Preclinical optimization of melanoma treatment

Melanoma is a malignancy that arises from melanocytes, the pigment-producing cells that can be predominantly found in the eye or the epidermal basal layer of the skin. Mainly due to increased UV exposure, the incidence of melanoma has doubled worldwide over the past three decades (200.000 new cases in 2008). Primary melanomas can be easily treated by surgical resection, leading to a good prognosis for stage I patients. However, metastasized melanoma is almost completely resistant to therapeutic modalities such as radio- and chemotherapy, resulting in a median overall survival of less than one year for this patient group. Despite considerable efforts, for over 20 years there was no melanoma treatment developed that could improve survival of stage IV patients. However, the treatment of unresectable metastasized melanoma has progressed markedly in recent years due to the development of both immunotherapies that stimulate anti-tumor immunity and targeted therapies that block oncogenic proteins. This thesis will focus on pre-clinical work concerning the optimization of melanoma treatment. In detail, it will address for both targeted therapies and immunotherapies factors that play a role in the identification of response-predictive biomarkers, the toxicity of treatments, and the potential efficacy of combination treatments.Sponsors drukkosten proefschrift: Nederlands Kanker Instituut Bayer Healthcare Boehringer IngelheimUBL - phd migration 201

The mouse B cell repertoire : antibody specificities and immunoglobulin (sub) class distribution

The total number of different immunoglobulin (I g) molecules that the immune system produces is often called the antibody specificity repertoire orB cell repertoire (Chapter 1). This repertoire can be subdivided into three categories: the potential, the available and the actual repertoires. The potential repertoire is determined by the number, structure and mechanisms of expression of the germl ine genes encoding lg molecules plus the possible somatic variants derived from them and can be regarded as what potentially can be made. The available repertoire is defined as the set of diverse antibody molecules that are expressed by immunocompetent but resting B lymphocytes and can be looked upon as what has been made and can be used. The actual repertoire is represented by that set of antibodies that is actually secreted by S cells and can be regarded as what is actually being used. Little is known about the regulatory mechanisms that enable the establishment, from the potential repertoire, of the available and functionally expressed repertoire of the immunocompetent resting B cell compartment. Similarly, the mechanisms that govern the establishment of the actual repertoire from the available repertoire are only partly known. Therefore~ the purpose of the studies presented in this thesis (as outlined in Chapter Ill) was to obtain more information concerning the regulatory mechanisms that are involved in the functional expression of the lg C and V genes by murine B cells. To this end, frequency analyses of B cells secreting particular lg heavy chain isotypes (C gene expression) and specific lgM antibodies (V gene expression) were performed among the progeny of B cells that had differentiated from pre-S cells in vitro. The same analyses were performed on in vivo generated mitogen-reactive S cells (available repertoire) and on the 1Spontaneously1 occurring ( 1background') lg-secreting cells (actual repertoire). The possible regulating influences studied include age, T cells and exogenous antigens. The latter became feasible, since, with the successful breeding of germfree mice fed an ultrafiltered solution of chemically defined low molecular weight nutrients, exogenous stimuli such as antigens and mitogens can be reduced to a minimum never attained before

Effect of an inhaled glucocorticoid on reactive oxygen species production by bronchoalveolar lavage cells from smoking COPD patients

Oxidative stress in the lung is important in the pathogenesis of COPD. Published data indicate that glucocorticoids inhibit blood cells in their capacity to produce reactive oxygen species (ROS). We investigated the effect of Fluticasone propionate (FP) on the ROS production capabilities of pulmonary cells. Bronchoalveolar lavage (BAL) was performed in smoking COPD patients, before and after a six month, placebo-controlled treatment with FP. BAL cells were stimulated with phorbol myristrate acetate (PMA) alone, and together with superoxide dismutase (SOD). From kinetic plots of ferricytochrome-c conversion we calculated the maximal rate of superoxide production: Vmax. We also examined BAL cell subsets and performed correlation analyses on ROS production and relevant clinical determinants. Paired results were obtained from 6 FP- and 9 placebo-treated patients. No significant change of Vmax was found in both patient groups. Also BAL cellularity was unchanged. Correlation analyses showed a significant (inverse) association of Vmax with the number of cigarettes smoked per day. We concluded that a potent inhaled glucocorticoid had no effect on the ROS production capability of BAL cells from smoking COPD patients. Apparently, heavy smoking impaired the ability of alveolar macrophages to produce ROS, which was not further decreased by FP