assay and the blasto-genesis assay, were used to insure that the beta-endorphin preparation used in this study were active. Natural killer cell activity had been reported to be enhanced by the addition of beta-endorphin. In the present study natural killer cell activity of human peripheral blood mononuclear cells (PBMC) was enhanced in the presence of betaendorphin, suggesting that the peptide was active. In addition, the blastogenic response of human PBMC was enhanced by the addition of beta-endorphin to cultures in the present study.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22121号 / 医科博第106号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 濵﨑 洋子, 教授 河本 宏, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

The purpose of this study was to identify the effects of the neurotransmitter, serotonin (SE), on the immune function of peripheral blood mononuclear cells (PBMC) of normal, healthy subjects. This was done as a preliminary investigation to studies on the association of SE with immune changes in autistic subjects. The PBMC isolated from normal male subjects were treated with various concentrations of SE for 48 hrs. Their incubation in SE at a concentration of 10-3 M induced about a 35% decrease in DNA synthesis. However, incubation of the cells in lower concentrations (10-4 to 10-10) of SE produced no significant effect. The ability of natural killer (NK) cells to lyse K562 target cells was also examined after incubation with SE for 48 hrs. The NK activity was almost completely eliminated following incubation in 10-3M of SE, but the activity was not significantly decreased by exposure to lower concentrations of SE. The viability of PBMC was not altered following incubation with SE under identical conditions as those utilized in the NK assay. Preliminary analysis using a fluorescence-activated cell sorter (FACS) of monoclonal antibodies directed against Tll (total T cell), T4 (helper T cell), T8 (suppressor and cytotoxic T cells), B-cell and NK cell markers indicated that the suppressive effect exerted by SE could be attributed to a decrease in the density of these markers or receptors on the cell surface. These findings provide additional evidence for a possible link between neurotransmitters, specifically SE, and immune function.

No description available.

Interferons comprise a class of structurally related proteins which exert several regulatory effects in responsive cells. These effects include the establishment of an antiviral state, the inhibition of cellular proliferation and the alteration of different immune reactions. In particular, the IFN:s rapidly augment the lytic activity of the natural killer (NK) cells. In the present thesis, some of the functional and molecular mechanisms by which IFN:s act on NK cells and other leucocytes are studied. A good correlation is found between the ability of different tumor cell lines to induce IFN production among peripheral blood lymphocytes and their sensitivity to NK cell cytotoxicity, indicating that IFN might regulate the activity of NK cells through a positive feed-back mechanism. When studying the interaction between the NK cells and two target cell lines it is demonstrated that the two cell lines are not recognized by the same receptors. The augmentation of NK cell cytotoxicity by IFN is shown to involve both alteration of receptor structures on the NK cell and enhancement of steps in their lytic machinery. The effects of IFN on the synthesis of individual proteins is then studied by two-dimensional electrophoresis. It is demonstrated that IFN-a and IFN-ß within 1.5 hours induce the synthesis of nine proteins (Mf80, 75, 62, 58, 53, 38, 36, 33 and 30 kD) in human lymphocytes. Tne induction is dependent on a rapid de novo RNA synthesis, which is initiated less than 30 minutes after the addition of IFN. The expression of the nine proteins is well correlated to the development of augmented NK cell cytotoxicity. Four of the proteins (Mr 80, 62, 38 and 33 kD) are found to be expressed in a panel of ten hematopoetic and two anchorage-dependent cell lines, whereas the remaining proteins seem to be expressed in leucocytes only. IFN induce the synthesis of the same proteins in both purified large granular lymphocytes (responsible for the main NK cell activity in man), T cells and monocytes, demonstrating that the augmentation of NK cell activity does not involve the formation of unique 1NK-cel11 specific proteins. Rather, the augmentation of the lytic activity of both NK cells, cytotoxic T cells and monocytes seem to involve common stages in their lytic mechanisms. In contrast to IFN-a and IFN-ß, IFN-y, does not induce any detectable proteins in either NK cells or T cells. This lack of effect of IFN-y on the protein synthesis is not a general phenomenon, since the effects of IFN-a and IFN-y are similar 1n a glioma cell line. These results demonstrate that there exists at least one pathway to augment the NK cell cytotoxicity which does not involve the increased synthesis of the nine IFN-a/IFN-ß inducible proteins and indicates that either these proteins are mainly involved in other effects of IFN, or that the augmentation by IFN-a/IFN-ß and IFN-y involve different pathways. When the effects of IFN-a on the synthesis of membrane-associated proteins is studied, it is demonstrated that only the 80 kD IFN-a inducible protein is associated with the cell membrane. In addition, IFN-a seems to induce three additional, me mb rane-as so ci a ted proteins (Mr 94, 76 and 66 kD) which are not detected in whole cell lysates. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1985, härtill 5 uppsatser.</p> / digitalisering@umu

Islet transplantation has the potential to be an effective treatment for patients with type 1 diabetes. However, a shortage of human donor islets and the need for continuous immunosuppressive therapy currently limit this therapy to patients with brittle type 1 diabetes. Neonatal pigs may provide an unlimited source of islets for transplantation; however, the barrier of islet xenograft rejection must still be overcome. Understanding the mechanism of neonatal porcine islet (NPI) rejection will help to develop targeted therapies to prevent rejection. This thesis studied the early immune cells and molecules involved in NPI xenograft rejection, compared the role of NK cells in two models of islet xenotransplantation and investigated the role of T cell co-stimulatory and adhesion pathways in NPI xenograft rejection. Targeting these aspects of the immune response to NPI xenografts with short-term therapies may play a role in improving NPI xenograft acceptance and induce long-term xenograft survival.