<p>Cancer immunotherapy aims at reversing the immunosuppressive tumor environment and enhancing anti-tumor immunity. This thesis comprises studies on murine models for prostate (TRAMP-C2) and bladder (MB49) cancer with the aim to explore if the introduction of an adenoviral vector expressing CD40 ligand (AdCD40L) can induce anti-tumor immune responses.</p><p>We show in subcutaneous mouse models that AdCD40L treatment suppresses tumor growth. Bladder cancer is known to secrete immunosuppressive IL-10 which may inhibit T cell function. We show that introducing AdCD40L into mouse bladder tumors inhibits IL-10 production and reverses immunosuppression. AdCD40L-transduced mouse prostate cancer cells showed caspase activation and reduced cell viability. Vaccination with CD40L-modified prostate cancer cells induces anti-tumor responses and protects mice against rechallenge with native TRAMP-C2 cells. In order to enhance AdCD40L therapy, we explored the possibility of combining it with the histone deacetylase inhibitor FK228, also known as depsipeptide. We show that FK228 upregulates coxsackie and adenovirus receptor expression and thereby enhances adenoviral-mediated CD40L expression in both murine and human prostate cancer cells. Increasing amounts of FK228 or AdCD40L reduces prostate cancer cell viability, while the combined treatment gives at least an additive therapeutic effect. Moreover, we show that AdCD40L transduction of prostate cancer cells induces endogenous CD40 expression and sensitize them for CD40L-mediated therapy.</p><p>In order to conduct prostate-specific gene therapy, prostate-specific promoters can be used to drive transgene expression. However, there are no reports on prostate-specific promoters that are transcriptionally active in mouse cells. Here we show that by using the two-step transcription activation system (TSTA), we can enhance the activity of a recombinant human promoter sequence and obtain activity in mouse prostate cancer cells as well. This finding paves the way for future studies of prostate-specific gene therapy in immunocompetent mouse models.</p>

<p>This thesis deals with recognition and elimination of tumours by T lymphocytes and their use in adoptive immunotherapy.</p><p>The first tumour-draining lymph node; the sentinel node, is identified by peritumoural injection of a tracer. This is the hypothesised location for the activation of tumour-reactive lymphocytes. Accordingly, proliferation and IFN-γ production in response to autologous tumour extract was detected in sentinel nodes from patients with colon and urinary bladder cancer. Reactivity in metastatic nodes was generally lower or absent, but the non-responsiveness could be subdued in long-term cultures by addition of tumour antigen and IL-2. A novel padlock-probe based method was developed for measuring the T cell receptor Vβ repertoire. Common Vβ gene expansions were detected in tumour-infiltrating lymphocytes and sentinel nodes. Thus, tumour antigens are recognised in sentinel nodes by Th1 lymphocytes, resulting in a clonally expanded cell population that can be further propagated <i>ex vivo</i>.</p><p>Regulatory T cells (Tregs) may contribute to tumour-induced immunosuppression. Immunohistochemical stainings against the pan-T cell marker CD3 and Treg marker FOXP3 was performed on tumour tissue from 20 historical urinary bladder cancer patients. The ratio of FOXP3⁺ to CD3⁺ cells was lower in patients alive 7 years post-cystectomy, suggesting that Tregs in bladder cancer have prognostic implications.</p><p>Lymphocytes were isolated from sentinel nodes from sixteen patients with advanced or high-risk colon cancer. <i>In vitro</i> expansion with addition of autologous tumour extract and IL-2 mainly promoted the outgrowth of CD4⁺ Th1 lymphocytes, which were safely re-transfused to the patients. Four patients responded with complete tumour regression. Survival time in the Dukes’ D patients was significantly increased compared with conventionally treated controls (2.6 versus 0.8 years; p=0.048).</p><p>In conclusion, human solid tumours are recognised in sentinel nodes and <i>in vitro</i> expanded sentinel node-acquired CD4⁺ T lymphocytes seem useful in the treatment of patients with disseminated cancer.</p>

Background: Urinary incontinence (UI) and urgency are common conditions and can have a profound influence on many aspects of life. Approximately one in four women has UI and one in ten has daily symptoms. Knowledge is lacking, however, on the impact of UI and urgency on the lives of affected women and their partners and on the situation of women with urinary leakage one year postoperatively. Aims: To study the consequences of female UI and urgency for patients and their partners on quality of life (QoL), the partner relationship, and their sexual lives. Also to evaluate the success rates of three operation methods: tension-free vaginal tape (TVT), tension-free vaginal tape-obturator (TVT-O), and transobturator tape (TOT) for stress urinary incontinence (SUI), with a particular focus on women who still have urinary leakage one year after surgery. Methods: Women seeking healthcare for UI and/or urgency and their partners were invited to answer questionnaires. The women completed disease-specific questionnaires and both the women (n = 206) and their partners (n = 109) answered questions about their psychosocial situation, partner relationship, and sexual life. Patient-reported outcomes one year after surgery with TVT, TVT-O, or TOT (n = 3334) were derived from the Swedish National Quality Register for Gynaecological Surgery. Results: Most of the women reported that their urinary problems negatively affected their physical activities, and almost half reported negative consequences for their social life. Women aged 25–49 years were less satisfied with their psychological health, sexual life, and leisure than women aged 50–74 years. One third of both the women and their partners (all the partners were men) experienced a negative impact on their relationship, and sexual life was negatively affected in almost half of the women and one in five of their men. Coital incontinence was reported in one third of the women. Most of their men did not consider this a problem, but the majority of the affected women did. Satisfaction with outcome of the operation did not differ between TVT, TVT-O, and TOT, but TVT showed a higher success rate for SUI than TOT did. Higher age, higher body mass index, a diagnosis of mixed urinary incontinence, and a history of urinary leakage in combination with urgency each constitute a risk for a lower operation success rate. After one year, 29% of the women still had some form of UI, but half of these were satisfied with the outcome and most reported fewer negative impacts on family, social, working, and sexual life than before the operation. Conclusions: Female UI and/or urgency impaired QoL, particularly in young women, and had negative effects on partner relationships and on some partners’ lives. Sexual life was also affected, more often in women with UI and/or urgency than in their partners. At one-year follow-up after surgery, about one third of the women still had some form of UI, but the negative impact on their lives was reduced. A challenge for health care professionals is to initiate a dialogue with women with urinary symptoms about sexual function and what surgery can realistically be expected to accomplish.

Cancer immunotherapy aims at reversing the immunosuppressive tumor environment and enhancing anti-tumor immunity. This thesis comprises studies on murine models for prostate (TRAMP-C2) and bladder (MB49) cancer with the aim to explore if the introduction of an adenoviral vector expressing CD40 ligand (AdCD40L) can induce anti-tumor immune responses. We show in subcutaneous mouse models that AdCD40L treatment suppresses tumor growth. Bladder cancer is known to secrete immunosuppressive IL-10 which may inhibit T cell function. We show that introducing AdCD40L into mouse bladder tumors inhibits IL-10 production and reverses immunosuppression. AdCD40L-transduced mouse prostate cancer cells showed caspase activation and reduced cell viability. Vaccination with CD40L-modified prostate cancer cells induces anti-tumor responses and protects mice against rechallenge with native TRAMP-C2 cells. In order to enhance AdCD40L therapy, we explored the possibility of combining it with the histone deacetylase inhibitor FK228, also known as depsipeptide. We show that FK228 upregulates coxsackie and adenovirus receptor expression and thereby enhances adenoviral-mediated CD40L expression in both murine and human prostate cancer cells. Increasing amounts of FK228 or AdCD40L reduces prostate cancer cell viability, while the combined treatment gives at least an additive therapeutic effect. Moreover, we show that AdCD40L transduction of prostate cancer cells induces endogenous CD40 expression and sensitize them for CD40L-mediated therapy. In order to conduct prostate-specific gene therapy, prostate-specific promoters can be used to drive transgene expression. However, there are no reports on prostate-specific promoters that are transcriptionally active in mouse cells. Here we show that by using the two-step transcription activation system (TSTA), we can enhance the activity of a recombinant human promoter sequence and obtain activity in mouse prostate cancer cells as well. This finding paves the way for future studies of prostate-specific gene therapy in immunocompetent mouse models.

This thesis deals with recognition and elimination of tumours by T lymphocytes and their use in adoptive immunotherapy. The first tumour-draining lymph node; the sentinel node, is identified by peritumoural injection of a tracer. This is the hypothesised location for the activation of tumour-reactive lymphocytes. Accordingly, proliferation and IFN-γ production in response to autologous tumour extract was detected in sentinel nodes from patients with colon and urinary bladder cancer. Reactivity in metastatic nodes was generally lower or absent, but the non-responsiveness could be subdued in long-term cultures by addition of tumour antigen and IL-2. A novel padlock-probe based method was developed for measuring the T cell receptor Vβ repertoire. Common Vβ gene expansions were detected in tumour-infiltrating lymphocytes and sentinel nodes. Thus, tumour antigens are recognised in sentinel nodes by Th1 lymphocytes, resulting in a clonally expanded cell population that can be further propagated ex vivo. Regulatory T cells (Tregs) may contribute to tumour-induced immunosuppression. Immunohistochemical stainings against the pan-T cell marker CD3 and Treg marker FOXP3 was performed on tumour tissue from 20 historical urinary bladder cancer patients. The ratio of FOXP3+ to CD3+ cells was lower in patients alive 7 years post-cystectomy, suggesting that Tregs in bladder cancer have prognostic implications. Lymphocytes were isolated from sentinel nodes from sixteen patients with advanced or high-risk colon cancer. In vitro expansion with addition of autologous tumour extract and IL-2 mainly promoted the outgrowth of CD4+ Th1 lymphocytes, which were safely re-transfused to the patients. Four patients responded with complete tumour regression. Survival time in the Dukes’ D patients was significantly increased compared with conventionally treated controls (2.6 versus 0.8 years; p=0.048). In conclusion, human solid tumours are recognised in sentinel nodes and in vitro expanded sentinel node-acquired CD4+ T lymphocytes seem useful in the treatment of patients with disseminated cancer.

Spinal cord injury (SCI) remains a significant cause for morbidity and mortality in North America. Bladder dysfunction following SCI is very common and could lead to severe complications including renal failure and autonomic dysreflexia (AD). AD involves life threatening episodes of hypertension in patients with SCI above T6 level. Current management protocols for AD are symptomatic and usually ineffective. Botulinum toxin-A (BTX-A), has been successfully used recently in SCI patients because it reduces the detrusor contractility via inhibiting acetylcholine release from efferent nerve endings. Recent evidence, however, suggests a sensory involvement via modulation of sensory neuropeptides, neurotransmitters, and receptors. It is still, however, unclear whether BTX-A can affect putative spinal neurons involved in AD. In this study we demonstrated that intravesical BTX-A treatment has blocked AD in rats with T4-SCI, and also provided a novel mechanism for the control of autonomic dysreflexia via a minimally invasive treatment modality.

Spinal cord injury (SCI) remains a significant cause for morbidity and mortality in North America. Bladder dysfunction following SCI is very common and could lead to severe complications including renal failure and autonomic dysreflexia (AD). AD involves life threatening episodes of hypertension in patients with SCI above T6 level. Current management protocols for AD are symptomatic and usually ineffective. Botulinum toxin-A (BTX-A), has been successfully used recently in SCI patients because it reduces the detrusor contractility via inhibiting acetylcholine release from efferent nerve endings. Recent evidence, however, suggests a sensory involvement via modulation of sensory neuropeptides, neurotransmitters, and receptors. It is still, however, unclear whether BTX-A can affect putative spinal neurons involved in AD. In this study we demonstrated that intravesical BTX-A treatment has blocked AD in rats with T4-SCI, and also provided a novel mechanism for the control of autonomic dysreflexia via a minimally invasive treatment modality.

<p>The urinary tract is one of the most intractable mucosal surfaces for pathogens to colonize. In addition to the natural barriers at this site, potential pathogens have to contend with the vigorous local innate immune response that is initiated by engagement of surveillance molecule TLRs. TLR4 appears to be not only exclusively expressed on superficial BECs but also critical to triggering robust local innate immune responses. TLR4 recognizes Gram-negative bacterial component LPS and initiates a series of intracellular NF-kappaB associated signaling events resulting in a cytokine response. We examined intracellular signaling events in human BECs leading to the production of IL-6, a major urinary cytokine, following activation by E. coli and isolated LPS, and observed that, in addition to the classical NF-kappaB associated pathway, BEC TLR4 triggers a distinct and more rapid signaling response involving, sequentially, Ca2+, AC3 generated cAMP, and the transcriptional factor CREB. This capacity of BECs to mobilize secondary messengers and evoke a more rapid IL-6 response might be critical in their role as first responders to microbial challenge in the urinary tract. </p><p>Here, we also report two additional distinct TLR4-mediated defense mechanisms in BECs. First, BEC TLR4 inhibits bacterial invasion, a necessary step for successful infection. TLR4-mediated suppression of bacterial invasion was linked to increased intracellular cAMP levels which negatively impacted Rac-1 mediated mobilization of the cytoskeleton. Additionally, we found that BECs continue to fight UPEC even after bacterial invasion by triggering bacterial exocytosis through a distinct TLR4-mediated mechanism following activation by LPS. In addition, we reveal that Caveolin-1, Rab27b, PKA, and MyRIP are components of the exocytic compartment and that they form a complex involved in the exocytosis of bacteria. The ability of TLR4 to mediate the rapid cytokine response, the inhibition of bacterial invasion, and the expulsion of intracellular bacteria from infected cells represents three previously unrecognized functions for this innate immune receptor.</p> / Dissertation

No description available.

INTRODUCTION Benign prostatic hyperplasia (BPH) is the most common pathological condition of aged men which significantly impairs the quality of life status. Though pharmacotherapy with adrenoblockers and 5-alpha reductase inhibitors markedly decreased the rate of surgical interventions, BPH surgery still constitutes the main workload (around ¼ of total) of urologists in the department. The results of the observational study performed by Barry et al. show that the probability of surgical treatment of BPH during the period of 4 years for subjects with mild symptoms equals to 10%, and in case of moderate and severe symptoms - 24% and 39%, respectively. According to the data of large multicenter study performed with 7,588 men in Asia and Australia, moderate and severe symptoms are present in 29%, 40%, and 56% of men in their fifth, sixth, and seventh decade of life, respectively. Symptoms are the main driving force of BPH treatment, but their correlation with residual urine, objective findings of uroflowmetry and invasive urodynamics is only poor. The association of various parameters with the outcomes of surgical treatment is complicated and still not completely investigated. Therefore finding parameters that predict the outcome of surgical BPH treatment is important. According to the literature, unfavorable outcomes of transurethral resection are present in around 15-30% of men with symptomatic BPH. This is mostly associated with inadequate preoperative evaluation, not fully... [to full text]